Jbc Hist Persp Glycobiology

HISTORICAL PERSPECTIVES

Glycobiology & Carbohydrates

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PROLOGUE

H1 JBC Historical Perspectives: Glycobiology and Carbohydrates.Nicole Kresge, Robert D. Simoni, and Robert L. Hill

CLASSICS

H3 Benedict’s Solution, a Reagent for Measuring Reducing Sugars:the Clinical Chemistry Work of Stanley R. Benedict

H5 The Discovery of Hyaluronan by Karl Meyer

H7 Otto Fritz Meyerhof and the Elucidation of the GlycolyticPathway

H10 Luis F. Leloir and the Biosynthesis of Saccharides

H13 Bernard L. Horecker’s Contributions to Elucidating the PentosePhosphate Pathway

H15 The Entner-Doudoroff Pathway for Glucose Degradation: theWork of Michael Doudoroff

H17 Albert Dorfman and the Biosynthesis of Hyaluronic Acid

H20 Hexosamine Metabolism, Sialic Acids, and thePhosphotransferase System: Saul Roseman’s Contributions toGlycobiology

H23 The Regulation of Glucose Uptake in Muscle: the Work ofCharles R. Park

H26 Plant Carbohydrates and the Biosynthesis of Lactose: the Workof William Zev Hassid

H29 The Control of Gluconeogenesis: the Work of John Exton

H31 Mycobacterial Glycophosphoinositides: the Work of Clinton E.Ballou

H34 Proteoglycans and Orchids: the Work of Vincent Hascall

H37 Lactose Synthesis in the Mammary Gland: Lactose Synthaseand the Work of Robert L. Hill

H40 Lysosomal Storage Disease Factors: the Work of Elizabeth F.Neufeld

H43 The Biosynthesis of Membrane Glycoproteins: the Work ofWilliam J. Lennarz

H45 Hepatic Carbohydrate Binding Proteins and GlycoproteinCatabolism: the Work of Gilbert G. Ashwell

H47 The Pathway of Complex Oligosaccharide Biosynthesis: theWork of Stuart A. Kornfeld

H49 The Isolation and Localization of Laminin by Rupert Timpl

H51 The Formation of N-Glycosidic Linkages: the Work of PhillipsW. Robbins

H53 The Transient Glucosylation of Glycoproteins: the Work ofArmando J. Parodi

REFLECTIONS

H56 Reflections on Glycobiology. Saul Roseman

H72 The Pentose Phosphate Pathway. Bernard L. Horecker

H79 My Brief Encounter with the Phosphoinositides and IP3. ClintonE. Ballou

H87 Lectins: Carbohydrate-specific Reagents and BiologicalRecognition Molecules. Nathan Sharon

H99 In Search of the Message. John H. Exton

H108 From the �-Glutamyl Cycle to the Glycan Cycle: A Road withMany Turns and Pleasant Surprises. Naoyuki Taniguchi

The Journal of Biological ChemistryTABLE OF CONTENTS

HISTORICAL PERSPECTIVES ON GLYCOBIOLOGYAND CARBOHYDRATES

2010

JOURNAL OF BIOLOGICAL CHEMISTRY i

JBC Historical Perspectives: Glycobiology and Carbohydrates*

Nicole Kresge, Robert D. Simoni, and Robert L. Hill

Defined in the broadest sense, glycobiology is the study ofthe roles of carbohydrates in cellular life. Carbohydrates arethe primary products of plant photosynthesis and the meta-bolic precursors of all other organic compounds. Often theyare covalently bound to proteins (glycoproteins) and lipids(glycolipids) to form glycoconjugates. Many glycoconjugateshave structural roles. However, the carbohydrate groups ofglycoconjugates also can be involved in cellular processesincluding adhesion, transformation, growth, endocytosis,and fertilization.The Classics and Reflections included in this collection trace

many of the discoveries that have led to our current knowledgeof these important molecules.The earliest Classic paper in this collection, published just 3

years after the founding of the Journal in 1908, is an article byStanley R. Benedict reporting an analytical method for deter-mining the reducing sugar content of biological fluids such asurine. As explained in theClassic, the resulting Benedict’s Solu-tion, and themany variants that evolved from it, was used as thereagent of choice formeasuring sugar content formore than 50years. Benedict also served as managing editor of the Journal ofBiological Chemistry (JBC) from 1926 until his death in 1936.

Carbohydrate Metabolism

Several of the articles in this collection explain the eventssurrounding milestones in carbohydrate metabolism. Forexample, Nobel laureate Otto Fritz Meyerhof publishedthree papers in the JBC in the mid-1940s detailing severalsteps in the glycolytic pathway, the process whereby glucoseis converted into pyruvate and ATP. A few years later, in1952, Michael Doudoroff published a JBC paper containingexperiments that eventually led to the formulation of theEntner-Doudoroff pathway, a series of reactions that catab-olize glucose to pyruvic acid using a set of enzymes differentfrom those used in either glycolysis or the pentose phosphatepathway. Finally, in the 1960s, JBC Associate Editor JohnExton published two papers on the control of gluconeogen-esis, the metabolic pathway that results in the generation ofglucose from non-carbohydrate carbon substrates such aslactate, glycerol, and glucogenic amino acids. Exton usedisolated, perfused rat liver, which allowed them to study theprocess and its regulation without the interference of otherchanges in the body.

Carbohydrate Biosynthesis

Carbohydrate biosynthesis is a topic that also figures prom-inently in the collection. In the early 1950s, Luis F. Leloir pub-lished three papers in the JBC detailing the discovery of the

nucleotide sugars uridine diphosphate glucose (UDPG),UDP-N-acetylglucosamine (UDPAG), and guanosine di-phosphate mannose (GDPM). The importance of this andLeloir’s other work on sugar nucleotides was recognizedwhen he received the Nobel Prize in Chemistry in 1970 forhis discovery of sugar nucleotides and their role in the bio-synthesis of carbohydrates.Another seminal carbohydrate biosynthesis paper published

in the early 1950s was Bernard L. Horecker’s article describingthe oxidation of 6-phosphogluconate and the metabolic inter-mediates involved in the formation of the pentose phosphatepathway. To obtain these results, Horecker had to purify6-phosphogluconate dehydrogenase from brewers’ yeast andshow that the first products in a TPN-dependent reaction werecarbon dioxide and ribulose 5-phosphate. The ribulose 5-phos-phate was then converted to ribose 5-phosphate.In 1960, Saul Roseman published a paper in the JBC on the

structures of one of the sialic acids, N-acetylneuraminic acid,and the N-acetylneuraminic acid aldolase from Clostridium

perfringens. He also described the enzymatic synthesis of CMP-sialic acid, the donor substrate in the synthesis of sialic acid-containing oligosaccharides, and the requirements for theacceptor substrate of CMP-sialic acids. About 10 years later,Roseman published another JBCpaper describing a novel seriesof phosphotransferase reactions that transport sugars acrossthe bacterial membrane. Central in this work was Robert D.Simoni, Deputy Editor of the JBC, who has made many JBCinnovations, including the JBC Classics.A little more than 15 years after Horecker’s paper was pub-

lished, Clinton E. Ballou explained the biosynthesis of glyco-phosphoinositides inmycobacteria in his Classic paper. His co-author Patrick Brennan, now at Colorado State University, is amember of the JBC editorial board and continues to work onthe biochemistry and genetics of glycophosphoinositides inthese organisms.Many advances in our knowledge about carbohydrate bio-

synthesis were made in the 1970s, including William J. Len-narz’s description of the enzymatic synthesis of mannolipids inMicrococcus lysodeikticus. As reported in his Classic, Lennarzfound that mannosyl phosphoryl undecaprenol participates inthis process. Subsequently, he elucidated the enzymatic trans-fer of mannose from GDP-mannose to endogenous acceptorsin bovine thyroid and hen oviduct that are associated withmembrane components of the cell. Stuart A. Kornfeld also pub-lished a Classic paper in 1978. His experiments described theglycosylation of glycoproteins containing asparagine-linkedoligosaccharides. Kornfeld noted that a lipid-linked oligo-saccharide is first formed and then transferred en bloc to anasparagine-linked oligosaccharide, which was then partiallydegraded before excess mannose residues were removed andouter sugars were added.

* To cite articles in this collection, use the citation information that appears inthe upper right-hand corner on the first page of the article.

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JOURNAL OF BIOLOGICAL CHEMISTRY

PROLOGUE This paper is available online at www.jbc.org

H1

Rounding out the biosynthesis collection is a 1982 paper byPhillips W. Robbins in which he reports on the pathway ofN-linked oligosaccharide glycosylation in mammals and alsocharacterizes temperature-sensitive yeast mutants that had adeficiency of the different enzymes involved in this process. Ayear later,Armando J.Parodi explained thedolichol-phosphate-dependent pathway of protein N-glycosylation in his ClassicJBC paper. Several protein-linked glycans were identified andinitially synthesized as dolichol-P-P-glycans.

Carbohydrates in Biology

Carbohydrates also play a prominent role in physiology anddisease, as exemplified by several other papers in the collection.Charles R. Park was very interested in the hormonal regulationof carbohydrate metabolism and performed a classic series ofexperiments studying the regulation of glucose uptake in mus-cle by insulin and other hormones. He used isolated perfusedrat heart, and his work resulted in the publication of a series ofsix Classic papers in the JBC in 1961. He determined that thelimiting step for glucose uptake was the transport of the sugaracross the cell membrane, whichwas accelerated by insulin andanoxia.In the mid-1960s, William Zev Hassid published two Classic

JBC papers reporting the first studies of lactose biosynthesis inmammary glands. He recognized that lactose was synthesizedfrom UDP-galactose and glucose and that partial purificationand some of the properties of the galactosyltransferase wereresponsible for synthesis of lactose. In the early 1970s, JBCAssociate Editor Robert L. Hill published several papers inwhich he reported the complete amino acid sequence and thelocation of four disulfide bonds in the milk protein �-lactalbu-min, one of two proteins that are required for lactose synthesis.Hill noted that the covalent structure of�-lactalbuminwas verysimilar to egg white lysozyme and proposed that the two pro-teins arose from a common ancestral gene. He also purified thesecond protein required for lactose synthesis, galactosyltrans-ferase, which normally catalyzes synthesis of N-acetylgalac-tosamine but, in the presence of �-lactalbumin, synthesizeslactose.The 1961 Gilbert G. Ashwell Classic describes membrane

proteins (lectins) that remove senescent glycoproteins fromblood. Ashwell identified two types, one from rabbit liver thatbinds terminal galactose and the other from chicken liver thatbinds proteinswith terminalN-acetylglucosamine.Hiswork onhepatic binding proteins has served as a stimulus for the iden-

tification of a host of carbohydrate-specific receptors on vari-ous cell surfaces and has inaugurated the current concept of a“cellular lectin.”The inability to remove carbohydrates from the body can

cause disease. For example, a group of lysosomal storage dis-eases called mucopolysaccharidoses (MPS) and related disor-ders result from the failure to properly store or metabolizemucopolysaccharides. Elizabeth F. Neufeld studied three ofthese diseases, Hurler syndrome, Sanfilippo syndrome, andHunter syndrome, and their corrective factors. She discoveredthat Hurler syndrome was corrected by �-L-iduronidase, San-filippo syndrome by heparan sulfate sulfatase, and Hunter syn-drome by iduronate sulfatase and published these resultsbetween 1971 and 1972 in three JBC papers.Hyaluronan (also called hyaluronic acid or hyaluronate) is an

anionic, non-sulfated glycosaminoglycan distributed widelythroughout connective, epithelial, and neural tissues. Many ofthe experiments leading to our current understanding of thismolecule were published in the JBC. In his 1934 paper, KarlMeyer described the repeating disaccharides that are the basicunit of the hyaluronan polymer, namely glucuronate-�-1,3-N-acetylglucosamine-�1,4. Nineteen years later, Albert Dorfmandetailed the synthesis of hyaluronic acid from UDP-glucuronicacid and UDP-N-acetylglucosamine in a cell-free system fromstreptococci. Sixteen years after Dorfman’s paper was pub-lished, JBC Associate Editor Vincent Hascall showed how theproteoglycan aggregates from cartilage were formed by non-covalent binding of glycosaminoglycans to hyaluronic acidwiththe aid of a small molecular weight link protein in three JBCpapers. Finally, in 1979, Rupert Timpl described the character-ization of extracellularmatrix glycoproteins in basementmem-branes in his JBC paper. A high molecular weight non-collage-nous glycoprotein that was a major constituent of tumors wasidentified and named “laminin.”

Reflections

The collection also contains six JBC Reflections articles writ-ten by several of the above scientists, including Clinton E. Bal-lou, John H. Exton, Bernard L. Horecker, and Saul Roseman,who explain many of the seminal discoveries in carbohydratebiochemistry in their own words. Also included are Reflectionsby Nathan Sharon and Naoyuki Taniguchi.This is just a brief overview of the many papers that we have

assembled in this collection.We hope you take the time to readthem, and that you find them both enjoyable and educational.

PROLOGUE: Glycobiology and Carbohydrates

JOURNAL OF BIOLOGICAL CHEMISTRYH2

Benedict’s Solution, a Reagent for Measuring ReducingSugars: the Clinical Chemistry of Stanley R. BenedictA Reagent For the Detection of Reducing Sugars(Benedict, S. R. (1908) J. Biol. Chem. 5, 485–487)

Stanley Rossiter Benedict was born in Cincinnati in 1884. While a student at the Universityof Cincinnati he worked with J. F. Snell, and together they published nine papers describingnew analytical methods in inorganic chemistry. This research experience as a college studentprovided the intellectual foundation for his career. After a mistaken year in medical school atCincinnati, he went to Yale, to the Department of Physiological Chemistry, to study withRussell Chittenden and Lafayette Mendel where he received training in metabolism andphysiology. He received his Ph.D. in 1908, 2 years after entering graduate school. (Currentstudents take note.) In 1910, he became Professor of Chemistry at Cornell University MedicalCollege, the position he held until his death in 1936 at the age of 52 (1).

In a biographical review of Benedict’s career, E. V. McCollum wrote, “It is not possible to givean accurate account of the scientific work of Stanley Benedict without at the same timediscussing the parallel researches of Otto Folin . . . they succeeded, through many years ofintensive investigations, in devising and refining analytical procedures for determination ofminute amounts of the principal non-protein constituents of blood and urine so that, for thefirst time, chemical analysis became a highly useful technic (sic) for the discovery of thechemical processes in the normal functioning of the body” (1).

Of Benedict’s relationship with Folin, Shaffer wrote, “Both excelled in designing very cleveranalytical methods of the widest usefulness, and in using these tools with rare success for thediscovery of new facts about metabolism. In spite of seventeen years difference in their age(Folin was the older), of the rivalry and controversy sometimes evident in their papers, thereearly developed between them a warm friendship which reveals the fine character of both.They were kindred spirits” (2). (We will present a classic paper by Otto Folin in a subsequentinstallment of JBC Classics, stay tuned.)

As McCollum and Shaffer described, Benedict’s major contributions to biochemistry were indevising analytical methods. Although he published many papers in the Journal of BiologicalChemistry (JBC), the paper reprinted here seemed appropriate to characterize a distinguishedcareer. It had been known for many years that the common sugars had carbonyl groups andwere therefore, “reducing sugars.” That is, they were oxidized by a variety of metal ions, Ag�,Fe3�, and Cu2�. Treatment with hot alkali fragments the sugars, and the resulting productsreduce Cu2� to Cu� with the formation of a precipitate of Cu2O. As noted in the paper,Benedict’s goal was to improve this general method to make the reagent less corrosive andmore stable. He accomplished this by substituting carbonate for hydroxide as the alkalicomponent, to reduce the corrosiveness, and by substituting citrate for tartrate as the agent tochelate the Cu2�, to make the reagent more stable.

Benedict’s Solution, or one of the many variants that evolved over the years, was used as thereagent of choice for measuring sugar content for more than 50 years. It was the most commontest for diabetes and was the standard procedure for virtually all clinical laboratories. SaulRoseman remembers that all inductees into the army during World War II had their urine

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 16, Issue of April 19, p. e5, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

ClassicsA PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005

JBC Centennial1905–2005

100 Years of Biochemistry and Molecular Biology

This paper is available on line at http://www.jbc.orgH3

tested for sugar with Benedict’s Solution.1 Although Benedict’s assay was the method of choicefor more than 50 years, it suffered from lack of sugar specificity and was eventually supplantedby the use of enzymatic methods such as glucose oxidase.

Benedict’s work on analytical methods was particularly important for clinical applications.There was, for many years, a very close relationship between basic biochemistry research andbiochemical clinical applications. Many biochemists were employed as clinical chemists be-cause academic jobs as biochemists were difficult to find. Many of the methods that have beentaken for granted for many years find their origins with biochemists working in clinicallaboratories.

Benedict was active in the Society and the JBC. He served as Secretary of the Society and,in 1919, served as President. It was during his tenure as President that the JBC wastransferred to the Society for management. (Officially the financial relationship between theSociety and the Journal Corporation was not finalized until 1942.) Benedict became a memberof the JBC Editorial Board and in 1926 became Managing Editor, a position he held until hisdeath in 1936. An interesting characterization of Benedict’s service as JBC Managing Editoris made in the obituary of Benedict written by Philip A. Shaffer (2), “With many othercontributor (sic), the present writer has occasionally smarted under sometimes sharp criticismof the editor; but rarely if ever were the criticisms unjustified. His standards were high, heexpected clarity, logic, and brevity in exposition, his opinions were definite and outspoken hisjudgements were based on essential facts and were impartial as to individuals.” (During thisperiod of the Journal, and later as well, all communication between authors and the Journalwas conducted personally by the Managing Editor.)

Robert D. Simoni, Robert L. Hill, and Martha Vaughan

REFERENCES1. McCollum, E. V. (1974) Memoir of Stanley Rossiter Benedict, Vol. 27, National Academy of Sciences, Washington,

D. C.2. Shaffer, P. A. (1937) Obituary for Stanley Rossiter Benedict. J. Biol. Chem. 117, 428

1 Much of the background information for sugar chemistry and clinical usage of Benedict’s Reagent was kindlyprovided by Professor Saul Roseman, Professor of Biology, Johns Hopkins University. Professor Roseman knewStanley R. Benedict personally.

Stanley R. Benedict. Photo courtesy of the National Library of Medicine.

Classics

H4

The Discovery of Hyaluronan by Karl MeyerThe Polysaccharide of the Vitreous Humor(Meyer, K., and Palmer, J. W. (1934) J. Biol. Chem. 107, 629–634)

Karl Meyer (1899–1990) was born in the village of Karpen, Germany, near Cologne. In 1917,he was drafted into the German army and served in the last year of World War I. After the war,he entered medical school at the University of Cologne and received the M.D. degree in 1924.He then went to Berlin for a year of study in medical chemistry and met several promisingyoung biochemists including Fritz Lippman, Hans Krebs, and Ernst Chain. To gain moretraining in chemistry, he enrolled as a graduate student with Otto Meyerhof. For his Ph.D.thesis work, he investigated lactic acid formation in yeast and muscle demonstrating therequirement for a “co-enzyme” later identified as ADP. His research career was launched.After three years as a Rockefeller Fellow in Zurich studying heme-catalyzed oxidation ofunsaturated compounds with Professor Kuhn, he was offered a position as Assistant Professorat the University of California at Berkeley with Herbert Evans.

In 1932, while at Berkeley, he attended a conference in Europe. During the conference, hereceived notice that Evans had terminated his position with the suggestion that he stay inGermany. He decided, no doubt in part because of the rising anti-Semitism in Europe and theincreasing probability of war, to go back to the United States. His return was facilitated byHans T. Clarke at Columbia University who provided interim fellowship support until 1933when he received a position as Assistant Professor in the Department of Ophthalmology at theCollege of Physicians and Surgeons. In part because of the mission of his department, Meyerbegan to study the lysozyme present in tears and undertook to identify a physiologicalsubstrate for the enzyme. Examination of the viscous vitreous humor of the eye as a plausiblesource of substrate quickly led to the discovery of hyaluronan, which is reported in thisJournal of Biological Chemistry (JBC) Classic.

Meyer and his assistant John Palmer isolated a novel, high molecular weight polysaccharideand reported that it was composed of “a uronic acid, an amino sugar, and possibly a pentose.”(The last is incorrect.) They proposed “for convenience, the name hyaluronic acid, from hyaloid(vitreous) � uronic acid.” Nearly 25 years of work were required to establish the structure ofthe repeating disaccharide that is the basic unit of the hyaluranan polymer, namelyglucuronate-�-1,3-N-acetylglucosamine-�1,4-.

Hyaluronan is one member of a family of glycosaminoglycans that includes chondroitin/dermatan sulfate, keratan sulfate, and heparin/heparan sulfate, each with a characteristicdisaccharide-repeat structure of an amino sugar, either glucosamine or galactosamine, plus anegatively charged sugar, a carboxylate and/or a sulfate. The polymers are found as cellsurface molecules and in the extracellular matrix. Glycosaminoglycans, with the exception ofhyaluronan, are covalently bound to proteins to form proteoglycans. These ubiquitous andstructurally diverse macromolecules are found as cell surface molecules and in the extracel-lular matrix. The multiplicity of their functions that is now recognized was not alwaysappreciated. In a symposium at the 1958 annual meeting of the American Society of BiologicalChemists entitled “Acid Mucopolysaccharides of Animal Origin,” which was chaired by Meyer,he states in an opening remark, “It is my opinion that the mucopolysaccharides will never bea highly popular field in biochemistry, but they will probably not be relegated again to theinsignificance and disregard in which they were held not so long ago.”

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 39, Issue of September 27, p. e27, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.




This paper is available on line at http://www.jbc.org H5

Meyer’s scientific contributions were not limited to the discovery of hyaluronan. He isconsidered the father of glycosaminoglycan chemistry and received many honors includingelection to the National Academy of Sciences in 1967. On the occasion of his induction hecommented as follows: “Looking back on my scientific career, I have often wondered whetherit was worthwhile to stick so tenaciously to a technically difficult and conceptually apparentlyunexciting field, while my colleagues and friends shifted over to more fashionable and reward-ing areas. The reasons for my persistence are manifold, among them a distaste for jumping inon ground broken by others. Besides, I felt committed to problems such as the biologicalfunctions of the mucopolysaccharides of connective tissues to their role in differentiation, incell membranes and in inherited diseases.”

His persistence was biochemistry’s gain.1

Robert D. Simoni, Robert L. Hill, Martha Vaughan, and Vincent Hascall

REFERENCES1. McDonald, J., and Hascall, V. C. (2002) J. Biol. Chem. 277, 4575–4579

1 A recent overview of glycosaminoglycan biochemistry and additional information about Karl Meyer are includedin the JBC Hyaluronan Minireview Series edited by John McDonald and Vincent Hascall (1).

Karl Meyer. Photo courtesy of the National Library of Medicine.

Classics

H6

Otto Fritz Meyerhof and the Elucidation of the GlycolyticPathwayThe Equilibria of Isomerase and Aldolase, and the Problem of the Phosphorylationof Glyceraldehyde Phosphate(Meyerhof, O., and Junowicz-Kocholaty, R. (1943) J. Biol. Chem. 149, 71–92)

The Origin of the Reaction of Harden and Young in Cell-free Alcoholic Fermentation(Meyerhof, O. (1945) J. Biol. Chem. 157, 105–120)

The Mechanism of the Oxidative Reaction in Fermentation(Meyerhof, O. and Oesper, P. (1947) J. Biol. Chem. 170, 1–22)

The elucidation of the glycolytic pathway, the process whereby glucose is converted intopyruvate and ATP, began in 1860 when Louis Pasteur observed that microorganisms wereresponsible for fermentation. Several years later, in 1897, Eduard Buchner made the signif-icant discovery that cell-free extracts could carry out fermentation. The next importantcontribution was from Arthur Harden and William Young in 1905. They realized that inor-ganic phosphate was necessary for glycolysis and that fermentation requires the presence ofboth a heat-labile component they called “zymase” and a low molecular weight, heat-stablefraction called “cozymase.” (It was later shown that zymase contains a number of enzymeswhereas cozymase consists of metal ions, ATP, ADP, and coenzymes such as NAD.) Buildingon these initial observations, the complete glycolytic pathway was elucidated by 1940 by thecombined efforts of several scientists including Otto Fritz Meyerhof (1884–1951).

Meyerhof was born in Hanover, Germany and grew up in Berlin. In 1909, he graduated asa doctor of medicine from the University of Heidelberg. Around this time, Ludolf von Krehl wasbuilding a small research program on metabolism at the University of Heidelberg MedicalClinic, and he offered Meyerhof a position in his laboratory. There, Meyerhof met OttoWarburg whose innovative ideas and confident approach inspired him to focus his career onphysiological chemistry.1

In 1912, Meyerhof took a position at the University of Kiel. A year later, he delivered alecture on the energetics of living cells, one of the very first adaptations of the physical lawsof thermodynamics to physiological chemistry. Meyerhof had recognized that after energy isinput as food it is transformed through a series of intermediate steps and finally dissipated asheat. He soon began using muscle to look at energy transformations and chemical changesduring cellular function. Meyerhof was also interested in analogies between oxygen respira-tion in muscle and alcoholic fermentation in yeast and proved, in 1918, that the coenzymesinvolved in lactic acid production were the same as the yeast coenzymes discovered by Hardenand Young, revealing an underlying unity in biochemistry.

Soon after World War I, Meyerhof began collaborating with Archibald Vivian Hill who wasinvestigating heat production in muscle. The pair worked to decipher metabolism in terms ofheat development, mechanical work, and cellular chemical reactions. Meyerhof determinedthat glycogen is converted to lactic acid in the absence of oxygen and showed that in thepresence of oxygen only a small portion of lactic acid is oxidized and the rest is converted backto glycogen. This discovery of the lactic acid cycle provided the first evidence of the cyclical

1 All biographical information on Otto Fritz Meyerhof was taken from Ref. 6.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 4, Issue of January 28, p. e3, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.





nature of energy transformation in cells. These results also confirmed and extended LouisPasteur’s theory (now called the Pasteur-Meyerhof effect) that less glycogen is consumed inmuscle metabolism in the presence of oxygen than in its absence. Meyerhof and Hill won theNobel Prize in Physiology or Medicine in 1922 for their analysis of the lactic acid cycle and itsrelation to respiration.

Two years after wining the Nobel Prize, Meyerhof joined the Kaiser Wilhelm Institutes inBerlin-Dahlem. Then, in 1929, he took charge of the newly founded Kaiser Wilhelm Institutefor Medical Research at Heidelberg.

By this time, it was clear that glycolysis was far more complicated than anyone hadimagined. The sheer number of components and their short lived nature made the task ofsorting out the pathway daunting. However, during his time at Heidelberg, Meyerhof’s groupwas extremely successful at breaking down glycolysis into its many separate components. In1932, Meyerhof made the first associations between the uptake of phosphate during thebreakdown of carbohydrates to lactic acid and the splitting of ATP. By 1934, Kurt Lohmann inMeyerhof’s laboratory provided direct evidence that ATP synthesis was the byproduct ofutilization of glucose. Lohmann also established that creatine phosphate is an energy sourcefor ATP phosphorylation, which led Meyerh of to the conclusion that the energy release fromATP hydrolysis was the primary event leading to muscle contraction.

By the 1930s Meyerhof had managed to isolate and purify the co-enzymes involved in theconversion of glycogen to lactic acid and had reconstructed the main steps of this set ofreactions in cell-free solution. All in all, Meyerhof’s group discovered more than one-third ofthe enzymes involved in glycolysis. In 1932, Gustav Embden constructed a detailed proposalfor reaction sequences for almost the entire glycolytic pathway. Over the next 5 years,Meyerhof, along with Warburg, Jacob Parnas, Carl Neuberg, Gerti and Karl Cori, and Hansvon Euler worked out the details of glycolysis, which is often referred to as the Embden-Meyerhof pathway.

With Adolf Hitler’s rise to power, Meyerhof left Germany in 1938 and became director of theInstitut de Biologie Physiochimique in Paris. In 1940, when the Nazis invaded France,Meyerhof fled to the United States where the post of Research Professor of PhysiologicalChemistry was created for him by the University of Pennsylvania and the Rockefeller Foun-dation. He remained at Pennsylvania where he continued to study metabolism until his death.The three Journal of Biological Chemistry (JBC) Classics reprinted here are from Meyerhof’stime at Pennsylvania.

Otto F. Meyerhof. Photo courtesy of the National Library of Medicine.

Classics

H8

The first paper deals with one of the intermediate reactions that occurs in glycolysis: thesplitting of hexose diphosphate (now known as fructose 1,6-bisphosphate) into two triosephosphate isomers, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, by zymo-hexase (fructose-1,6-bisphosphate aldolase). Triose-phosphate isomerase then converts dihy-droxyacetone phosphate into glyceraldehyde 3-phosphate. In the next step of glycolysis,glyceraldehyde 3-phosphate is oxidized and phosphorylated to become 1,3-diphosphoglycericacid. Warburg and Christian (1, 2) and Negelein and Bromel (3, 4) proposed that this stepoccurs through the intermediate 1,3-diphosphoglyceraldehyde with the aid of an oxidizingenzyme and cozymase. If this were true, then inorganic phosphate could be used to removeglyceraldehyde 3-phosphate from the hexose diphosphate reaction.

To investigate this matter further, Meyerhof and Renate Junowicz-Kocholaty redeterminedthe equilibrium constant for the isomerase and aldolase reactions in the presence and absenceof inorganic phosphate, cozymase, and Warburg’s oxidizing enzyme. They found that theirvalues agreed with those previously determined and that equilibrium is not influenced by thepresence of inorganic phosphate, cozymase, or Warburg’s enzyme. They were also unable todetect the formation of any substance that would break down into glyceraldehyde phosphateand phosphate, prompting them to write that Warburg’s claims of a diphosphoglyceraldehydeintermediate may have been “premature.”

The second Classic deals with the next two steps of glycolysis shown as Reactions 1 and 2.

Glyceraldehyde 3-phosphate � phosphate � cozymase ^

1,3-diphosphoglyceric acid � dihydrocozymase

REACTION 1

1,3-Diphosphoglyceric acid � ADP ^ 3-phosphoglyceric acid � ATP

REACTION 2

Harden and Young stated that during fermentation, one sugar molecule is fermented to CO2

and alcohol while a second is esterfied to hexose diphosphate (5). In a cell-free system, thisreaction can be divided into two phases, a rapid “phosphate period” and a slower phase thatdepends on the rate of hexose diphosphate fermentation. Meyerhof proposed that the rate ofthe hexose diphosphate reaction was much slower in cell-free systems than in live yeastbecause the majority of the enzyme needed to split ATP, adenylpyrophosphatase (apyrase),was lost during the extraction process. He backed up his claim by studying the distribution ofapyrase in the yeast cell and showing that it remains mainly with solid elements that are notused in cell-free systems. Meyerhof also purified apyrase from potatoes and added it to cell-freepreparations to prove that it raises the rate of hexose diphosphate fermentation.

The final JBC Classic revisits the phosphorylation of glyceraldehyde 3-phosphate and itssubsequent oxidation. In this paper, Meyerhof and Peter Oesper use a Beckman spectropho-tometer to follow the reaction and provide further proof that a diphosphoglyceric aldehydeintermediate does not exist. They also alter the equation for this step of glycolysis to reflect thefact that the reduction of cozymase is accompanied by the formation of an H� ion.


REFERENCES1. Warburg, O., and Christian, W. (1939) Biochem. Z. 301, 2012. Warburg, O., and Christian, W. (1939) Biochem. Z. 303, 403. Negelein, E., and Bromel, H. (1939) Biochem. Z. 301, 1354. Negelein, E., and Bromel, H. (1939) Biochem. Z. 303, 1325. Harden A., and Young, W. J. (1908) Proc. R. Soc. Lond. Ser. B Biol. Sci. 80, 2996. States, D. M. Otto Meyerhof and the Physiology Department: the Birth of Modern Biochemistry. A History of the

Max Planck Institute for Medical Research (http://sun0.mpimf-heidelberg.mpg.de/History/Meyerhof.html)

Classics

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Luis F. Leloir and the Biosynthesis of SaccharidesIsolation of the Coenzyme of the Galactose Phosphate-Glucose PhosphateTransformation(Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184,333–350)

Uridine Diphosphate Acetylglucosamine(Cabib, E., Leloir, L. F., and Cardini, C. E. (1953) J. Biol. Chem. 203, 1055–1070)

Guanosine Diphosphate Mannose(Cabib, E., and Leloir, L. F. (1954) J. Biol. Chem. 206, 779–790)

Luis Federico Leloir (1906–1987) was born in Paris but moved to Buenos Aires with hisArgentine parents when he was 2 years old. He attended the University of Buenos Aires andgraduated with an M.D. in 1932. Leloir got a job at the University hospital but left the bedsidefor the bench 2 years later. As he recalled, “When I practiced medicine, except for surgery,digitalis, and a few other active remedies, we could do little for our patients. Antibiotics,psychoactive drugs, and all the new therapeutic agents were unknown. It was therefore notstrange in 1932 that a young doctor such as I should try to join efforts with those who weretrying to advance medical knowledge” (1).1

The most active research laboratory in town was run by Bernardo A. Houssay, who wouldlater be awarded the Nobel Prize with Carl and Gerty Cori for their work on the role of thepituitary gland in carbohydrate metabolism. Leloir joined Houssay’s laboratory as a graduatestudent and studied the role of the adrenals in carbohydrate metabolism.

After Leloir finished his thesis work, Houssay advised him to study abroad. So, in 1936Leloir moved to England to work at the Biochemical Laboratory of Cambridge University.There, he collaborated with Malcolm Dixon on the effect of cyanide and pyrophosphate onsuccinic acid dehydrogenase, Norman L. Edson on ketogenesis using liver slices, and David E.Green on the purification and properties of �-hydroxybutyrate dehydrogenase.

Leloir returned to Buenos Aires after his time at Cambridge and started investigating theoxidation of fatty acids in the liver with J. M. Munoz. They managed to produce an activecell-free system, which was an accomplishment since at that time it was thought that oxidationcould only occur in intact cells. Leloir also worked with E. Braun Menendez, Juan CarlosFasciolo, and A. C. Taquini on the mechanism of renal hypertension and the formation ofangiotensin.

In 1944, Leloir left Buenos Aires again. This time he went to Washington University in St.Louis to work with Carl and Gerty Cori, who were featured in a previous Journal of BiologicalChemistry (JBC) Classic (2). While in the States, Leloir reunited with Green and spent sometime at the College of Physicians and Surgeons at Columbia University working on thepurification of aminotransferases.

After his stay in the United States, Leloir returned to the Institute of Physiology in BuenosAires. He worked there for a time and then left for a private institution recently created, theInstituto de Investigaciones Bioquimicas Fundacion Campomar (now Fundacion InstitutoLeloir), where he remained until his death. In collaboration with Ranwel Caputto, Carlos E.

1 All biographical information on Luis F. Leloir was taken from Refs. 1 and 8. We thank Armando J. Parodi, Ph.D.,of the Fundacion Instituto Leloir, for helpful comments in the preparation of this JBC Classic Introduction.

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Cardini, Raul Trucco, and Alejandro C. Paladini, Leloir started to work on the metabolism ofgalactose. The project was initiated when Caputto presented some preliminary results thatindicated that mammary gland homogenates could produce lactose when incubated withglycogen. The group performed many experiments with mammary gland extracts but gener-ally got ambiguous results, mainly due to their lack of a reliable method for lactose detection.Discouraged, they decided to focus on the breakdown of lactose by Saccharomyces fragilis,hoping that this would give them information on the mechanism of lactose synthesis.

Leloir and his colleagues isolated lactase from the yeast and determined that galactose wasphosphorylated to produce galactose 1-phosphate. They synthesized glucose 1-phosphate andgalactose 1-phosphate and observed that the esters were used when incubated with enzymesfrom galactose-adapted yeast. At first they thought that only one factor was required for thisreaction, but soon realized that two factors were involved: one for the conversion of galactose1-phosphate into glucose 1-phosphate and another for the formation of glucose 6-phosphate, asshown in the following reaction.

Galactose 1-phosphate 3 glucoseFactor 1

1-phosphate 3 glucose 6-phosphateFactor 2

REACTION 1

The group first concentrated on finding Factor 2 and eventually determined that it wasglucose 1,6-diphosphate (3). Next, they turned their attention to identifying Factor 1. Thepurified factor absorbed light at 260 nm and had a spectrum similar to that of adenosine, withsome differences. They were stumped on the identity of this factor for quite some time untilCaputto came in one morning with an issue of the JBC that showed a spectrum identical totheirs. The spectrum was that of uridine. The group published their results in a preliminarycommunication in Nature (4) and then in the JBC, which is the first classic reprinted here. Inaddition to uridine, the co-factor was found to contain glucose and two phosphates and hencewas named uridine diphosphate glucose (UDPG). The presence of uridine in a co-factor wasrather novel as, until then, all known factors (ATP, NAD, FAD) only contained the nucleotideadenosine. The occurrence of a sugar derivative combined with a nucleoside was also novel.Eventually, Leloir determined that UDPG acts as a glucose donor in the synthesis of trehalose(5), sucrose (6), and glycogen (7).

Another result of the discovery of UDPG was the isolation and characterization of the sugarnucleotides UDP-N-acetylglucosamine (UDPAG) and guanosine diphosphate mannose(GDPM), which are the subjects of the remaining two JBC Classics reprinted here. UDPAGwas originally detected as an impurity in UDPG concentrates and was called UDP-X untilLeloir was able to identify the sugar moiety as N-acetylglucosamine. Similarly, GDPM wasfirst detected by paper chromatography of UDPG preparations that were purified by anionexchange. UDPAG and GDPM are now known to be involved in the biosynthesis of numerousglycoconjugates.

Luis F. Leloir. Photo courtesy of the National Library of Medicine.

Classics

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Leloir’s extensive work on sugar nucleotides and his contributions to biochemistry receivedthe recognition they deserved when he was awarded the Nobel Prize in Chemistry in 1970, “forhis discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.”


REFERENCES1. Leloir, L. F. (1983) Far away and long ago. Annu. Rev. Biochem. 52, 1–152. JBC Classics: Cori, C. F., and Cori, G. T. (1928) J. Biol. Chem. 79, 321–341; Cori, G. T., Colowick, S. P., and Cori,

C. F. (1938) J. Biol. Chem. 124, 543–555; Cori, G. T., Colowick, S. P., and Cori, C. F. (1939) J. Biol. Chem. 127,771–782; Green, A. A., and Cori, G. T. (1943) J. Biol. Chem. 151, 21–29; Cori, G. T., and Green, A. A. (1943)J. Biol. Chem. 151, 31–38 (http://www.jbc.org/cgi/content/full/277/29/e18)

3. Cardini, C. E., Paladini, A. C., Caputto, R., Leloir, L. F., and Trucco, R. E. (1949) Arch. Biochem. 22, 874. Cardini, C. E., Paladini, A. C., Caputto, R., and Leloir, L. F. (1950) Uridine diphosphate glucose: the coenzyme of

the galactose-glucose phosphate isomerization. Nature 165, 191–1935. Leloir, L. F., and Cabib, E. (1953) The enzymic synthesis of trehalose phosphate. J. Am. Chem. Soc. 75, 5445–54466. Cardini, C. E., Leloir, L. F., and Chiriboga, J. (1955) The biosynthesis of sucrose. J. Biol. Chem. 214, 149–1557. Leloir, L. F., and Cardini, C. E. (1957) Biosynthesis of glycogen from uridine diphosphate glucose. J. Am. Chem.

Soc. 79, 63408. Leloir, L. F. (1971) Two decades of research on the biosynthesis of saccharides. Science 172, 1299–1302

Classics

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Bernard L. Horecker’s Contributions to Elucidating thePentose Phosphate PathwayThe Enzymatic Conversion of 6-Phosphogluconate to Ribulose-5-Phosphate andRibose-5-Phosphate(Horecker, B. L., Smyrniotis, P. Z., and Seegmiller, J. E. (1951) J. Biol. Chem. 193,383–396)

Bernard Leonard Horecker (1914) began his training in enzymology in 1936 as a graduatestudent at the University of Chicago in the laboratory of T. R. Hogness. His initial projectinvolved studying succinic dehydrogenase from beef heart using the Warburg manometricapparatus. However, when Erwin Hass arrived from Otto Warburg’s laboratory he askedHorecker to join him in the search for an enzyme that would catalyze the reduction ofcytochrome c by reduced NADP. This marked the beginning of Horecker’s lifelong involvementwith the pentose phosphate pathway.

During World War II, Horecker left Chicago and got a job at the National Institutes ofHealth (NIH) in Frederick S. Brackett’s laboratory in the Division of Industrial Hygiene. Aspart of the wartime effort, Horecker was assigned the task of developing a method to deter-mine the carbon monoxide hemoglobin content of the blood of Navy pilots returning fromcombat missions. When the war ended, Horecker returned to research in enzymology andbegan studying the reduction of cytochrome c by the succinic dehydrogenase system.

Shortly after he began these investigation changes, Horecker was approached by futureNobel laureate Arthur Kornberg, who was convinced that enzymes were the key to under-standing intracellular biochemical processes. Kornberg suggested they collaborate, and thetwo began to study the effect of cyanide on the succinic dehydrogenase system. Cyanide hadpreviously been found to inhibit enzymes containing a heme group, with the exception ofcytochrome c. However, Horecker and Kornberg found that cyanide did in fact react withcytochrome c and concluded that previous groups had failed to perceive this interactionbecause the shift in the absorption maximum was too small to be detected by visualexamination.

Two years later, Kornberg invited Horecker and Leon Heppel to join him in setting up a newSection on Enzymes in the Laboratory of Physiology at the NIH. Their Section on Enzymeseventually became part of the new Experimental Biology and Medicine Institute and was laterrenamed the National Institute of Arthritis and Metabolic Diseases.

Horecker and Kornberg continued to collaborate, this time on the isolation of DPN and TPN.By 1948 they had amassed a huge supply of the coenzymes and were able to present OttoWarburg, the discoverer of TPN, with a gift of 25 mg of the enzyme when he came to visit.Horecker also collaborated with Heppel on the isolation of cytochrome c reductase from yeastand eventually accomplished the first isolation of the flavoprotein from mammalian liver.

Along with his lab technician Pauline Smyrniotis, Horecker began to study the enzymesinvolved in the oxidation of 6-phosphogluconate and the metabolic intermediates formed in thepentose phosphate pathway. Joined by Horecker’s first postdoctoral student, J. E. Seegmiller,they worked out a new method for the preparation of glucose 6-phosphate and 6-phosphoglu-conate, both of which were not yet commercially available. As reported in the Journal ofBiological Chemistry (JBC) Classic reprinted here, they purified 6-phosphogluconate dehydro-genase from brewer’s yeast (1), and by coupling the reduction of TPN to its reoxidation by

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pyruvate in the presence of lactic dehydrogenase, they were able to show that the first productof 6-phosphogluconate oxidation, in addition to carbon dioxide, was ribulose 5-phosphte. Thispentose ester was then converted to ribose 5-phosphate by a pentose-phosphate isomerase.They were able to separate ribulose 5-phosphate from ribose 5-phosphate and demonstratetheir interconversion using a recently developed nucleotide separation technique called ion-exchange chromatography. Horecker and Seegmiller later showed that 6-phosphogluconatemetabolism by enzymes from mammalian tissues also produced the same products.

Over the next several years, Horecker played a key role in elucidating the remaining stepsof the pentose phosphate pathway. His total contributions included the discovery of three newsugar phosphate esters, ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose4-phosphate, and three new enzymes, transketolase, transaldolase, and pentose-phosphate3-epimerase. The outline of the complete pentose phosphate cycle was published in 1955 (2).Horecker’s personal account of his work on the pentose phosphate pathway can be found in hisJBC Reflection (3).1

Horecker’s contributions to science were recognized with many awards and honors includingthe Washington Academy of Sciences Award for Scientific Achievement in Biological Sciences(1954) and his election to the National Academy of Sciences in 1961. Horecker also served aspresident of the American Society of Biological Chemists (now the American Society forBiochemistry and Molecular Biology) in 1968.


REFERENCES1. Horecker, B. L., and Smyrniotis, P. Z. (1951) Phosphogluconic acid dehydrogenase from yeast. J. Biol. Chem. 193,

371–3812. Gunsalus, I. C., Horecker, B. L., and Wood, W. A. (1955) Pathways of carbohydrate metabolism in microorganisms.

Bacteriol. Rev. 19, 79–1283. Horecker, B. L. (2002) The pentose phosphate pathway. J. Biol. Chem. 277, 47965–47971

1 All biographical information on Bernard L. Horecker was taken from Ref. 3.

FIG. 1

Classics

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The Entner-Doudoroff Pathway for Glucose Degradation:the Work of Michael DoudoroffGlucose and Gluconic Acid Oxidation of Pseudomonas saccharophila(Entner, N., and Doudoroff, M. (1952) J. Biol. Chem. 196, 853–862)

Michael Doudoroff (1911–1975) was born in St. Petersburg, Russia but moved to SanFrancisco when he was 12 years old. He entered Stanford University in 1929, planning tomajor in biology and specialize in entomology. However, as his exposure to different types ofscience broadened, he became fascinated with bacteriology and protozoology. As a result, hestudied the survival of Paramecium at elevated temperatures for his Master’s thesis with A. C.Giese and the adaptation of E. coli to elevated salt concentrations for his Ph.D. thesis withC. B. van Niel at the Hopkins Marine Station.

While assisting van Niel in a course in general microbiology at the Marine Station, Dou-doroff was introduced to the physiological and biochemical diversity of the microbial world. Hesubsequently started to study luminous bacteria, resulting in his discovery that riboflavin isdirectly involved in bacterial luminescence. Doudoroff also isolated a new species of H2-oxidizing bacteria, Pseudomonas saccharophila, which could oxidize a number of mono-, di-,and polysaccharides. This was a surprising discovery because most bacteria only oxidize di-and polysaccharides after first hydrolyzing them to monosaccharides. P. saccharophila, in fact,oxidized sucrose much more rapidly than its constituent monosaccharides, glucose and fruc-tose. This anomaly was later shown to be caused by the lack of permeases in P. saccharophilafor monosaccharides.

In 1940, Doudoroff joined the bacteriology department at the University of California,Berkeley. There he discovered that P. saccharophila extracts catalyze the reversible formationof glucose 1-phosphate and fructose from sucrose and inorganic phosphate. He used thereverse reaction to synthesize sucrose, a compound that had not yet been made by chemical orenzymatic methods. Doudoroff also used the synthetic reaction to make novel analogues ofsucrose by replacing fructose with D-ketoxylose and L-sorbose.

He subsequently purified sucrose phosphorylase from P. saccharophila and studied its modeof action using radioactive inorganic phosphate. He learned that the enzyme is a transgluco-sidase that transfers the glucosyl residue from a suitable donor such as sucrose or glucose1-phosphate to an appropriate acceptor such as fructose or orthophosphate. This was some ofthe first evidence for the formation of a substrate-enzyme complex as an intermediate in anenzymatic reaction. Doudoroff also discovered a second type of phosphorolytic enzyme, maltosephosphorylase, in Neisseria meningitides.

Doudoroff and his associates soon began to study the oxidative degradation of other sugarsby P. saccharophila. He and Nathan Entner examined the enzymatic oxidation of glucoselabeled with C14, as reported in the Journal of Biological Chemistry (JBC) Classic reprintedhere. They determined that glucose is phosphorylated to glucose 6-phosphate, which is oxi-dized to 6-phosphogluconic acid. This compound is then split to give rise to pyruvic acid andglyceraldehyde phosphate. 2-Keto-3-deoxy-6-phosphogluconate was thought to be an interme-diate in this reaction, and this was subsequently confirmed by Doudoroff and Joseph MacGeewho isolated and characterized the compound in 1954 (1). The enzyme that cleaved the ketoacid, ketodeoxyphosphogluconate aldolase, was later purified and crystallized in 1967 (2).






These experiments eventually led to the formulation of the Entner-Doudoroff pathway, aseries of reactions that catabolize glucose to pyruvic acid using a different set of enzymes fromthose used in either glycolysis or the pentose phosphate pathway. The novel feature of thispathway is the cleavage of 6-phosphogluconate to yield pyruvate and glyceraldehyde 3-phos-phate. Other sugars were shown to be metabolized by similar, but divergent, pathways.

Later in his career, Doudoroff studied assimilatory processes in aerobic and photosyntheticbacteria and showed that poly-�-hydroxybutyric acid is an important energy reserve that isutilized by both intracellular and extracellular enzymes. He was also involved in an extensiveclarification of taxonomic and phylogenetic relationships in Pseudomonas and other aerobicbacteria. The first publication resulting from this collaboration was a massive survey of 169phenotypic characters of 267 strains of Pseudomonas (3).

Doudoroff also had a profound influence on how bacteriology was taught at Berkeley. Whenhe joined the faculty in 1940, the bacteriology courses emphasized the medical and paramed-ical aspects of the subject. Doudoroff reorganized the curriculum to present bacteria and othermicroorganisms as creatures whose structures, behaviors, and metabolic activities were wor-thy of study independent of their roles in agriculture, industry, or disease. He eventually,along with Roger Y. Stanier and Edward A. Adelberg, wrote the popular textbook, TheMicrobial World, based on the Berkeley courses.

In recognition of Doudoroff’s contributions to microbiology and biochemistry he received thefirst Sugar Research Award from the National Academy of Sciences in 1945 with Horace A.Barker and William Z. Hassid (both of whom will be featured in future JBC Classics). He alsobecame a J. S. Guggenheim Foundation fellow in 1949 and was elected to membership in theNational Academy of Sciences in 1962.1


REFERENCES1. MacGee, J., and Doudoroff, M. (1954) A new phosphorylated intermediate in glucose oxidation. J. Biol. Chem. 210,

617–6262. Shuster C. W., and Doudoroff, M. (1967) Purification of 2-keto-3-deoxy-6-phosphohexonate aldolases of Pseudo-

monas saccharophila. Arch. Mikrobiol. 59, 279–2863. Stanier, R. Y., Palleroni, N. J., and Doudoroff, M. (1966) The aerobic pseudomonads: a taxonomic study. J. Gen.

Microbiol. 43, 159–2714. Barker, H. A. (1993) Biographical Memoir of Michael Doudoroff, Vol. 62, pp. 118–141, National Academy of

Sciences, Washington D. C.

1 All biographical information on Michael Doudoroff was taken from Ref. 4.

Classics

H16

Albert Dorfman and the Biosynthesis of Hyaluronic AcidThe Biosynthesis of Hyaluronic Acid by Group A Streptococcus. I. Utilization of1-C14-Glucose(Roseman, S., Moses, F. E., Ludowieg, J., and Dorfman, A. (1953) J. Biol. Chem. 203,213–225)

Albert Dorfman (1916–1982) was born and raised in Chicago. While in high school, hebecame interested in science because of his older brother who was studying chemistry at theUniversity of Illinois. After graduating, Dorfman obtained a scholarship to the University ofChicago where he enrolled as a chemistry major; however, during his senior year he switchedto biochemistry and entered the University of Chicago School of Medicine. He eventually foundbiochemistry so appealing that he dropped out of medical school after 2 years to pursuegraduate work. His thesis research was on the identification of nicotinamide as a growthrequirement for Shigella dysenteriae and the synthesis of various nicotinic acid derivatives tocorrelate structure with biological activity.

Dorfman received his Ph.D. from the University of Chicago in 1939. He remained at theUniversity as a research associate and started to study the role of bacterial growth factors inmetabolism. These studies led to Dorfman’s development of the technique of growing deficientcells to determine the role of growth factors in metabolism. He also elucidated the roles ofpantothenic acid in pyruvate metabolism and of biotin in aspartic acid biosynthesis.

With the arrival of World War II and lack of an academic position, Dorfman returned tomedical school and graduated in 1944. This experience rekindled his interest in medicine,particularly pediatrics. An encounter with a child with rheumatic fever also sparked aninterest in the mechanism of action of aspirin and would profoundly affect Dorfman’s subse-quent career.

When Dorfman finished medical school he got an internship in internal medicine at BethIsrael Hospital and then became a resident in pediatrics at the University of Chicago. Aftercompleting his residency, he served 2 years in the U. S. Army where he was assigned to theArmy Medical School and was able to resume research in biochemistry. Around this time astudy emerged claiming that aspirin exerted its antirheumatic effect by inhibiting hyaluron-idase. Dorfman promptly initiated studies on connective tissue polysaccharides, an area ofresearch he would pursue for the next 30 years. He started by studying the biosynthesis ofhyaluronic acid in group A streptococci, which led to the development of quantitative methodsfor assays of hyaluronidase, the discovery that chondroitin sulfate is a substrate for testicularhyaluronidase, and the recognition that hyaluronidase is unusually stable to heat and acid pH.

After his 2 years in the army, Dorfman returned to the University of Chicago as an assistantprofessor of pediatrics and continued to study the biosynthesis of hyaluronic acid. His goal wasto determine the origins of the 14 unique carbon atoms of the polysaccharide using specificallylabeled precursors. Dorfman embarked on this project with his postdoc, Saul Roseman, whowas an author in a previous Journal of Biological Chemistry (JBC) Classic on Karl Paul Link(1) and will be featured in his own Classic in the future. Dorfman and Roseman, along withJulio Ludowieg and Frances Moses, synthesized [1-14C]glucose and incorporated it into me-dium upon which they could grow streptococcus. They devised a method for isolating theradioactive hyaluronic acid from the streptococcus filtrate and then analyzed its components.It became immediately evident that glucose was the major carbon precursor of hyaluronic acid.The glucose was then converted to the glucosamine and glucuronic acid portions of the

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molecule without cleavage of the carbon chain. These results are presented in the JBC Classicreprinted here, which is the first in a series of JBC papers Dorfman published on thebiosynthesis of hyaluronic acid by group A streptococcus.

Dorfman and his colleagues subsequently synthesized [6-14C]glucose and [1-14C]acetic acidand used those compounds to establish that acetate is a precursor of the acetyl group ofN-acetylglucosamine and that glucosamine but not N-acetylglucosamine serves as a precursorof the N-acetylglucosamine residue in hyaluronic acid. These results were also published in theJBC series (2, 3).

The discovery of uridine nucleotide sugars by Luis Leloir, as reported in a previous JBCClassic (4), suggested to Dorfman that these compounds might be intermediates in polysac-charide synthesis. Together with J. A. Cifonelli, Dorfman established that streptococci containtwo uridine nucleotide sugars, UDP-N-acetylglucosamine and UDP-glucuronic acid, which arerequisite for the biosynthesis of hyaluronic acid (5). Using labeled nucleotides, they were ableto demonstrate the synthesis of hyaluronic acid in a cell-free preparation of streptococci. Thiswas published as the final paper in Dorfman’s hyaluronic acid series in the JBC (6).

In addition to his work on hyaluronic acid, Dorfman also contributed significantly tounderstanding the biosynthesis of other glycosaminoglycans. As well, he discovered the causeof Hurler’s syndrome, a genetic disease that affects bones and cartilage and results in mentalretardation. He deduced that the condition results from elevated levels of dermatan sulfateand heparin sulfate due to a defect in �-L-iduronidase, an enzyme needed for the normalcatabolism of the two glycosaminoglycans.

In 1967 Dorfman became the Richard T. Crane Distinguished Service Professor of Pediatricsand Biochemistry and acted as Chairman of the Department of Pediatrics from 1962 to 1972.He also served as Director of the La Rabida University of Chicago Institute (1957–1972) andDirector of the Joseph P. Kennedy, Jr. Mental Retardation Research Center (1967–1982). Inaddition to his research activities, Dorfman was President of the Society for Glycobiology(1975) and President of the Pediatric Society (1979). His contributions to science were recog-nized with his election to the National Academy of Sciences in 1973.1


REFERENCES1. JBC Classics: Campbell, H. A., and Link, K. P. (1941) J. Biol. Chem. 138, 21–33; Stahmann, M. A., Huebner, C. F.,

and Link, K. P. (1941) J. Biol. Chem. 138, 513–527; Overman, R. S., Stahmann, M. A., Huebner, C. F., Sullivan,

1 All biographical information on Albert Dorfman was taken from Ref. 7.

Albert Dorfman. Photo courtesy of the Office of NIH History, National Institutes of Health.

Classics

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W. R., Spero, L., Doherty, D. G., Ikawa, M., Graf, L., Roseman, S., and Link, K. P. (1944) J. Biol. Chem. 153,5–24 (http://www.jbc.org/cgi/content/full/280/8/e5)

2. Roseman, S., Ludowieg, J., Moses, F. E., and Dorfman, A. (1954) The biosynthesis of hyaluronic acid by group AStreptococcus. II. Origin of the glucuronic acid. J. Biol. Chem. 206, 665–669

3. Dorfman, A., Roseman, S., Moses, F. E., Ludowieg, J., and Mayeda, M. (1955) The biosynthesis of hyaluronic acidby group A Streptococcus. III. Origin of the N-acetylglucosamine moiety. J. Biol. Chem. 212, 583–592

4. JBC Classics: Caputto, R., Leloir, L. F, Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333–350;Cabib, E., Leloir, L. F., and Cardini, C. E. (1953) J. Biol. Chem. 203, 1055–1070; Cabib, E., and Leloir, L. F.(1954) J. Biol. Chem. 206, 779–790 (http://www.jbc.org/cgi/content/full/280/19/e16)

5. Cifonelli, J. A., and Dorfman, A. (1957) The biosynthesis of hyaluronic acid by group A Streptococcus. V. Theuridine nucleotides of group A Streptococcus. J. Biol. Chem. 228, 547–557

6. Markovitz, A., Cifonelli, J. A., and Dorfman, A. (1959) The biosynthesis of hyaluronic acid by group A Strepto-coccus. VI. Biosynthesis from uridine nucleotides in cell-free extracts. J. Biol. Chem. 234, 2343–2350

7. Schwartz, N. B., and Roden, L. (1997) Biographical Memoir of Albert Dorfman, Vol. 72, pp. 70–87, NationalAcademy of Sciences, Washington, D. C.

Classics

H19

Hexosamine Metabolism, Sialic Acids, and thePhosphotransferase System: Saul Roseman’sContributions to GlycobiologyThe Sialic Acids. I. The Structure and Enzymatic Synthesis of N-AcetylneuraminicAcid(Comb, D. G., and Roseman, S. (1960) J. Biol. Chem. 235, 2529–2537)

Sugar Transport. I. Isolation of a Phosphotransferase System from Escherichia coli(Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1393–1406)

Saul Roseman was born in Brooklyn, New York, in 1921. He received his Bachelor of Sciencein Chemistry from the City College of New York in 1941 and began his graduate studies in theBiochemistry Department at the University of Wisconsin, earning his masters degree in 1944.He then served for 2 years as an infantryman in Europe in World War II. Upon his return, hecompleted his Ph.D. in 1947, studying biochemistry with Karl Paul Link and organic chem-istry with Homer Atkins. Roseman’s graduate work focused on the synthesis and metabolismof coumarin derivatives (e.g. Dicumarol), which Link had discovered and which was the subjectof a previous Journal of Biological Chemistry (JBC) Classic (1). It was during his graduatestudies that Roseman’s life-long interest in carbohydrates began, when he started working onthe metabolism of 4-hydroxycoumarin, which is secreted into the urine of dogs as the glucu-ronide or glucuronic acid derivative. A growing interest in complex carbohydrates led Rosemanto do his postdoctoral studies with Albert Dorfman at the University of Chicago School ofMedicine, who was also featured in a previous JBC Classic (2). With Dorfman, Rosemandeveloped new methods of glycan radioisotopic labeling to study hyaluronic acid and chon-droitin sulfate biosynthesis and showed that the carbon chain of glucose was converted in vivodirectly, with no cleavage, to that of glucosamine. He remained at Chicago until 1953 and waspromoted to Assistant Professor.

Roseman then became Assistant Professor of Biological Chemistry at the University ofMichigan Medical School and Chemist of the Rackham Arthritis Research Unit. In the nextfew years, he rose in academic rank to Professor. While at Michigan, Roseman started to workwith enzymes and cell-free extracts, which took him to glucosamine metabolism, which, inturn, led to the sialic acids. His work on the identification of the structure and metabolism ofsialic acids is the subject of the first JBC Classic reprinted here.

In the Classic, Roseman and one of his first postdoctoral fellows, Donald G. Comb (founderand President of New England Biolabs), report on their studies on the structure of one sialicacid in particular, N-acetylneuraminic acid, and the properties of the bacterial enzyme,N-acetylneuraminic acid aldolase, that cleaves the sialic acid into N-acetyl-D-mannosamine(which was not previously known to occur naturally) and pyruvate. Using N-acetylneuraminicacid purified from both human blood and Escherichia coli and N-acetylneuraminic acidaldolase from Clostridium perfringens, Roseman and Comb established the products of thecleavage reaction, showed that it was reversible (yielding NAN), and characterized the en-zyme. They also used 1-[14C]- and 6-[14C]glucose to follow the biosynthesis of N-acetylneura-minic acid and concluded that it is the product of a 3-carbon and 6-carbon condensation.

At that time, and over a period of 3 decades, over 11 possible structures had been suggestedfor N-acetylneuraminic acid. Roseman and Comb’s work was critical in establishing the correct

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structure. Subsequently, Roseman and Comb isolated, characterized, and enzymatically syn-thesized a unique sugar nucleotide, CMP-sialic acid. This led to a series of novel studies onglycosyltransferases. Roseman and colleagues showed that families of glycosyltransferasesexisted, in which each family transferred a specific sugar, and that each member of the familyhad specific requirements for the acceptor and/or the linkage of the newly synthesized glyco-sidic bond. The sum of these studies was to establish the individual steps in the metabolicpathways between fructose-6-P and complex carbohydrates such as the gangliosides (withBasu), the oligosaccharide chains in the mucins (with Schachter), and the carbohydratetermini in the blood glycoproteins (with Jourdian, Carlson, and others).

In 1965 Roseman was recruited to the McCollum-Pratt Institute and Department of Biologyat the Johns Hopkins University, where he later served as Director and Chairman for twoterms (1969–1973 and 1988–1990). Just before leaving Michigan for Baltimore, Roseman,while working on N-acetylmannosamine metabolism, discovered what turned out to be a novelsugar transport system in bacteria, the PTS (phosphotransferase system). The system involvesa series of sequential phosphotransfer reactions between proteins and simultaneously phos-phorylates and translocates its sugar substrates across the membrane. Surprisingly, thesource of the phosphoryl group is not ATP or another nucleoside triphosphate but ratherphosphoenolpyruvate.

Roseman’s isolation of a PTS from E. coli is the subject of the second JBC Classic reprintedhere. In the paper, Roseman and Werner Kundig purify the two enzymes (Enzyme I and II)and one protein (HPr) that comprise the system and determine that Enzyme I and HPr aresoluble while Enzyme II is part of the cell membrane. Using 32P and 14C to assay the activityof the enzymes, they conclude that Enzyme I catalyzes the transfer of phosphate fromphosphoenolpyruvate to HPr and Enzyme II catalyzes the transfer of phosphate from phospho-HPr to the carbohydrate. The phosphate is linked to HPr via an imidazole nitrogen atom on ahistidine residue. In two subsequent JBC papers (3, 4), Roseman and Kundig further charac-terized the enzymes that constitute the PTS, and somewhat later, with Simoni, establishedthat it was, in fact, a sugar transport system, not just a novel kinase.

Still at Johns Hopkins, Roseman retains his position of Professor in the Department ofBiology. His laboratory continues to make key contributions to the molecular understanding ofcomplex carbohydrate glycosyltransferases, bacterial sugar transport, and intercellular adhe-sion. Most recently, he has made novel forays into the complex metabolism of chitin, the secondmost abundant organic compound in nature. More information about the history of glycobiol-ogy can be found in Roseman’s JBC Reflections (5).

Saul Roseman

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In recognition of his contributions to glycobiology, Roseman has been the recipient of manynational and international awards and honors, among which was his election, in 1972, to theNational Academy of Sciences, and the degree of Doctor of Medicine Honoris causa from theUniversity of Lund. He has also received the Sesquicentennial award from the University ofMichigan (1967), the T. Duckett Jones Memorial award from the Helen Hay Whitney Foun-dation (1973), the Rosenstiehl award from Brandeis University (1974), the Internationalaward from the Gairdner Foundation (1981), and the Karl Meyer award from the Society ofGlycobiology (1993). Roseman is also a former member of the JBC editorial board and haspublished 136 papers in the journal over a 60-year period.



and Link, K. P. (1941) J. Biol. Chem. 138, 513–527; Overman, R. S., Stahmann, M. A., Huebner, C. F., Sullivan,W. R., Spero, L., Doherty, D. G., Ikawa, M., Graf, L., Roseman, S., and Link, K. P. (1944) J. Biol. Chem. 153,5–24 (http://www.jbc.org/cgi/content/full/280/8/e5)

2. JBC Classics: Roseman, S., Moses, F. E., Ludowieg, J., and Dorfman, A. (1953) J. Biol. Chem. 203, 213–225(http://www.jbc.org/cgi/content/full/280/31/e28)

3. Kundig, W., and Roseman, S. (1971) Sugar transport. II. Characterization of constitutive membrane-boundEnzymes II of the Escherichia coli phosphotransferase system. J. Biol. Chem. 246, 1407–1418

4. Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) Sugar transport. III. Purification and properties ofa phosphocarrier protein (HPr) of the phosphoenolpyruvate-dependent phosphotransferase system of Esche-richia coli. J. Biol. Chem. 246, 7023–7033

5. Roseman, S. (2001) Reflections on glycobiology. J. Biol. Chem. 276, 41527–41542

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The Regulation of Glucose Uptake in Muscle: the Work ofCharles R. ParkRegulation of Glucose Uptake in Muscle. I. The Effects of Insulin and Anoxia onGlucose Transport and Phosphorylation in the Isolated, Perfused Heart of NormalRats(Morgan, H. E., Henderson, M. J., Regen, D. M., and Park, C. R. (1961) J. Biol. Chem.236, 253–261)

Charles Rawlinson “Rollo” Park was born in Baltimore in 1916. He attended HarvardCollege and began medical studies at Johns Hopkins School of Medicine in 1941. Afterinterning at Johns Hopkins, he was appointed Assistant Resident and Chief Resident at thePeter Bent Brigham Hospital in Boston. In 1944, Park served in the U.S. Army Medical Corpsat Fort Knox, Kentucky, where he studied thermoregulation during the acclimatization ofmilitary personnel exposed to desert-like conditions. Park jocularly refers to this as his frontline experience during the war.

In 1947, Park joined the laboratory of Carl and Gerty Cori at Washington University in St.Louis. The Coris, who were featured in a previous Journal of Biological Chemistry (JBC)Classic (1), won the Nobel Prize for Physiology or Medicine that year, and many Nobellaureates emerged from their laboratory. Park’s work in St. Louis focused on the effects ofgrowth hormone, hypophysectomy, and adrenalectomy on glucose uptake by isolated ratdiaphragms. He relates his consternation when Carl Cori looked at some of his early resultsand commented in his German-accented English, “I do not like these data!” Despite this“encouraging comment,” Park enjoyed interacting with the stellar group of scientists workingin the laboratory at that time.

In 1952, at a relatively young age, Park was appointed to the Chair of Physiology atVanderbilt University Medical School. At that time, the department consisted of three phys-iologists, a technician, a graduate student, and a secretary, and the rooms were full ofabandoned, dusty, obsolete equipment. However from these humble beginnings, Park built oneof the most distinguished physiology departments in the country with a stellar reputation forresearch.

Park developed a vigorous research program in the hormonal regulation of carbohydratemetabolism and recruited faculty with a strong research background. The most notable ofthese was his wife, Jane Harting Park, who also came from the Cori department. Anotherrecruit was Howard Morgan, who was an obstetrician working at the Fort Campbell ArmyBase in Clarksville, Tennessee. In an effort to relieve the tedium of deliveries, he applied toPark who, in an enlightened choice, took him on as a researcher. Morgan turned out to be avery gifted investigator who later became Chair of Physiology at Pennsylvania State MedicalSchool, President of the American Physiological Society, and President of the American HeartAssociation. In an interesting turn of events, Park’s son Edwards A. Park later obtained hisPh.D. under Morgan.

Together with Morgan, Margaret J. Henderson, Robert Post, and David M. Regen, Parkbegan a classic series of experiments studying the regulation of glucose uptake in muscle byinsulin and other hormones. The experimental system was the isolated perfused rat heart, andthe work resulted in the publication of a series of six classic papers in the JBC in 1961 (2–6),the first of which is reprinted here as a JBC Classic. The papers showed that the limiting step






for glucose uptake was the transport of the sugar across the cell membrane and that this wasaccelerated by insulin and anoxia. Diabetes was shown to decrease this transport and alsoreduce its sensitivity to insulin. Hypophysectomy reduced basal glucose transport but made itmore sensitive to insulin, whereas growth hormone treatment in vivo had the opposite effect.Diabetes was also shown to decrease glucose phosphorylation, which was relieved by hypoph-ysectomy or adrenalectomy and restored by treatment with growth hormone or cortisol. Allthese findings were of fundamental relevance to human diabetes, and Park later won theBanting Award of the American Diabetes Association.

In 1963, Park recruited Earl Sutherland from Western Reserve University. Sutherland hadinitiated seminal studies of the hormonal regulation of glycogen phosphorylase in the Corilaboratory, which led to the discovery of cyclic AMP and the award of a Nobel Prize in 1971.This was the subject of a previous JBC Classic (7). Also in 1963, a postdoc named John Extonarrived from New Zealand and began a new area of research with Park: the hormonalregulation of hepatic gluconeogenesis. The research utilized the isolated perfused rat liver andagain resulted in a series of Classic papers published in the JBC (8).

Park’s scientific career didn’t stop there but extended to studies of the regulation of cyclicAMP-dependent protein kinase, cyclic AMP phosphodiesterase, fatty acid transport, and, incollaboration with Jane Harting Park, studies of muscular dystrophy and muscle metabolism.In addition to the Banting Award, Park has won many distinctions and awards at VanderbiltUniversity and was elected to the National Academy of Sciences in 1980. He was listed amongthe 10 most frequently cited authors in Physiology during 1965–1978.

The Parks have a long history with the JBC and the American Society for Biochemistry andMolecular Biology (ASBMB). Jane Harting Park was Treasurer of the American Society ofBiological Chemists (now the ASBMB) from 1979 to 1982, and Edwards A. Park served on theJBC editorial board from 1998 to 2003 and started another 5-year term in 2006. Rollo and Janewere also both JBC editorial board members in the 1960s and 1970s.

Charles Rawlinson “Rollo” Park

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Rollo Park has a relaxed, aristocratic demeanor and can properly be called a gentleman anda scholar. He is notable for treating the lowliest members of society the same as the accom-plished. His prowess as a fisherman, runner, and kayaker is legendary, and he has terrifiedmany famous scientists as he fearlessly guided his kayak through the rapids. His attitude topublishing and deadlines was relaxed, and priority was not a concern. When an anguishedpostdoctoral fellow would protest when a manuscript lay on his desk for many months or Parkspent almost a day studying a single result, he would say, “Don’t worry, the best paper winsin the end!” Park’s record proves he was right.

John H. Exton, Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES1. JBC Classics: Cori, C. F., and Cori, G. T. (1928) J. Biol. Chem. 79, 321–341; Cori, G. T., Colowick, S. P., and Cori,

C. F. (1938) J. Biol. Chem. 124, 543–555; Cori, G. T., Colowick, S. P., and Cori, C. F. (1939) J. Biol. Chem. 127,771–782; Green, A. A., and Cori, G. T. (1943) J. Biol. Chem. 151, 21–29; Cori, G. T., and Green, A. A. (1943)J. Biol. Chem. 151, 31–38 (http://www.jbc.org/cgi/content/full/277/29/e18)

2. Morgan, H. E., Cadenas, E., Regen, D. M., and Park, C. R. (1961) Regulation of glucose uptake in muscle. II.Rate-limiting steps and effects of insulin and anoxia in heart muscle from diabetic rats. J. Biol. Chem. 236,262–268

3. Post, R. L., Morgan, H. E., and Park, C. R. (1961) Regulation of glucose uptake in muscle. III. The interaction ofmembrane transport and phosphorylation in the control of glucose uptake. J. Biol. Chem. 236, 269–272

4. Henderson, M. J., Morgan, H. E., and Park, C. R. (1961) Regulation of glucose uptake in muscle. IV. The effect ofhypophysectomy on glucose transport, phosphorylation, and insulin sensitivity in the isolated, perfused heart.J. Biol. Chem. 236, 273–277

5. Henderson, M. J., Morgan, H. E., and Park, C. R. (1961) Regulation of glucose uptake in muscle. V. The effect ofgrowth hormone on glucose transport in the isolated, perfused rat heart. J. Biol. Chem. 236, 2157–2161

6. Morgan, H. E., Regen, D. M., Henderson, M. J., Sawyer, T. K., and Park, C. R. (1961) Regulation of glucose uptakein muscle. VI. Effects of hypophysectomy, adrenalectomy, growth hormone, hydrocortisone, and insulin onglucose transport and phosphorylation in the perfused rat heart. J. Biol. Chem. 236, 2162–2168

7. JBC Classics: Rall, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232, 1065–1076; Sutherland, E. W., andRall, T. W. (1958) J. Biol. Chem. 232, 1077–1092 (http://www.jbc.org/cgi/content/full/280/42/e39)

8. JBC Classics: Exton, J. H., and Park, C. R. (1967) J. Biol. Chem. 242, 2622–2636; Exton, J. H., and Park, C. R.(1968) J. Biol. Chem. 243, 4189–4196 (http://www.jbc.org/cgi/content/full/282/10/e7)

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Plant Carbohydrates and the Biosynthesis of Lactose: theWork of William Zev HassidA Soluble Lactose-synthesizing Enzyme from Bovine Milk(Babad, H., and Hassid, W. Z. (1964) J. Biol. Chem. 239, 946–948)

Soluble Uridine Diphosphate D-Galactose:D-Glucose �-4-D-Galactosyltransferasefrom Bovine Milk(Babad, H., and Hassid, W. Z. (1966) J. Biol. Chem. 241, 2672–2678)

Zev Hassid (1899–1974) was born in Jaffa, Palestine. He added “William” to his name afterhe came to the United States in 1920. Hassid was educated in Palestine at a Hebrew languageschool and then at an Agricultural High School, from which he graduated in 1916. He thenworked as a farm laborer until 1918 when he joined the British army to help liberate Palestinefrom the Turks. While in the army, Hassid was never involved in combat; instead he guardedprisoners and supplies in transit, which allowed him to travel to places like Alexandria inEgypt. It was in Alexandria that Hassid first heard about the University of California from afellow soldier who had studied there.

After leaving the army, Hassid decided to use his savings to go to California to studyagronomy at the University, intending to return to Palestine to assist in the development ofscientific agriculture. He arrived in Berkeley in 1920 and registered at the University ofCalifornia. However, his knowledge of English was so limited that he could not follow thelectures or read the textbooks. After a week of frustration he took a leave of absence from theUniversity and moved to Fresno where he attended Fresno State Teachers College, majoringin Letters and Science with an emphasis on Chemistry, French Language, and Mathematics.In August 1924, he returned to UC Berkeley to major in Chemistry, but he changed his majorto general literature and obtained a Bachelor of Arts degree in 1925. He then enrolled ingraduate studies at the School of Education and graduated in 1926, at the same time earninga General Secondary School Credential from the State Board of Education. However, insteadof teaching, he worked as a chemical analyst for a year.

In 1927, Hassid was offered a position as a research assistant with D. R. Hoagland in theDivision of Plant Nutrition at Berkeley’s Agricultural Experiment Station. Working withHoagland, Hassid analyzed plant materials and soils for a variety of inorganic constituents.This renewed his interest in plant research, and he enrolled at UC Berkeley as a graduatestudent in Plant Nutrition. He earned his Ph.D. in 1934, investigating the structure ofpolysaccharides in marine algae for his thesis. After graduating Hassid joined the staff of theDivision of Plant Nutrition as a junior chemist and rose to the rank of Professor of PlantBiochemistry in 1947. In 1959 he transferred to the Biochemistry Department and in 1965 hebecame Emeritus.

Hassid’s independent scientific research started with his investigation of the structure of agalactan that was a major component of the fleshy marine alga, Iridea laminarioides. Inelucidating the structure he applied methylation methods that had recently been developed bythe English chemist and Nobel laureate, Walter N. Haworth. Later he used the same methodsto establish the primary structures of several other types of starch and glycogen, includingcanna starch, dog liver glycogen, the dextran formed from sucrose by Betacoccus arabinosa-ceus, an insoluble polysaccharide derived from Saccharomyces cerevisiae, and glycogen and






starch derived from sweet corn. These initial investigations led to an interest in the biochem-istry of carbohydrates that remained with Hassid throughout his career.

Hassid started collaborating with Samuel Ruben and Martin D. Kamen in 1939 on the firstapplication of 11C to the study of photosynthesis. When 14C became available in 1946, Hassidand his students pioneered in the development of biological methods for the preparation ofuniformly 14C-labeled carbohydrates from plant tissue, including D-glucose, D-fructose, D-galactose, sucrose, and starch. He generously supplied the radioactive sugars to many otherinvestigators before they became commercially available.

In 1943, Hassid initiated a collaboration with Michael Doudoroff and Horace A. Barker, bothauthors of previous Journal of Biological Chemistry (JBC) Classics (1, 2), as well as Nathan O.Kaplan. They investigated the biosynthesis of sucrose by sucrose phosphorylase, an enzymefrom Pseudomonas saccharophila. Their demonstration of the first enzymatic synthesis ofsucrose caught the attention of officials at the Coca-Cola Company who were having troubleobtaining sucrose because of wartime rationing. The company sent a representative to Berke-ley to offer them $500,000 for research on sucrose phosphorylase if a commercial process forsucrose synthesis seemed feasible. Unfortunately, Hassid and his associates were away at thetime, and the representative could only discuss the problem with Hoagland who was pessi-mistic about the method. Due to Hoagland’s lack of optimism, Coca-Cola did not end upproviding funding for sucrose phosphorylase research.

Subsequent efforts to show the presence of a similar enzyme in plants were unsuccessfuluntil Luis Leloir and his associates discovered uridine diphosphate D-glucose (UDPG) anddemonstrated the synthesis of sucrose from UDPG and fructose, as reported in a previous JBCClassic (3). This prompted Hassid and his colleagues to undertake a systematic investigationof the occurrence of nucleoside diphosphate sugars in plants. They isolated nucleoside diphos-phate derivatives of D-xylose, L-arabinose, D-galactose, D-galacturonic acid, D-mannuronic acid,and 2-acetamido-2-deoxy-D-glucose and established the roles of several of these compounds insugar interconversions and polysaccharide formation.

Hassid’s reputation attracted scientists from around the world to work in his laboratory.One of these scientists was Winifred N. Watkins, who embarked on a study of the biosynthesisof lactose in mammary tissue with Hassid. Using guinea pig and bovine mammary glands,they established that lactose was synthesized according to the following reaction.

William Zev Hassid, professor of biochemistry at the University of California Berkeley. Credit: Bob Lackenbach,University of California Berkeley (1951 or earlier).

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UDP-D-galactose � D-glucose 3 lactose � UDP

REACTION 1

They also discovered that mammary tissue contained an enzyme activity that transfersD-galactose to N-acetyl-D-glucosamine.

This led to Hassid’s isolation of lactose synthetase with Helene Babad, which is the subjectof the two JBC Classics reprinted here. The first Classic is a communication that describeshow Hassid and Babad used centrifugation and ammonium sulfate fractionation to obtain a“soluble enzyme preparation from bovine milk capable of catalyzing synthesis of lactose fromUDP-D-galactose and D-glucose.” They confirmed that their preparation contained lactosesynthetase activity using [1-14C]UDP-D-galactose and �-D-[14C]glucose 1-phosphate. The sec-ond Classic describes the partial purification and some of the properties of the galactosyl-transferase responsible for the synthesis of lactose. It was later discovered that lactosesynthetase is composed of two proteins: galactosyltransferase and �-lactalbumin, which in-creases the affinity of galactosyltransferase.

Hassid’s numerous contributions to understanding plant carbohydrates were recognized byseveral awards and honors. He was given the first Sugar Research Award (1945) of theNational Academy of Sciences (jointly with Doudoroff and Barker), the Charles Reid BarnesHonorary Life Membership Award of the American Society of Plant Physiologists (1964), andthe C. S. Hudson Award of the American Chemical Society (1967). In 1972 he was honored atthe Sixth International Symposium on Carbohydrate Chemistry as one of three outstandingsenior American carbohydrate chemists. He was a member of the National Academy ofSciences and the American Academy of Arts and Sciences, Chairman of the Division ofCarbohydrate Chemistry of the American Chemical Society (1949–1950), and a member ofnumerous editorial boards including that of the JBC.1


REFERENCES1. JBC Classic: Entner, N., and Doudoroff, M. (1952) J. Biol. Chem. 196, 853–862 (http://www.jbc.org/cgi/

content/full/280/27/e24)2. JBC Classic: Brady, R. O., Castanera, E. G., and Barker, H. A. (1962) J. Biol. Chem. 237, 2325–2332

(http://www.jbc.org/cgi/content/full/280/33/e30)3. JBC Classics: Caputto, R., Leloir, L. F, Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333–350;

Cabib, E., Leloir, L. F., and Cardini, C. E. (1953) J. Biol. Chem. 203, 1055–1070; Cabib, E., and Leloir, L. F.(1954) J. Biol. Chem. 206, 779–790 (http://www.jbc.org/cgi/content/full/280/19/e16)

4. Ballou, C. and Barker, H. A. (1979) Biographical Memoir of William Zev Hassid, Vol. 50, pp.196–231, NationalAcademy of Sciences, Washington, D. C.

1 All biographical information on William Zev Hassid was taken from Ref. 4.

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The Control of Gluconeogenesis: the Work of John ExtonControl of Gluconeogenesis in Liver. I. General Features of Gluconeogenesis in thePerfused Livers of Rats(Exton, J. H., and Park, C. R. (1967) J. Biol. Chem. 242, 2622–2636)

Control of Gluconeogenesis in Liver. II. Effects of Glucagon, Catecholamines, andAdenosine 3�,5�-Monophosphate on Gluconeogenesis in the Perfused Rat Liver(Exton, J. H., and Park, C. R. (1968) J. Biol. Chem. 243, 4189–4196)

John H. Exton was born in Auckland, New Zealand in 1933. He received his medical degreefrom the University of New Zealand in 1958 and his Ph.D. in biochemistry from the Universityof Otago, New Zealand in 1963. Exton then left New Zealand for Nashville, Tennessee to dopostdoctoral research in the Department of Physiology at the Vanderbilt University School ofMedicine with Charles R. Park and Journal of Biological Chemistry (JBC) Classic author EarlSutherland (1).

Along with Park, who will be featured in an upcoming JBC Classic, Exton worked onelucidating the pathways of gluconeogenesis, the formation of glucose from non-sugar carbonsubstrates like pyruvate, lactate, glycerol, and certain amino acids. Gluconeogenesis occursprincipally in the liver and is essential for survival in starvation; it also plays a major role inthe disposal of lactate and maintenance of glucose during exercise. The process is controlleddirectly or indirectly by many hormones, including insulin, glucagon, catecholamines, andglucocorticoids. Exton and Park published a series of papers in the JBC on the control ofgluconeogenesis using the isolated, perfused rat liver. This allowed them to study the processand its regulation without the interference of other changes in the body. Two of those papersare reprinted here as JBC Classics.

The first paper studied the basic process of gluconeogenesis (i.e. the conversion of lactate,pyruvate, glycerol, alanine, and a mixture of amino acids to glucose) and demonstrated thatphysiological increases in these substrates alone led to increased glucose production by theliver, indicating the importance of substrate supply in the regulation of gluconeogenesis.Comparison of the rates of gluconeogenesis from lactate, pyruvate, fructose, and dihydroxy-acetone suggested that the rate-limiting step for lactate gluconeogenesis was located betweenpyruvate and triose phosphate in the gluconeogenic pathway. Measurements of the conversionof [14C]pyruvate to glucose and CO2 were used in a mathematical analysis to determine theflow of isotope from this substrate into the gluconeogenic pathway and the Krebs cycle.

In the second paper, the stimulatory effects of glucagon and catecholamines on lactategluconeogenesis were analyzed. Physiological concentrations of glucagon were effective instimulating gluconeogenesis. However, blood levels of epinephrine were not, leading to theproposal that the stimulatory effects of the sympathetic nervous system were due to therelease of norepinephrine from adrenergic nerve endings in the liver. Exogenous cyclic AMPstimulated gluconeogenesis, consistent with this being the mediator of the effect of glucagon.Studies of gluconeogenesis from fructose indicated that glucagon/cyclic AMP stimulated thepathway somewhere between pyruvate and phosphoenolpyruvate in the gluconeogenicpathway.

In 1966, Exton became an Assistant Professor of Physiology at Vanderbilt and was promotedto Associate Professor in 1968. Later, in 1970, he became Professor of Molecular Physiology

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and Biophysics and in 1989 he became a Professor of Pharmacology. Exton remains atVanderbilt where he studies signal transduction.

Exton has received many honors for his research including the Lilly Award from theAmerican Diabetes Association, a Doctor Honoris Causa from the Autonomous University ofBarcelona, the Drummond Award from the University of Calgary, and the Sutherland Award,Vanderbilt’s highest research award. He is also a University National Scholar, New Zealand,and a Commonwealth Scholar, United Kingdom, as well as a fellow of the American Associ-ation for the Advancement of Science and a member of the National Academy of Science. Hewas named an investigator of the Howard Hughes Medical Institute in 1976 and has served asan Associate Editor for the Journal of Biological Chemistry since 1988.


REFERENCES1. JBC Classics: Rall, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232, 1065–1076; Sutherland, E. W., and

Rall, T. W. (1958) J. Biol. Chem. 232, 1077–1092 (http://www.jbc.org/cgi/content/full/280/42/e39)

John Exton

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Mycobacterial Glycophosphoinositides: the Work ofClinton E. BallouBiosynthesis of Mannophosphoinositides by Mycobacterium phlei. The Family ofDimannophosphoinositides(Brennan, P., and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046–3056)

Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. Enzymatic Acy-lation of the Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1968) J. Biol. Chem. 243, 2975–2984)

Clinton Edward Edgerton Ballou was born inKing Hill, Idaho in 1923. After graduating fromhigh school, he enrolled in a premed program atBoise Junior College, but his interests quicklyturned to chemistry after he dissected a poorlyembalmed cat in a comparative anatomy course.After 2 years at Boise, Ballou pursued a degree inchemistry at Oregon State College in Corvalliswhere he became involved in two researchprojects: the first during his junior year synthe-sizing new antimalarial drugs with Bert Chris-tensen, and the second during his senior yearstudying the guinea pig “antistiffness factor” withWillem van Wagtendonk.

The military draft was in effect as Ballou en-tered his last year of college so he decided to jointhe U. S. Navy after graduating in 1944. He wasdischarged 2 years later and decided to apply forgraduate study in biochemistry with Karl PaulLink at the University of Wisconsin-Madison. Asdetailed in a previous Journal of BiologicalChemistry (JBC) Classic (1), Link’s research cen-tered on blood anticoagulants, and when Ballouarrived in his laboratory in 1946, the primary

focus was the structure-function relationship of coumarin anticoagulants. Ballou was imme-diately intrigued when he learned of a failed attempt to synthesize the glucoside of dicumarolbecause the acetylated intermediate was degraded in the alkali conditions used for deacety-lation. Because glycosides are acetals, which are typically acid-labile and alkali-stable, Balloudecided to study a variety of synthetic compounds to try to understand the structural basis foralkali sensitivity. This research formed the core of his doctoral dissertation, and his exposureto carbohydrate chemistry influenced the direction of his career.

After earning his Ph.D. in 1950, Ballou did a year-long postdoctoral fellowship with E. L.Hirst in the Department of Chemistry at the University of Edinburgh. There he studied thestructure of maple sapwood starch. At the end of the year, Ballou returned to the U. S. to workwith Hermann O. L. Fischer (the son of Nobel laureate Hermann Emil Fischer) at theUniversity of California, Berkeley. Ballou explained his choice: “I was attracted to Fischer in

Clinton E. Ballou






part because of his research on phosphorylated sugars but also because during graduate schoolI had drawn heavily on the published works of his father, Emil Fischer. I guess the idea ofbeing associated with the son of Emil Fischer just seemed ‘real cool’ to me” (2).

The 1950s was a time of active research on biosynthetic pathways involving short chainphosphorylated sugars, and collaborating with Fischer and Donald MacDonald, Ballou under-took the syntheses of several such metabolic intermediates, including D-glyceric acid 2-phos-phate, D-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid3-phosphate, and D-erythrose 4-phosphate. He also became interested in inositol chemistry asa result of studies on the cyclitols in sugar pine heartwood.

In 1955, Ballou was appointed to the biochemistry faculty at Berkeley and went about settingup an independent research program. He decided to work on inositol-containing phospholipidsand was able to synthesize and characterize D-myoinositol 1-phosphate. He also spent severalyears isolating and characterizing myoinositol polyphosphates from beef brain phosphoinositide.This culminated in his discovery of D-myoinositol l,4,5-trisphosphate or IP3. More information onBallou’s studies of these phosphoinositides can be found in his JBC Reflections (2).

Ballou became eligible for sabbatical leave in 1961 and decided to spend a year at theNational Center for Scientific Research (CNRS) in France studying the glycophosphoinositidesof mycobacteria with Edgar Lederer. There he collaborated with Erna Vilkas on experimentsto establish the linkages of both the phosphatidyl and the mannosyl groups to the myoinositolring (3). Upon his return to Berkeley, Ballou and Yuan Chuan Lee determined the structuresof the family of mannosyl phosphoinositides in Mycobacterium smegmatis (4–6). Ballou andhis postdoctoral fellow Patrick Brennan then began to look at the biosynthesis of the intactdimannophosphoinositides on Mycobacterium phlei, which is the subject of the two JBCClassics reprinted here.

In the first Classic, Ballou and Brennan used subcellular fractions of M. phlei to catalyzethe biosynthesis of several mannosyl derivatives of phosphatidylmyoinositol and reportedthat the major products are a family of three dimannophosphoinositides (A, B, and C) thatdiffer in the number of fatty acyl groups they contain (four, three, and two, respectively). Onthe basis of their results, they proposed that guanosine diphosphate mannose acts as the sugardonor in the conversion of phosphatidylmyoinositol to phosphatidylmyoinositol dimannoside(dimannophosphoinositide C), which is then acylated in a two-step process to first yielddimannophosphoinositide B and then dimannophosphoinositide A.

In the second JBC Classic, Ballou and Brennan provide further evidence for their proposedbiosynthesis scheme by using an enzyme from M. phlei to specifically incorporate labeled fattyacids into the dimannophosphoinositides. They showed that label from [14C]palmityl-CoA isincorporated into dimannophosphoinositide C to yield dimannophosphoinositide B. After ashort incubation period, this molecule is converted to dimannophosphoinositide A, but withlonger incubation periods the product is deacylated to isomeric forms of dimannophosphoi-nositides B and C.

Brennan continued to work on these glycophosphoinositides after completing his postdoc-toral fellowship with Ballou and eventually showed that the lipoglycans (lipomannan (LM) andlipoarabinomannan (LAM)) were multiglycosylated extensions of Ballou’s phosphatidylinositolmannosides and are very important in the pathogenesis of tuberculosis and leprosy. Morerecent research has defined the biochemistry and genetics of synthesis of these molecules.Brennan is currently University Distinguished Professor in the Department of Microbiology,Immunology, and Pathology at Colorado State University. He served on the editorial board ofthe Journal of Biological Chemistry for several years and was also named Colorado StateUniversity Researcher of the Year in 1992.

In 1991, Ballou became Professor Emeritus of Biochemistry at the University of California,Berkeley, although he continued research and teaching for a few years. In recognition of hiscontributions to science, Ballou has received many awards and honors including election to theNational Academy of Sciences (1975), the American Chemical Society’s Claude Hudson Awardin Carbohydrate Chemistry (1981), the Welch Foundation Lectureship (1972), the Universityof Notre Dame Reilly Lectureship (1976), the Duke University Belfort Lectureship (1977), aNational Science Foundation Senior Fellowship (1961), and a University of California Berke-ley Citation (1992). Ballou also served as an editorial board member for the Journal ofBiological Chemistry.


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and Link, K. P. (1941) J. Biol. Chem. 138, 513–527; Overman, R. S., Stahmann, M. A., Huebner, C. F., Sullivan,W. R., Spero, L., Doherty, D. G., Ikawa, M., Graf, L., Roseman, S., and Link, K. P. (1944) J. Biol. Chem. 153, 5–24(http://www.jbc.org/cgi/content/full/280/8/e5)

2. Ballou, C. E. (2004) My brief encounter with the phosphoinositides and IP3. J. Biol. Chem. 279, 54975–549823. Ballou, C. E., Vilkas, E., and Lederer, E. (1963) Structural studies on the myo-inositol phospholipids of Mycobac-

terium tuberculosis (var. bovis, strain BCG). J. Biol. Chem. 238, 69–764. Lee, Y. C., and Ballou, C. E. (1964) Structural studies on the myo-inositol mannosides from the glycolipids of

Mycobacterium tuberculosis and Mycobacterium phlei. J. Biol. Chem. 239, 1316–13275. Ballou, C. E., and Lee, Y. C. (1964) The structure of myoinositol mannoside from Mycobacterium tuberculosis

glycolipid. Biochemistry 3, 682–6856. Lee, Y. C., and Ballou, C. E. (1965) Complete structures of the glycophospholipids of mycobacteria. Biochemistry 4,

1395–1404

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Proteoglycans and Orchids: the Work of Vincent HascallProteinpolysaccharide Complex from Bovine Nasal Cartilage. A Comparison of Lowand High Shear Extraction Procedures(Sajdera, S. W., and Hascall, V. C. (1969) J. Biol. Chem. 244, 77–87)

Proteinpolysaccharide Complex from Bovine Nasal Cartilage. The Function ofGlycoprotein in the Formation of Aggregates(Hascall, V. C., and Sajdera, S. W. (1969) J. Biol. Chem. 244, 2384–2396)

Aggregation of Cartilage Proteoglycans. III. Characteristics of the Proteins Isolatedfrom Trypsin Digests of Aggregates(Heinegård, D., and Hascall, V. C. (1974) J. Biol. Chem. 249, 4250–4256)

Vincent C. Hascall, Jr. was bornin Burwell, Nebraska in 1940. Heattended the California Institute ofTechnology and earned a B.S. in1962. After graduating he enrolledat Rockefeller University, where heearned his Ph.D. in 1969. While atRockefeller, Hascall met Alan Kap-uler who had won a WestinghousePrize in high school for using a newmethod for meristem cultures on anorchid species. As a result Kapulerwas taken to Bogota, Colombia tolearn how specimens were collectedand preserved.

“Alan suggested that we take asummer and go collecting orchids inCentral America and Colombia,” re-calls Hascall. “Rockefeller Univer-sity agreed to let us do it, and Alan’sparents gave us an old Renault Dau-phin 2-cylinder car, and we drovefrom Manhattan down through Cen-tral America to Panama, put the caron a boat and went on to Colombia.In Colombia, on the Pacific Oceanside, we explored a region that hadlarge, moss ridden trees alongside aroad. In the moss of one of the trees,

I noticed some small orange dots. They turned out to be what is likely the smallest orchid inthe world. The flower is the size of the head of a pin. It was a new species.” Kapuler and Hascallnamed the orchid Platystele acutilingua (1) (see Fig. 1). The genus Platystele was known, butthe species name, acutilingua, meaning sharp tongue, was their contribution.

Vincent Hascall

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However, for his thesis research,Hascall chose to focus on cartilagerather than orchids. “I ended up inthe basement of one of the Rockefellerresearch buildings next to the EastRiver in a small lab with Stan Sa-jdera, a fellow graduate student,” re-calls Hascall. “We taught as lab techsin a summer biochemistry lab courseheaded by Dominic Dziewiatkowski,who had cartilage experiments going.Both of us then continued to workwith cartilage for our Ph.D. work.”

Dziewiatkowski accepted a positionat the University of Michigan before

Hascall and Sajdera finished theirresearch at Rockefeller. The two stu-dents continued their research with-out Dziewiatkowski and were able todevelop an extraction and purifica-tion procedure for proteoglycans ontheir own. This work is the subject ofthe first and second Journal of Bio-logical Chemistry (JBC) Classics re-printed here.

At the time this research was pub-lished, scientists were using highspeed homogenization in low ionicstrength salt solutions to extractproteinpolysaccharides (now calledproteoglycans) from tissue. How-ever, this method introduced shearartifacts and denatured and depoly-merized the macromolecules. In aserendipitous experiment, Sajdera,who was exploring the effects of re-ducing agents and denaturants on

the isolation and activity of the proteoglycans, used 4 M guanidine HCl as a control for theextraction of cartilage slices. To Hascall and Sajdera’s surprise, almost all of the proteoglycanswere released into the solution whereas the slices remained intact. In a series of follow-upexperiments, they showed that this extraction method caused proteoglycan aggregates in thecartilage to dissociate, thereby allowing the proteoglycan monomer (now called aggrecan) todiffuse into the extraction solvent. This procedure, which they termed “the dissociativeextraction method” for proteoglycans, is still used today. The Classic paper became the basisfor Sajdera’s Ph.D thesis.

In the second Classic paper, Hascall and Sajdera showed that cartilage proteoglycans in thedissociative extracts reformed aggregates when dialyzed into lower, associative solvent con-centrations (0.4 M guanidine HCl). They also showed that a protein they named the glycopro-tein link protein was required for successful aggregation. This paper became the basis forHascall’s Ph.D. thesis. What Hascall and Sajdera did not know at the time, however, was thathyaluronan was present in the extracts and also required for aggregation.

After receiving his Ph.D., Hascall joined the faculty of the University of Michigan as anassistant professor. A couple years later, in 1972, he met Dick Heinegård at a GordonConference. Heinegård invited Hascall to spend a year in his laboratory at the University ofLund in Sweden. Hascall accepted the offer and was off to Sweden the next year. When hearrived, he learned that Hardingham and Muir had just discovered that by adding smallamounts of hyaluronan to a solution of proteoglycan monomer, they could induce the formation

FIG. 2. Electron micrograph and model of a proteoglycan aggregatepurified from calf epiphyseal cartilage. Micrograph kindly provided byJoseph Buckwalter, University of Iowa. Model and supplemental three-dimensional, rotational view kindly provided by Mark Sabo (Art Depart-ment) and Vincent Hascall, Cleveland Clinic Foundation.

FIG. 1. Platystele acutilinqua.

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of proteoglycan aggregates (2). Curious about this phenomenon, Heinegård and Hascall begantheir own series of investigations.

The final JBC Classic paper reprinted here is the last in a series of three that the twoscientists published (3, 4), which finally put all the pieces together to define the role ofhyaluronan and the link protein in forming the cartilage proteoglycan (aggrecan) aggregates.By digesting aggregated proteoglycan with proteases, Heinegård and Hascall were able toshow that two proteins were bound to hyaluronan. One protein was the link protein, and theother was derived from a domain of the core protein of aggrecan, now referred to as the G1

domain. These results led to a model for the proteoglycan aggregates in which the link proteinand the G1 domain were bound to each other as well as to hyaluronan (see Fig. 2).

After returning from Sweden, Hascall spent 2 more years at the University of Michigan asan associate professor. In 1975, he became Senior Staff Fellow in the Laboratory of Biochem-istry at the National Institute of Dental Research, National Institutes of Health. He eventuallybecame Chief of the Proteoglycan Chemistry Section in the Laboratory of Biochemistry at theNational Institute of Dental Research, a position he held until he left the NIH in 1994 to jointhe Department of Biomedical Engineering at the Cleveland Clinic Foundation. From 2001 to2005, Hascall was co-director of the Orthopaedic Research Center at the Cleveland Clinic.Today, he remains at the Cleveland Clinic and also holds a position as a professor in theDepartment of Biological Chemistry at Case Western Reserve University.

In addition to his scientific research, Hascall is an Associate Editor for the Journal ofBiological Chemistry, a position he has held since 1995. He also received the NIH Merit Awardin 1979, was President of the Society for Complex Carbohydrates in 1987, and was awardedthe Karl Meyer Award for Glycoconjugate Research from the Society for Complex Carbohy-drates in 1992.1


REFERENCES1. Hascall, V. C., and Kapuler, A. M. (1966) Collecting along a transect: from the rain forest to the Andes in Choco,

Colombia. American Orchid Society Bulletin 35, 540–5442. Hardingham T. E., and Muir, H. (1972) The specific interaction of hyaluronic acid with cartilage proteoglycans.

Biochim. Biophys. Acta 279, 401–4053. Hascall, V. C., and Heinegård, D. (1974) Aggregation of cartilage proteoglycans. I. The role of hyaluronic acid.

J. Biol. Chem. 249, 4232–42414. Hascall, V. C., and Heinegård, D. (1974) Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors

of the proteoglycan-hyaluronic acid interaction. J. Biol. Chem. 249, 4242–42495. Hascall, V. C. (2000) Hyaluronan, a common thread. Glycoconj. J. 17, 607–616

1 Biographical information on Vincent Hascall was taken from Ref. 5.

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Lactose Synthesis in the Mammary Gland: LactoseSynthase and the Work of Robert L. HillThe Complete Amino Acid Sequence of �-Lactalbumin(Brew, K., Castellino, F. J., Vanaman, T. C., and Hill, R. L. (1970) J. Biol. Chem. 245,4570–4582)

The Disulfide Bonds of Bovine �-Lactalbumin(Vanaman, T. C., Brew, K., and Hill, R. L. (1970) J. Biol. Chem. 245, 4583–4590)

The Purification and Properties of the A Protein of Lactose Synthetase(Trayer, I. P., and Hill, R. L. (1971) J. Biol. Chem. 246, 6666–6675)

Robert L. Hill was born in Kansas City, Missouri in 1928. He earned a B.A. in chemistry in1949 and a Ph.D. in biochemistry in 1954 from the University of Kansas. After receiving hisPh.D., he went to the University of Utah as a National Institutes of Health (NIH) postdoctoralfellow, where he worked with Emil L. Smith, who was featured in two Journal of BiologicalChemistry (JBC) Classics (1, 2). Although Hill’s Ph.D. thesis research concerned bacterialmetabolism, his postdoctoral studies introduced him to protein and enzyme chemistry, re-search areas he pursued during his subsequent career. At Utah, he published papers onproteolytic enzymes and human hemoglobin and myoglobin. Hill remained at the Universityof Utah as a faculty member of the Department of Biochemistry until 1961. He then joined thefaculty of the Department of Biochemistry at Duke University School of Medicine, where heremains today.

At Duke, Hill continued his work on human hemoglobins, including several abnormalvariants, but soon turned his attention to the structure-function relationships of other proteinsand enzymes, including human fibrinogen and other blood coagulation factors, immunoglobu-lins, egg white lysozyme, bacterial acyl carrier protein, and lactose synthase. In subsequentyears he studied glycosyltransferases and worked in several areas of glycobiology. Hill’s workon lactose synthase is the subject of the three JBC Classics reprinted here.

Lactose synthase consists of a catalytic galactosyltransferase in the endoplasmic reticulumof the mammary gland and a regulatory protein, �-lactalbumin, secreted in milk. Without�-lactalbumin, galactosyltransferase cannot synthesize lactose and instead catalyzes theattachment of galactose to N-acetylglucosamine units on glycoproteins. The presence of �-lac-talbumin changes the specificity of galactosyltransferase so that it can transfer galactose toglucose.

In the first JBC Classic, Hill, along with Keith Brew, Francis J. Castellino, and Thomas C.Vanaman, reports the complete amino acid sequence of bovine �-lactalbumin. They deducedthe sequence by characterizing the tryptic, chymotryptic, and peptic peptides of �-lactalbumincleaved with cyanogen bromide. Because the amino acid sequence of �-lactalbumin is verysimilar to that of egg white lysozyme, Hill and his colleagues aligned the two sequences andfound that 49 of the residues were identical and 23 were conservative replacements. Thus, theyconcluded that the three-dimensional structures of the two proteins were probably very similar(3) and that they most likely arose from a common ancestral gene.

Hill, Brew, and Vanaman confirmed this structural similarity in the second Classic in whichthey describe the locations of the four disulfide bonds in �-lactalbumin. They found that thedisulfide bonds are arranged in a manner similar to those in egg white lysozyme and proposed

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a three-dimensional structure for �-lactalbumin based on the three-dimensional structure oflysozyme, as shown in Fig. 2. Later, Hill, Brew, and Vanaman showed how �-lactalbuminserves as a regulatory protein for lactose synthase and permits the enzyme to synthesizelactose in the mammary gland.

In the final Classic reprinted here, Hill and Ian P. Trayer describe the purification of thegalactosyltransferase component of lactose synthase or what they refer to as the “A protein.”A key step to their purification was the use of columns of �-lactalbumin attached covalently toSepharose. In the presence of glucose, galactosyltransferase would bind to the column andcould then be removed by omission of glucose from the elution buffer. Using this method, Hilland Trayer were able to purify galactosyltransferase from bovine milk about 12,000-fold.

In 1969 Hill became the chairman of the Department of Biochemistry, and in 1974, theJames B. Duke Professor. He served as chairman until 1993. He was President (1976) andSecretary (1972–1975) of the American Society of Biological Chemistry and was also on theEditorial Board (1965–1970, 1972–1977) and an Associate Editor (1988-present) of the JBC.Hill served on the FASEB Board (1972–1978), was General Secretary of the InternationalUnion of Biochemistry (1982–1991), and chair of the Organizing Committee of the 17thInternational Congress of Biochemistry and Molecular Biology in San Francisco in 1997. Hewas elected to the National Academy of Sciences in 1975, the Institute of Medicine in 1978, andthe American Academy of Arts and Sciences in 1974. Hill received the Rose Award from theAmerican Society for Biochemistry and Molecular Biology in 1991, the North Carolina GoldMedal (Science-1985), and the Karl Meyer Award from the Society for Glycobiology (2001).

Each of the coauthors on these Classic papers was a student or postdoctoral fellow in Hill’slaboratory, and all have had productive, independent careers in biochemistry. Thomas Vana-man was a Ph.D. student who subsequently became Professor of Biochemistry and Chair at theUniversity of Kentucky. The others were postdoctoral fellows. Keith Brew went to the Uni-versity of Leeds (UK) before returning to the U. S. where he became Professor of Biochemistryat the University of Miami. He is now at Florida Atlantic University. Francis J. Castellino iscurrently Professor of Biochemistry at the University of Notre Dame, and Ian P. Trayer was

Robert L. Hill

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Professor and Head of the School of Chemistry at the University of Birmingham (UK). Brewand Castellino served on the JBC editorial board. Vanaman also served on the JBC board andcurrently is an Associate Editor.

Nicole Kresge and Robert D. Simoni

REFERENCES1. JBC Classics: Smith, E. L., and Bergmann, M. (1944) J. Biol. Chem. 153, 627–651 (http://www.jbc.

org/cgi/content/full/279/47/e6)2. JBC Classics: DeLange, R. J., Fambrough, D. M., Smith, E. L., and Bonner, J. J. (1969) J. Biol. Chem. 244,

5669–5679 (http://www.jbc.org/cgi/content/full/280/36/e33)3. Browne, W. J., North, A. C., Phillips, D. C., Brew, K., Vanaman, T. C., and Hill, R. L. (1969) A possible

three-dimensional structure of bovine �-lactalbumin based on that of hen’s egg-white lysozyme. J. Mol. Biol. 42,65–86

Fig. 2

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Lysosomal Storage Disease Factors: the Work ofElizabeth F. NeufeldThe Hurler Corrective Factor. Purification and Some Properties(Barton, R. W., and Neufeld, E. F. (1971) J. Biol. Chem. 246, 7773–7779)

The Sanfilippo A Corrective Factor. Purification and Mode of Action(Kresse, H., and Neufeld, E. F. (1972) J. Biol. Chem. 247, 2164–2170)

The Hunter Corrective Factor. Purification and Preliminary Characterization(Cantz, M., Chrambach, A., Bach, G., and Neufeld, E. F. (1972) J. Biol. Chem.247, 5456–5462)

Elizabeth Fondal Neufeld was born in 1928 in Paris, France. Her parents were Russianrefugees who had settled in France after the Russian revolution. However, the impendingoccupation of France by the Germans forced the Fondal family to move to New York in 1940.Consequently, Neufeld attended Queens College in New York, where she received her B.S. in1948. She then worked briefly as a research assistant to Elizabeth Russell at the JacksonMemorial Laboratory in Bar Harbor, Maine, before enrolling in graduate school at theUniversity of Rochester. Due to personal reasons Neufeld moved to Maryland in 1951, whereshe served as a research assistant to Nathan Kaplan and Sidney Colowick at the McCollum-Pratt Institute at Johns Hopkins University. Colowick’s research on NADH was the subject ofa previous Journal of Biological Chemistry (JBC) Classic (1), and Kaplan will be featured inan upcoming JBC Classic.

In 1952, Neufeld enrolled in graduate school again, this time at the University of California,Berkeley, where she studied with JBC Classic author William Zev Hassid (2). She received aPh.D. in 1956 for her work on nucleotides and complex carbohydrates. Neufeld then did severalyears of postdoctoral research at Berkeley, first working with Dan Mazia on non-proteinsulfhydryl compounds in mitosis and then returning to Hassid’s laboratory to pursue researchon substituted sugars, polysaccharides, and glycoproteins.

In 1963, Neufeld moved to the National Institutes of Health (NIH), where she became aresearch biochemist at the National Institute of Arthritis, Metabolism, and Digestive Dis-eases. It was during her time at the NIH that Neufeld began her research on mucopolysac-charidoses (MPS) disorders, a group of lysosomal storage diseases in which mucopolysaccha-rides cannot be stored or metabolized properly. Hurler syndrome and Hunter syndrome aretwo MPS disorders in which partially degraded dermatan sulfate and heparan sulfate accu-mulate in lysosomes. Affected individuals usually die from the diseases by age 15. It wasaccepted dogma in the early 1960s that mucopolysaccharides accumulate in these diseasesbecause of aberrant regulation of their synthesis resulting in overproduction.

At the NIH, Neufeld began to study mucopolysaccharide turnover in cultured fibroblastsfrom people with Hurler or Hunter syndrome by incorporating radioactive sulfate into newlysynthesized mucopolysaccharides and studying their degradation with chase experiments. Sheshowed that the defects in Hurler and Hunter syndromes were due to decreased degradationof the mucopolysaccharides and their resulting accumulation in lysosomes rather than anoverproduction of the sugars (3).

Subsequently, Neufeld found that Hurler syndrome could be corrected by a macromolecularfactor derived from fibroblasts or urine from donors who did not have Hurler syndrome. Her

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purification and characterization of this factor is discussed in the first JBC Classic reprintedhere. The factor was purified 1000-fold from normal human urine. From her experiments,Neufeld surmised that the Hurler factor accelerates degradation of stored sulfated mucopo-lysaccharides. When an assay became available for �-L-iduronidase in 1972, Neufeld was ableto show that the corrective factor for Hurler syndrome was, in fact, �-L-iduronidase and thatthis syndrome, as well as the Scheie syndrome, was due to a deficiency of that enzyme (4).

As reported in the second JBC Classic, Neufeld found a similar factor for Sanfilipposyndrome, a lysosomal storage disease marked by impaired degradation of heparan sulfate.The life span of children with this disorder does not usually extend beyond late teens to earlytwenties. Sanfilippo patients fall into four subgroups, designated A through D, deficient in oneof four factors. Neufeld purified the Sanfilippo A corrective factor 850-fold from normal humanurine. She found that incubation of stored mucopolysaccharide with the purified factor re-sulted in release of inorganic sulfate, suggesting that the Sanfilippo A factor was a heparansulfate sulfatase. This proved to be correct.

In the final JBC Classic, Neufeld returned to her research on Hunter syndrome andpresented the purification of the Hunter corrective factor from normal human urine. She foundthat the factor accelerates the degradation of labeled dermatan sulfate as well as exogenouslyadded proteodermatan [35S]sulfate. Neufeld eventually determined that the Hunter correctivefactor was iduronate sulfatase (5).

In 1973 Neufeld was named chief of the NIH Section of Human Biochemical Genetics, andin 1979 she was named chief of the Genetics and Biochemistry Branch of the National Instituteof Arthritis, Diabetes, and Digestive and Kidney Diseases (NIADDK). She served as deputydirector of NIADDK’s Division of Intramural Research from 1981 to 1983. In 1984 Neufeldreturned to the University of California, this time the Los Angeles campus, as chair of theBiological Chemistry Department. She continues to do research at UCLA today.

Neufeld has chaired the Scientific Advisory Board of the National MPS Society since 1988and was president of the American Society for Biochemistry and Molecular Biology in 1992.She was elected to both the National Academy of Sciences and the American Academy of Artsand Sciences in 1977 and was named a fellow of the American Association for Advancement inScience in 1988. In recognition of her scientific achievements she was awarded the HildebrandAward in 1975, the Gairdner Foundation Award in 1981, the Lasker Award in 1982, theInternational Society for Clinical Enzymology J. Henry Wilkinson Memorial Award in 1983,

Elizabeth F. Neufeld

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the Franklin Institute’s Elliot Cresson Medal in 1984, the Wolf Prize in Medicine in 1988, theNational Medal of Science in 1994, and the Christopher Columbus Discovery Award forBiomedical Research in 1992. She was named California Scientist of the Year in 1990.1


REFERENCES1. JBC Classics: Pullman, M. E., San Pietro, A., and Colowick, S. P. (1954) J. Biol. Chem. 206, 129–141

(http://www.jbc.org/cgi/content/full/280/39/e36)2. JBC Classics: Babad, H., and Hassid, W. Z. (1964) J. Biol. Chem. 239, 946–948; Babad, H., and Hassid, W. Z.

(1966) J. Biol. Chem. 241, 2672–2678 (http://www.jbc.org/cgi/content/full/280/34/e31)3. Fratantoni, J. C., Hall, C. W., and Neufeld, E. F. (1968) The defect in Hurler’s and Hunter’s syndromes: faulty

degradation of mucopolysaccharide. Proc. Natl. Acad. Sci. U. S. A. 60, 699–7064. Bach, G., Friedman, R., Weissmann, B., and Neufeld, E. F. (1972) The defect in the Hurler and Scheie syndromes:

deficiency of �-L-iduronidase. Proc. Natl. Acad. Sci. U. S. A. 69, 2048–20515. Bach, G., Eisenberg, F., Cantz, M., and Neufeld, E. F. (1973) The defect in the Hunter syndrome: deficiency of

sulfoiduronate sulfatase. Proc. Natl. Acad. Sci. U. S. A. 70, 2134–21386. Hirschhorn, K. (1983) The William Allan Memorial Award. Am. J. Hum. Genet. 35, 1077–1080

1 Biographical information on Elizabeth F. Neufeld was taken from Ref. 6.

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The Biosynthesis of Membrane Glycoproteins: the Work ofWilliam J. LennarzMembrane Glycoproteins. I. Enzymatic Synthesis of Mannosyl Phosphoryl Polyiso-prenol and Its Role as a Mannosyl Donor in Glycoprotein Synthesis(Waechter, C. J., Lucas, J. J., and Lennarz, W. J. (1973) J. Biol. Chem. 248, 7570–7579)

William Joseph Lennarz was born in New York City in 1934. He received his B.S. inchemistry from Pennsylvania State University in 1956 and his Ph.D. in organic chemistryfrom the University of Illinois in 1959. Subsequently, he carried out postdoctoral research onfatty acid biosynthesis at Harvard University with Journal of Biological Chemistry (JBC)Classic author Konrad Bloch (1). In 1962 Lennarz moved to Baltimore where he was appointedAssistant Professor in the Department of Physiological Chemistry at the Johns HopkinsSchool of Medicine. He was later promoted to Associate Professor of Biochemistry in 1966 andProfessor in 1971.

The focus of Lennarz’s work at Johns Hopkins was lipids and bacterial cell surfaces. Hefound that mannosyl phosphoryl undecaprenol participates in the biosynthesis of mannolipidsin Micrococcus lysodeikticus (2). This work showed that lipid-linked sugars were biosyntheticprecursors of envelope-associated bacterial polysaccharides. Curious as to whether polyisopre-nol phosphates also served as glycosyl carriers in mammalian tissue, Lennarz and his col-leagues looked at the enzymatic transfer of mannose from GDP-mannose to endogenousacceptors in cell-free preparations of bovine thyroid and hen oviduct. This is the subject of theJBC Classic reprinted here.

Lennarz found that the preparations did catalyze the transfer of mannose to severalendogenous acceptors including mannosyl phosphoryl polyisoprenol. Two other receptors werediscovered and named “soluble mannosylated endogenous acceptor” (mannosyl s-acceptor) and“residual mannosylated endogenous acceptor” (mannosyl r-acceptor). Lennarz and his col-leagues then showed that mannosyl phosphoryl polyisoprenol serves as the mannosyl donor forthe synthesis of both mannosyl s- and r-acceptor. He further postulated that mannosyls-acceptor mediates the transfer of mannosyl residues from mannosyl phosphoryl polyisopre-nol to glycoproteins. From these results Lennarz concludes, “it seems possible that in eukary-otic systems, as in bacterial systems, activated lipid-linked sugars mediate the synthesis ofglycose-containing macro-molecules that are associated with the membranous components ofthe cell.”

Lennarz left Baltimore for Texas in 1983 when he was appointed Robert A. Welch Professorand Chairman of the Department of Biochemistry and Molecular Biology at the University ofTexas Cancer Center, M. D. Anderson Hospital in Houston. In 1989, he joined the faculty of theState University of New York at Stony Brook and became Distinguished Professor andChairman of the Department of Biochemistry and Cell Biology, a title he still holds today. In1990 he founded and became Director of the Institute for Cell and Developmental Biology atStony Brook.

Lennarz continues to focus on glycoproteins, and his more recent efforts have been on thestructure, biosynthesis, and function of cell surface glycoproteins and the role of cell surfaceproteins in fertilization and embryonic development in the sea urchin and frog. Currently, heis studying the steps involved in glycoprotein synthesis, including N-glycosylation and proteinfolding, as well as the functions of the glycan chains.






Lennarz served as president of the American Society for Biochemistry and MolecularBiology in 1989 and was also president of both the Biochemistry Chairman’s Organization andthe Society for Glycobiology. He was awarded the Society for Glycobiology’s Karl Meyer Awardin 2004. Lennarz was a member of the Executive Committee of the International Union ofBiochemistry and Molecular Biology for almost a decade. He served as co-editor-in-chief for theEncyclopedia of Biological Chemistry and was a member of the editorial board for Biochemicaland Biophysical Research Communications. He was elected to the National Academy ofSciences in 1989.


REFERENCES1. JBC Classics: Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625–636; Rittenberg, D., and Bloch, K.

(1945) J. Biol. Chem. 160, 417–424; Bloch, K. (1945) J. Biol. Chem. 157, 661–666(http://www.jbc.org/cgi/content/full/280/10/e7)

2. Lahav, M., Chiu, T. H., and Lennarz, W. J. (1969) Studies on the biosynthesis of mannan in Micrococcuslysodeikticus. II. The enzymatic synthesis of mannosyl-1-phosphoryl-undecaprenol. J. Biol. Chem. 244,5890–5898

William J. Lennarz

Classics

H44

Hepatic Carbohydrate Binding Proteins and GlycoproteinCatabolism: the Work of Gilbert G. AshwellThe Isolation and Properties of a Rabbit Liver Binding Protein Specific forAsialoglycoproteins(Hudgin, R. L., Pricer, W. E., Jr., Ashwell, G., Stockert, R. J., and Morell, A. G. (1974)J. Biol. Chem. 249, 5536–5543)

Isolation and Characterization of an Avian Hepatic Binding Protein Specific forN-Acetylglucosamine-terminated Glycoproteins(Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252, 6536–6543)

G. Gilbert Ashwell was born in Jersey City, New Jersey, in 1916. He attended the Universityof Illinois where he earned his B.A. in 1938 and his M.S. in 1941. Ashwell then went toColumbia University and received his M.D. in 1948. After graduating, he remained at Colum-bia University as a research fellow for 2 years. In 1950, Ashwell joined the National Instituteof Arthritis, Metabolism, and Digestive Diseases at the National Institutes of Health. TheInstitute later split into the National Institute of Arthritis and Musculoskeletal and SkinDiseases and the National Institute of Diabetes and Digestive and Kidney Diseases, whereAshwell remains today as emeritus scientist.

Ashwell is perhaps best known for his work with Anatol G. Morell in which they proposedthat membrane lectins remove senescent circulating glycoproteins and discovered one of theearliest known carbohydrate receptors. Ashwell met Morell when he was on sabbatical leaveat Columbia University in 1965. Morell, who was at the Albert Einstein College of Medicine inthe Bronx, was interested in devising a method for labeling serum glycoproteins to study therole of ceruloplasmin in Wilson disease. Together, Ashwell and Morell devised a labelingprocedure (1) that involved enzymatic removal of the glycoprotein’s terminal sialic acidresidue, thereby exposing galactose which was then treated with galactose oxidase andtritiated borohydride, resulting in the incorporation of tritium into the protein.

When they injected their radioactive ceruloplasmin into rabbits, Ashwell and Morell noticedthat the asialoglycoproteins rapidly disappeared from the serum and appeared in parenchymalcells in the liver (2). Further investigations showed that this phenomenon occurred with avariety of naturally occurring plasma glycoproteins (3) and that the plasma membranes of theliver were the primary site of binding for the circulating glycoproteins (4). This led to thehypothesis that the exposure of terminal, nonreducing galactosyl residues by the removal ofsialic acid provides a means by which the liver recognizes and removes the defective moleculesfrom circulation as part of their normal catabolic pathway.

As described in the first Journal of Biological Chemistry (JBC) Classic reprinted here,Ashwell and Morell eventually isolated the asialoglycoprotein binding protein from rabbit liverusing an affinity column composed of asialoorosomucoid covalently linked to Sepharose 4B.

Several years later, Ashwell and Toshisuke Kawasaki isolated an avian hepatic bindingprotein that was specific for terminal N-acetylglucosamine residues on glycoproteins. This isthe subject of the second JBC Classic reprinted here. They compared the avian and rabbitproteins and found that they had many properties in common, such as similar carbohydrateconstituents and a requirement for calcium. However, the two proteins also differed in manyways. For example, the avian protein, in contrast to the mammalian protein, exhibited onlyminimal binding activity for asialoglycoproteins but interacted strongly with agalactoglyco-

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proteins. The structures of the two proteins also differed. The rabbit protein consisted of twodifferent subunits that were 48,000 and 40,000 daltons. The avian protein contained a singlesubunit with an estimated molecular weight of 26,000.

Ashwell’s work on hepatic binding proteins has served as a stimulus for the identification ofa host of carbohydrate-specific receptors on various cell surfaces and has inaugurated thecurrent concept of a “cellular lectin.” In recognition of his contributions to science, Ashwell hasreceived many awards including the Gairdner Foundation International Award in 1982, theMerck Prize from the American Society for Biological Chemists (now the American Society forBiochemistry and Molecular Biology) in 1984, and the Senior Scientist Award from theAlexander von Humboldt Foundation in 1989. He was elected to the National Academy ofSciences in 1979.


REFERENCES1. Morell, A. G., Van Den Hamer, C. J. A., Scheinberg, I. H., and Ashwell, G. (1966) Physical and chemical studies

on ceruloplasmin. IV. Preparation of radioactive, sialic acid-free ceruloplasmin labeled with tritium on terminalD-galactose residues. J. Biol. Chem. 241, 3745–3749

2. Morell, A. G., Irvine, R. A., Sternlieb, I., Scheinberg, I. H., and Ashwell, G. (1968) Physical and chemical studieson ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J. Biol. Chem. 243, 155–159

3. Morell, A. G., Gregoriadis, G., Scheinberg, I. H., Hickman, J., and Ashwell, G. (1971) The role of sialic acid indetermining the survival of glycoproteins in the circulation. J. Biol. Chem. 246, 1461–1467

4. Pricer, W. E., and Ashwell, G. (1971) The binding of desialylated glycoproteins by plasma membranes of rat liver.J. Biol. Chem. 246, 4825–4833

Gilbert G. Ashwell Anatol G. Morell

Classics

H46

The Pathway of Complex Oligosaccharide Biosynthesis:the Work of Stuart A. KornfeldProcessing of High Mannose Oligosaccharides to Form Complex Type Oligosaccha-rides on the Newly Synthesized Polypeptides of the Vesicular Stomatitis Virus GProtein and the IgG Heavy Chain(Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716–722)

The Synthesis of Complex-type Oligosaccharides. I. Structure of the Lipid-linkedOligosaccharide Precursor of the Complex-type Oligosaccharides of the VesicularStomatitis Virus G Protein(Li, E., Tabas, I., and Kornfeld, S. (1978) J. Biol. Chem. 253, 7762–7770)

The Synthesis of Complex-type Oligosaccharides. II. Characterization of the Proc-essing Intermediates in the Synthesis of the Complex Oligosaccharide Units of theVesicular Stomatitis Virus G Protein(Kornfeld, S., Li, E., and Tabas, I. (1978) J. Biol. Chem. 253, 7771–7778)

Stuart A. Kornfeld was born in St. Louis in 1936. He attended Dartmouth College andreceived his A.B. in 1958 and then went to the Washington University School of Medicinewhere he earned an M.D. in 1962. Kornfeld then spend a year as an intern at Barnes Hospitalin St. Louis, 2 years as a research associate at the National Institute of Arthritis and MetabolicDiseases, National Institutes of Health, and an additional year as an assistant resident atBarnes Hospital. In 1966, he joined the faculty of the Washington University School ofMedicine where he has remained since. He became a professor of medicine in 1972 and ofbiochemistry in 1976, the same year he began co-directing the hematology division. Kornfeldalso directed the Medical Scientist Training Program from 1991 to 1997. He is currently theDavid C. and Betty Farrell Distinguished Professor of Medicine at Washington University.

In his early research at Washington University, Kornfeld focused on the synthesis ofcomplex oligosaccharides. This is the subject of the three Journal of Biological Chemistry(JBC) Classics reprinted here. At the time the Classics were published, it was believed that theglycosylation of glycoproteins containing asparagine-linked oligosaccharides occurred by enbloc transfer of preformed oligosacchride chains from a lipid-linked oligosaccharide interme-diate to an asparagine residue in a newly synthesized polypeptide chain. As reported in thefirst Classic, Kornfeld and his student Ira Tabas, in collaboration with the virologist SondraSchlesinger, were able to isolate this lipid-linked oligosaccharide intermediate from vesicularstomatitis virus-infected Chinese hamster ovary cells. They determined that the intermediatewas a high molecular weight mannose-rich oligosaccharide and that as the viral glycoproteinmatured in the cell, the excess mannose residues were removed and the outer sugars wereadded. This indicated a single pathway for the formation of both high mannose and complex-type glycans on glycoproteins.

In the second JBC Classic, Kornfeld and his students Ellen Li and Ira Tabas determined thecomplete structure of the lipid-linked oligosaccharide intermediate. They did this by growingcells on radioactive precursors, isolating the lipid-linked oligosaccharide, and then specificallydigesting the molecule. Prior to this paper, only partial structures of the molecule werepublished. The structure of the oligosaccharide is conserved from yeast to man.

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After the lipid-linked intermediate is transferred to the nascent protein, the oligosaccharideis processed to give rise to the completed complex oligosaccharide units. In the final JBCClassic, Kornfeld and his students characterized the major processing intermediates andproposed a scheme for the pathway of complex oligosaccharide biosynthesis.

Kornfeld has received numerous honors, including the Passano Award in 1991, the E.Donnall Thomas Lectureship and Prize in 1992, the Karl Meyer Award from the Society ofGlycobiology in 1999, the UCSD/Nature Medicine “Mentorship Award” in 2002, the Washing-ton University Gerty & Carl Cori Faculty Recognition Award in 2002, and the WashingtonUniversity Second Century Award in 2002. An author or co-author of more than 200 scientificarticles, he is a member of several honorary societies including the National Academy ofSciences, the Institute of Medicine, the American Academy of Arts and Sciences, and theAssociation of American Physicians. Kornfeld was an Associate Editor for the JBC from 1982to 1987 and Editor of the Journal of Clinical Investigation from 1981 to 1982. He has alsoserved on numerous editorial boards including those of the Archives of Biochemistry andBiophysics, the Proceedings of the National Academy of Sciences, the Journal of Cell Biology,and Molecular Biology of the Cell.


Stuart A. Kornfeld

Classics

H48

The Isolation and Localization of Lamininby Rupert TimplLaminin—a Glycoprotein from Basement Membranes(Timpl, R., Rohde, H., Robey, P. G., Rennard, S. I., Foidart, J. M., and Martin, G. R.(1979) J. Biol. Chem. 254, 9933–9937)

Rupert Timpl (1936–2003) was widely known for his work on extracellular matrix proteins.As a graduate student Timpl, who already had an impressive list of highly cited publications,led a group of connective tissue immunologists started by Carl Steffen at the Institute forGeneral and Experimental Pathology at the University of Vienna Medical School. In this role,Timpl supervised three postdoctoral fellows. He and his colleagues isolated collagen type I andpublished several papers on the production and specificity of the first antibodies to extracel-lular matrix proteins.

Timpl earned his Ph.D. in chemistry from the University of Graz in 1966, and in 1967 hebecame an Assistant in the Department of Immunology at the University of Vienna, Austria.In 1969 he moved to Germany to become Head of the Research Group in the Department ofConnective Tissue Research at the Max-Planck-Institut for Biochemistry in Martinsried/Munich. Timpl remained at Max-Planck for the rest of his scientific career, eventuallybecoming Scientific Member and Director of the Department of Protein Chemistry in 1992.Timpl also served as the Executive Director of the Max-Planck-Institut from 1995 to 1997.

At the Max-Planck-Institut, Timpl continued to study extracellular matrix proteins, focus-ing on the identification of epitopes of collagenous and non-collagenous extracellular matrixproteins, and became one of the first scientists to apply immunofluorescence to the analysis ofnormal and fibrotic tissues. For example, he showed that the tissue distribution of procollagentype I differs from that of mature type I collagen and also clarified that type III collagenproduction precedes that of type I collagen in any type of fibrosis.

The Journal of Biological Chemistry (JBC) Classic reprinted here is the result of a collab-oration between Timpl and George R. Martin in which they delineated basement membranesunder normal and pathologic conditions. For their studies, Timpl and Martin used a trans-plantable mouse tumor, the EHS sarcoma, which produced an extracellular matrix of base-ment membrane. From the tumor, they extracted type IV collagen and, using antibodies,localized it to the basement membrane of normal tissues (1, 2). In this paper, Timpl, Martin,and their colleagues isolated a high molecular weight non-collagenous glycoprotein that wasalso a major constituent of the tumors. They determined that the protein, which they namedlaminin, consisted of at least two polypeptide chains joined to each other by disulfide bonds.Using purified antibody against laminin, they showed that the glycoprotein is produced by avariety of cultured cells and is a constituent of the basement membranes of these tissues.

In addition to his research on collagen and laminin, Timpl performed some unorthodoxexperiments. These included analyzing the distribution of extracellular matrix proteins in1500-year-old Peruvian mummies, in tissues of the Tyrolean Iceman (Otzi), and in 50 million-year-old fossils using immunohistochemical and immunofluorescent methods, thus creating anew scientific discipline, paleoimmunology.

In recognition of his scientific achievements, Timpl received many honors. These includethe1984 Barbara Robert Medal, the 1991 Max Planck Research Award, the 1997 Wenner-Gren






Distinguished Lectureship, and the 1998 Lennox K. Black Award from Thomas JeffersonUniversity in Philadelphia.1

Timpl’s collaborator on the Classic, George R. Martin, is known for his studies on thestructure and function of connective tissue and alterations with disease. At the time the paperwas written, Martin was chief of the National Institute of Dental Research’s Laboratory ofDevelopmental Biology and Anomalies. In 1988 he was named Scientific Director of theNational Institute on Aging, a position he held until 1994. Martin has been involved in twobiotech startups, including the South San Francisco-based FibroGen.

Martin received his undergraduate degree in chemistry from Colgate University and hisPh.D. from the University of Rochester. He has been the recipient of several honors includingthe International Association of Dental Research Award in Basic Science, the Department ofHealth and Human Services Distinguished Service Award, the Alexander von HumboldtSenior Scientist Award, and the Federal Meritorious Executive Rank Award in 1987.


REFERENCES1. Timpl, R., Martin, G. R., Bruckner, P., Wick, G., and Wiedemann, H. (1978) Nature of the collagenous protein in

a tumor basement membrane. Eur. J. Biochem. 84, 43–522. Timpl, R., Martin, G. R., and Bruckner, P. (1979) Frontiers in Matrix Biology, Vol. 7, pp. 130–141, Karger, Basel3. Wick, G. (2004) Rupert Timpl—a Personal Account. Int. Arch. Allergy Immunol. 134, 89–92

1 Biographical information Rupert Timpl on was taken from Ref. 3.

Rupert Timpl. Photo reprinted from Ref. 3, published by S. Karger AG, Basel.

Classics

H50

The Formation of N-Glycosidic Linkages: the Work ofPhillips W. RobbinsTemperature-sensitive Yeast Mutants Deficient in Asparagine-linked Glycosylation(Huffaker, T. C., and Robbins, P. W. (1982) J. Biol. Chem. 257, 3203–3210)

Phillips Wesley Robbins was bornin 1930, in Barre, Massachusetts. Hegraduated from DePauw Universityin 1952 with an A.B. and received hisPh.D. in biochemistry from the Uni-versity of Illinois in 1955. After finish-ing his Ph.D. research, he became aresearch associate for Nobel Laureateand Journal of Biological Chemistry(JBC) Classic author Fritz Lipmann(1) at the Massachusetts GeneralHospital. Working with Lipmann,Robbins studied how ATP was used inthe activation and transfer of sulfateand showed that the thermodynamicpotential for the process came directlyfrom ATP. This discovery made alarge contribution to the general un-derstanding of the global sulfur cycle,and as a result Robbins received theEli Lilly Award in Biochemistry in1956. The next year, Lipmann movedto the Rockefeller Institute, and Rob-bins joined him as Assistant Professorwhere he continued his work on sul-fur activation.

In 1960, Robbins became AssistantProfessor of Biochemistry at the Mas-sachusetts Institute of Technology.

There, he met Nobel Laureate Salvador Luria, who had just discovered that the structure ofSalmonella lipopolysaccharide (LPS) was controlled by lysogenic bacteriophage. Over the nextseveral years, Robbins solved the structure of LPS and showed that the repeating units of thepolysaccharide were preassembled on a polyisoprenoid lipid carrier that was eventually namedbactoprenol. Robbins then elucidated the cycle involved in polysaccharide assembly.

After working on bactoprenol, Robbins turned to mammalian N-linked glycosylation. Theformation of N-glycosidic linkages of eukaryotic glycoproteins was known to involve thetransfer of a common precursor oligosaccharide from a lipid carrier (dolichol) to an asparagineresidue in the nascent polypeptide chain. In many organisms, the lipid-linked precursor hadthe composition Glc3Man9GlcNAc2. Robbins set out to identify and characterize the genesinvolved in the early steps of the dolichol-linked oligosaccharide assembly. To do this, he used

Phillips W. Robbins






Saccharomyces cerevisiae mutants that were blocked in the synthesis of the lipid-linkedoligosaccharide, its co-translational transfer to protein, and the first steps of post-translationalprocessing.

The JBC Classic reprinted here describes Robbins’ procedures for isolating temperature-sensitive mutants in asparagine-linked glycosylation as well as his characterization of one ofthese mutants (algl-1). Robbins and Tim C. Huffaker showed that algl-1 cells were able tosynthesize GlcNAc2-lipid but were unable to synthesize any mannose-containing oligosaccha-ride-lipids. The algl-1 cells were also unable to elongate exogenous GlcNAc2-lipid but were ableto convert Man1GlcNAc2-lipid to Man5GlcNAc2-lipid. These results indicated that the algl-1mutant was blocked at the addition of the fist mannose residue to the oligosaccharide-lipid.Characterization of the Glc3Man9GlcNAc2-lipid had shown that only the mannose residueattached to GlcNAc exists in a �-D-linkage, thus indicating that the mutant had a deficiencyin the enzyme involved in this process.

Robbins subsequently identified and characterized several other genes in this pathway.These yeast genes were eventually shown to have orthologs in mammals. The enzymology andgenetics of the dolichol pathway enzymes represent classical pieces of glycobiology history.

Robbins’ later research turned to the dynamics of yeast cell wall synthesis and remodeling,focusing on chitin synthesis. In 1998, he joined the newly formed Department of Molecular andCell Biology at the Boston University School of Dental Medicine (BUSDM) where he continuedhis research on chitin synthesis as well as the evolution of N-linked glycosylation.

In recognition of his contributions to science, Robbins has received many awards and honors.These include the Karl Meyer Award for Lifetime Achievement in Glycobiology (2000) andelection to the National Academy of Sciences (1986).


REFERENCES1. JBC Classics: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)

Classics

H52

The Transient Glucosylation of Glycoproteins: the Work ofArmando J. ParodiProtein Glycosylation in Trypanosoma cruzi. The Mechanism of Glycosylation andStructure of Protein-bound Oligosaccharides(Parodi, A. J., Lederkremer, G. Z., and Mendelzon, D. H. (1983) J. Biol. Chem. 258,5589–5595)

Transient Glucosylation of Protein-bound Man9GlcNAc2, Man8GlcNAc2, andMan7GlcNAc2 in Calf Thyroid Cells. A Possible Recognition Signal in the Processingof Glycoproteins(Parodi, A. J., Mendelzon, D. H., and Lederkremer, G. Z. (1983) J. Biol. Chem. 258,8260–8265)

Evidence That Transient Glucosylation of Protein-linked Man9GlcNAc2,Man8GlcNAc2, and Man7GlcNAc2 Occurs in Rat Liver and Phaseolus vulgaris Cells(Parodi, A. J., Mendelzon, D. H., Lederkremer, G. Z., and Martin-Barrientos, J. (1984)J. Biol. Chem. 259, 6351–6357)

Armando J. Parodi was born in Bue-nos Aires, Argentina, in 1942. As ateenager in a Latin American countryin the late 1950s, he was much moreinterested in politics than in sciencebut nonetheless enrolled at the Schoolof Sciences at the University of Bue-nos Aires after graduating from highschool. The professors at the univer-sity were mostly young, dynamic scien-tists who had just returned from post-doctoral training in the United Statesand Europe, and their enthusiasm wascontagious. As a result, Parodi decidedto obtain a Ph.D. after graduating fromthe university. His father, who hadbeen a graduate student along withLuis Leloir in Bernardo A. Houssay’slaboratory at the University of BuenosAires, encouraged his son to attend theInstituto de Investigaciones Bioquımi-cas Fundacion Campomar (now theFundacion Instituto Leloir), a privateinstitute created by Leloir in 1947. Be-ing an obedient son, Parodi followed hisfather’s advice.

Once at the Campomar Institute,Parodi joined Leloir’s research group.Armando J. Parodi






Leloir, who was featured in a previous Journal of Biological Chemistry (JBC) Classic (1), wasinterested in the biosynthesis of saccharides, and Parodi was assigned a problem relating tothe synthesis of high molecular weight glycogen. He was able to publish five papers based onthis research and earned his Ph.D. in 1970, the same year that Leloir was awarded the NobelPrize in Chemistry.

Several years prior to 1970, Philips Robbins (who will be featured in an upcoming JBCClassic) and JBC Classic author Jack Strominger (2) had shown that lipid (polyprenol)-linkedglycans were intermediates in the synthesis of several components of the bacterial cell wall.Leloir decided to study whether or not this phenomenon occurred in eukaryotes as well andwas able to show that incubating rat liver microsomes with UDP-Glc led to the synthesis ofdolichol-P-Glc. After defending his Ph.D. thesis, Parodi joined this project and was part of ateam that elucidated the role of dolichol in transferring glycans to asparagine residues duringprotein N-glycosylation.

After spending 2 years as a postdoctoral fellow at the Pasteur Institute in Paris, Parodireturned to the Leloir Institute where he remains today. Between 1975 and 1978 he was ableto show that the dolichol-P-dependent pathway of protein N-glycosylation was also present inyeast. A report in 1980 claiming that the pathway was not present in trypanosomatid protozoa,based on the fact that neither free nor sugar-bound dolichol-P was present in the organisms,led Parodi to challenge this claim. By incubating trypanosomatids with [14C]glucose, he wasable to show that the protozoans did indeed synthesize dolichol-P-P-glycans. However, theglycans that were formed, Man9GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2, lacked glucose.

Parodi decided to further investigate the processing of protein-linked glycans in the trypano-some Trypanosoma cruzi, which is the subject of the first JBC Classic reprinted here. He andhis colleagues found that short pulses with [14C]glucose produced three protein-linked glycansthat were identified as Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2. Thesecompounds disappeared upon chasing cells with unlabeled glucose, and after a certain period,Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2 were found in mature, fullyprocessed glycoproteins. Because an unglucosylated glycan was transferred to protein in thisprotozoon, the only way in which Glc1Man9GlcNAc2 could have been synthesized was by thetransfer of glucose units to protein-linked Man9GlcNAc2.

The same glucosylated compounds, Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, andGlc1Man7GlcNAc2, had been detected by other groups in mammalian cells. Curious as towhether or not glucose was transferred to Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 toform the respective monoglucosylated compounds in these organisms, Parodi carried outstudies with calf thyroid slices that confirmed that transient glucosylation of glycoproteinsoccurred not only in trypanosomatids but in mammalian cells as well. As reported in thesecond JBC Classic reprinted here, after pulsing the slices with [14C]glucose, label in theglucose of Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 appeared instanta-neously whereas, as in T. cruzi, label in the mannoses of the last two compounds appearedafter a considerable delay.

Similar results were found in experiments with rat liver slices and Phaseolus vulgaris cells,as reported in the third JBC Classic. This confirmed that protein-bound Glc1Man9GlcNAc2,Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 were formed by glucosylation of unglucosylatedoligosaccharides. Parodi’s experiments also indicated that UDP-Glc was the donor of theglucose residues and that the rough endoplasmic reticulum was the main subcellular site ofprotein glucosylation.

These three JBC Classics established the occurrence of transient glucosylation of glycopro-teins in the endoplasmic reticulum. Parodi later showed that the glucosyltransferase involvedin these reactions preferentially used acceptor glycoproteins not displaying their native three-dimensional structure, suggesting that the enzyme might be involved in the quality control ofglycoprotein folding. It was later discovered that the endoplasmic reticulum resident chaper-one calnexin interacted specifically with glycoproteins bearing monoglucosylated glycans, thatis, with the structures created by the endoplasmic glucosyltransferase, thus confirming therole of transient glucosylation in quality control of glycoprotein folding.

In recognition of his contributions to science, Parodi has received many awards and honorsincluding the 1994 Award in Biology from the Third World Academy of Sciences. He hasreceived fellowships from the Eleanor Roosevelt-International Union against Cancer and theGuggenheim Memorial Foundation. He was elected a member of the Third World Academy of

Classics

H54

Sciences in 1997, a foreign associate of the U. S. National Academy of Sciences in 2000, aforeign member of the Brazilian Academy of Sciences and a fellow of the American Academyof Microbiology in 2001, and a member of the National Academy of Sciences (Argentina) in2003.1,2


REFERENCES1. JBC Classics: Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333–350;

Cabib, E., Leloir, L. F., and Cardini, C. E. (1953) J. Biol. Chem. 203, 1055–1070; Cabib, E., and Leloir, L. F.(1954) J. Biol. Chem. 206, 779–790 (http://www.jbc.org/cgi/content/full/280/19/e16)

2. JBC Classics: Suginaka, H., Blumberg, P. M., and Strominger, J. L. (1972) J. Biol. Chem. 247, 5279–5288;Blumberg, P. M., and Strominger, J. L. (1972) J. Biol. Chem. 247, 8107–8113 (http://www.jbc.org/cgi/content/full/282/31/e25)

3. Parodi, A. J. (2007) How I became a biochemist. IUBMB Life 59, 361–363

1 Biographical information on Armando J. Parodi was taken from Ref. 3.2 We would like to thank Armando J. Parodi for providing background material for this Introduction.

Classics

H55

Reflections on Glycobiology*1

Published, JBC Papers in Press, September 11, 2001, DOI 10.1074/jbc.R100053200Saul RosemanFrom the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University,Baltimore, Maryland 21218

Glycobiology has become a “hot” subject,2 a timely one for “Reflections.” The primary reason,I think, is illustrated in Fig. 1, which shows the surface of an erythrocyte in cross-section. Justoutside the plasma membrane of this and nearly all cells is a coat of fuzzy material called theglycocalyx, consisting of a myriad of carbohydrate-rich molecules, polysaccharides, proteogly-cans, glycoproteins, and glycolipids. If the cell shown here was a fibroblast or an intestinalepithelial cell that secretes polysaccharides or mucins, it would be difficult to determine thelocation of the cell boundary; the polymers begin on the cytoplasmic face of the lipid bilayer,within it, or on its periphery, but it is not clear where they end. These extensive, complexstructures must serve essential roles in cell surface phenomena, but we are only beginning tounderstand what some of these functions are. I believe that the glycoconjugates or glycans canserve as important informational macromolecules.

In this remarkable age of genomics, proteomics, and functional proteomics, I am often askedby my colleagues why glycobiology has apparently lagged so far behind the other fields. Thesimple answer is that glycoconjugates are much more complex, variegated, and difficult tostudy than proteins or nucleic acids. To understand where we are and to appreciate what it hastaken to get here requires some background, so this article will briefly survey the history ofglycobiology from early studies on fermentation to the beginning of the contemporary era.

The PastAlthough glycobiology antedates biochemistry by many millenniums, their histories are

inextricably linked. The principal foundations of both fields lie in the development of organicchemistry during the 19th century and in studies on the process of fermentation of glucose andsucrose.

* This work was supported by Grant GM51215 from the National Institutes of Health.1 The original title of this article was: “Glycobiology: Past, Present, and a Very Bright Future.” It was intended to

show something of the development of major concepts and to recognize the excellent contributions of pioneers in thefield. I had no problem until the modern era, when I realized that it would take volumes to adequately describe thediversity of glycans and to cover, however briefly, current work and investigators. So, with regret, I am limited to thepast, where I tried to capture some of the flavor of the field, the origins of some contemporary ideas, and how they maytie to the future. Insofar as the chemistry is concerned, I have chosen to emphasize the cell surface and extracellularmatrix because these are where most of the glycoconjugates are found. The focus is on two examples, the cartilageaggrecan aggregate because it illustrates the enormous complexity that is possible with glycans and the erythrocytesurface and the ABO blood groups, which in some respects, at least, may be a model for other cell surfaces.

2 A recent issue of Science features glycobiology (1), and the April, 2001 meeting of the Carbohydrate Division of theAmerican Chemical Society emphasizes glycobiology as a major subject; their prestigious C. S. Hudson Award waspresented to a well known glycobiologist, Y. C. Lee. How times have changed! In the 1950s, glycobiology was not apopular subject. There were a few interested biochemists at the meetings, and we had an annual lunch (Karl Meyer,Al Dorfman, Dick Winzler, Roger Jeanloz, Ward Pigman and a few others). After lunch, one might as well go home.My papers (glucosamine metabolism) were invariably scheduled as either last or next to last on Friday afternoon atthe Federation Meetings in Atlantic City. The most hilarious incident was when my paper (next to last) wasannounced at one of these sessions. When I reached the platform, the chairman of the session apologized because hehad to leave to make a train. My audience consisted of the next speaker and the slide projectionist. I stayed for thelast paper, but unfortunately I never asked the projectionist how he liked the presentations. I had the same experience

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 45, Issue of November 9, pp. 41527–41542, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Fermentation—Fermentation was known to the cave man and has been the subject ofintense study ever since. The Old Testament has many references to wine and libations, thefirst being to Noah (Genesis, 9:20–21): “Noah, the husbandman, began and planted a vineyard.And he drank of the wine and was drunk.”3 Treatises and philosophical discourses werepublished on the process during and after the Middle Ages.4 Fermentation was not confined tomaking alcohol but has been used for thousands of years to make cheese, soy sauce, etc.

The first chapter of the biochemical classic Alcoholic Fermentation by Arthur Harden (1stedition, 1911) reviews the history. (a) The most important early study was that of Lavoisier(1789) who quantitatively established the stoichiometry of the process and concluded that thesugar was split into two parts, one of which was oxidized (carbonic acid) at the expense of theother (alcohol). Furthermore, “if it were possible to recombine these two substances, sugarwould result.” The methodology was insufficient to permit him to see an increase in the weightof the yeast or of other products that were formed. (b) Yeast at the time was regarded as acatalyst, much like alumina or diatomaceous earth, and during the first 60 years of the 19thcentury, this was the prevailing view of the leading chemists and journal editors of the time(Liebig, Berzelius, Wohler). This was despite the fact that in 1837 three independent inves-tigators, Cagniard-Latour, Schwann, and Kutzing, presented evidence that yeast was a livingorganism, an idea that was ridiculed by the establishment. (c) In 1860, the pivotal experimentsof Louis Pasteur finally laid this ghost to rest (5), and he showed unequivocally that yeast wasa living organism. He also did careful stoichiometry. The balance sheet showed that about 95%of the C,H,O of the sugar was converted to CO2 and ethanol. The remainder, from 1.6 to 5%,

at the American Chemical Society meetings. Starch chemistry was a big thing for members of the CarbohydrateDivision, and they walked out on papers devoted to the glycoconjugates or hexosamines. At one such meeting, myaudience consisted of other members of the laboratory waiting to drive back to Ann Arbor with me. It was, however,a great time to do this kind of research. There was virtually no pressure. The handful of us in this country who workedin the field were supported by the National Institutes of Health. I can capture a little of the intellectual flavor of thetimes by my experience when I submitted my first independent application. It stated that I would work on theenzymatic synthesis of one of the monosaccharides in the glycoconjugates, but I did not know which to choose. I thenlisted about four monosaccharides (glucosamine, fucose, glucuronic acid, and galactosamine) and possible preliminaryexperiments for each; I would work on whichever problem appeared most fruitful. I was funded, and a short time laterI met one of the members of the Study Section (Ef Racker) who told me that it was the best application he had read.What would happen today with an application that was so “unfocused” and with such nonspecific aims? Equallyimportant to the National Institutes of Health support, we received unsparing help from a number of farsightedphysicians such as Walter Bauer at Massachusetts General Hospital, who not only created a high caliber research unit(Roger Jeanloz, Jerome Gross, Karl Schmid, Morris Soodak, and others) but was also instrumental in the Helen HayWhitney Foundation. In my case, it was William Robinson and Ivan Duff at the Rackham Arthritis Unit at theUniversity of Michigan. Only once (when I was first interviewed) did I have to explain to Bill Robinson how work onEscherichia coli might relate to arthritis. Thus, we had the luxury of following our noses and serendipity wherever thework took us. We started with studies on the intermediary metabolism of glucosamine, which led in turn to thestructure of the sialic acids and the discovery of N-acetylmannosamine, to the intermediary metabolism of thesecompounds, to CMP-sialic acid and its enzymatic synthesis, to the glycosyltransferases, and finally to the phospho-transferase system for sugar uptake by bacteria (reviewed in Refs. 2 and 3). In recent years, the complex process ofchitin catabolism by marine bacteria has become a major project (4).

3 This reference was kindly called to my attention by Dr. Michael Edidin.4 One that struck a chord was a 74-page treatise by John Richardson (1790) entitled “Theoretic Hints on an

Improved Practice of Brewing Malt-liquors . . . ”. He defines fermentation as: “A spontaneous internal motion ofconstituent parts, which occasions a spontaneous separation and removal from their former order of combination, anda remarkable alteration in the subject, by a new arrangement and re-union.” Not a bad definition of intermediarymetabolism and the thermodynamics of glycolysis.

FIG. 1. The erythrocyte glycocalyx. This is revealed in electron microscopy by special staining methods. It is upto 1400 Å thick, and the oligosaccharide filaments are 12–25 Å in diameter. (Taken from Voet and Voet, Biochemistry,with permission of the publisher. Original was by courtesy of Harrison Latta, UCLA).

Reflections: Reflections on Glycobiology

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consisted of substances that the “yeast had taken from the sugar.” The result of this andsubsequent work by Pasteur led to his famous dictum, “no fermentation without life.” In anextension of this work, he came to the conclusion (1875) that fermentation was the result of lifewithout oxygen, the cells being able under anaerobic conditions to avail themselves of theenergy liberated by the decomposition of substances containing combined oxygen (i.e. anaer-obic glycolysis). (d) Enzymes (called ferments), generally hydrolases, were known during the19th century; indeed, invertase (i.e. sucrase) had been extracted from yeast. In 1858, Traubeproposed that fermentation resulted from the action of ferments secreted by cells on sugar.Many attempts were made to extract yeast cells and obtain cell-free fermentation of sugar butwithout success. Finally, while attempting to preserve yeast extracts for therapeutic purposes,Eduard Buchner succeeded in 1897. The preservative was sugar, and he noted that carbondioxide was formed. This fortunate and serendipitous result marks the beginning of biochem-istry as we know it today. It is interesting to note that the Journal of Biological Chemistry wasfounded only a few years later by Christian Herter.

The story would not be complete without summarizing what was learned between Buchner’slandmark result in 1897 and the publication of Harden’s monograph in 1911. Kinetic exper-iments were conducted using yeast extracts and glucose, and the rate of fermentation wasfollowed by measuring the rate of CO2 evolution. The following results, especially by Hardenand Young, were obtained. (i) Fructose and mannose were fermented as well as glucose, butthe yeast had to be “trained” (i.e. adapted) for the extract to ferment galactose. They speculatedthat different ferments were required for galactose utilization. (ii) Inorganic phosphate wasrequired. (iii) A hexose diphosphate was isolated, characterized as fructose-di-P, and wasshown to be an intermediate in the process. (iv) The extract was pressure filtered through agelatin film, giving a dialysate and a “residue.” Neither alone supported fermentation, but itwas restored by mixing the two. The residue contained the heat-labile zymase, and thedialysate contained the heat-stable coenzyme(s) or cozymase. Soon after, it was shown thatyeast anaerobic glycolysis was closely connected to anaerobic glycolysis by muscle and muscleextracts. The cozymase, of course, was the source of ATP, NAD�, etc.

Development of Organic and Carbohydrate Chemistry—The close connection between thedevelopment of organic chemistry and biochemistry in the 19th century is summarized in anexemplary, early textbook (6). However, carbohydrate history goes back many centuriesearlier. Cellulose in the form of cotton, for instance, was known from ancient times, andsucrose was one of the first organic substances to be crystallized (300 A.D., from the juice ofsugar cane in India). Because the climate in Europe was not favorable for growing sugar cane,alternative sweetening agents were sought early in the 19th century, leading to the discoveryof new sugars (glucose, fructose, mannose, galactose, etc.), all with the same elementarycomposition (CH2O)n. Clarifying the structural relationships between these compounds occu-pied carbohydrate chemists for most of the century. Finally, the structure of D-glucose wasestablished by Emil Fischer in 1891, which marks the beginning of modern carbohydratechemistry. Fischer’s multitudinous and brilliant contributions were likewise in the fields ofamino acid and purine/pyrimidine chemistry. It is worth reminding the reader thatchromatography and electrophoresis were unknown at the time, and substances were purifiedby fractional crystallization and characterized by elemental analyses and theirphysical properties (melting point, optical rotation, solubility, etc.).5 In this age of

5 My interest in carbohydrate chemistry began as a graduate student working in the laboratory of Karl Paul Linkat the University of Wisconsin. He was both a carbohydrate and natural products chemist, with very high standardsand an ability to inspire the best in us. The laboratory had isolated and characterized dicumarol as the hemorrhagicfactor in spoiled sweet clover hay prior to my arrival (warfarin is a synthetic analogue). My project was to study themetabolism of its parent compound, 4-OH-coumarin, which was not toxic. Four large dogs used as subjects were fedthe drug and maintained in very large metabolic cages so that their urine could be collected. (In those days, graduatestudents took complete and very good care of their animals, including feeding, exercising them, and cleaning theircages.) The metabolic product turned out to be 4-OH-coumarin �-D-glucuronide. However, this had to be establishedby synthesis and also by elemental analysis. I spent a very muggy, frustrating summer in Madison recording theswings on a microbalance and learning how to do microanalyses before the standards finally came out right.Somewhat later, I developed considerable experience with fractional crystallization, particularly of anomeric glyco-sides. They were being synthesized for Joshua Lederberg, a young faculty member in a neighboring department(genetics), who was using them for assaying the expression of glycosidases, such as �-galactosidase in E. coli.Fractional crystallization, like elemental analysis, is tedious work, but above all it is a real art and when it works, itis most gratifying. In doing this kind of work, we even invoked the help of the Lord. To this day my children rememberthat my wife Martha (who is not a scientist) concluded the evening prayers over the Sabbath candles with thefollowing phrase: “and may Daddy have crystals.” It worked!


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electronics and the internet, one always thinks that science moves forward too slowly, but itis mind boggling to realize how far we have come since the 1890s (Fischer, Buchner).

Glycobiology in the 20th Century: ChemistryStructural Studies on Glycosaminoglycans (Mucopolysaccharides)—Although mucins from

various sources were studied by organic chemists as early as 1846 (see reviews by Blix,Gottschalk, and Morgan (7)) and were thought to contain sugars, there was always anunresolved question of purity. In 1925, the distinguished chemist, P. A. Levene, who had madefundamental contributions to the structures of the nucleic acids, published a monographentitled “Hexosamines and Mucoproteins.” Chondroitin sulfate had been isolated in 1884 fromcartilage, but the nature of its monosaccharides and structure were controversial until Leveneshowed conclusively that the constituents were D-glucuronic acid, chondrosamine (D-galactos-amine), acetic acid, and sulfuric acid in equimolar ratios. He depicted the structure as GalNAclinked to GlcUA and sulfated at C-6 on the GalNAc. As might be expected from the availablemethodology and misinformation on sugar ring structures, there were major errors in thestructural assignment, including the fact that it was a tetrasaccharide. Similarly, mucoitinsulfate (i.e. hyaluronic acid) was depicted as a tetrasaccharide containing GlcNAc but alsosulfate. He also questioned whether substances such as ovalbumin were “glucosidoproteins” orwhether such substances even existed.

In 1934 (8), hyaluronic acid was isolated in pure form from vitreous humor, and its correctcomposition was determined. This groundbreaking paper was the first of many from KarlMeyer’s laboratory, creating a science from chaos. His laboratory subsequently isolated andcharacterized the chondroitin sulfates, keratan sulfate, and various hyaluronidases.6

Establishing the structures of heteropolysaccharides can be exceptionally difficult, and theproblems can be summarized as follows: (i) identification and quantitation of the monosac-charides; (ii) D- or L-configurations; (iii) branched or unbranched; (iv) sequence; (v) � or �anomers; (vi) pyranose or furanose rings; (vii) positions of the linkages; (viii) many of thesepolymers are derivatized (e.g. phosphate, sulfate, acetate, etc.), and polymers with differentbiological and chemical properties are formed, depending on the position of the linkage in thederivative; and (ix) to complicate matters even further, some of the polymers and oligosaccha-rides are covalently linked to proteins or lipids.

One of the major problems confronting workers in this field was protein and how to get ridof it because it was regarded as a contaminant of the “mucopolysaccharides,” now calledglycosaminoglycans or GAGs.7 Protein was not easily removed.8 Meyer, for instance, thoughtthat the protein formed ionic bonds with the polysaccharides. In the 1950s, Maxwell Schubert’slaboratory showed that cartilage chondroitin sulfate was linked to protein, thus opening a newchapter in the chemistry of these polymers, now called proteoglycans. The next essential stepwas to characterize the linkage region between the GAG and the protein. Work on differentpolymers around the same time (late 1950s) by Pigman (mucins), Kabat (blood groupsubstances), and Muir (chondroitin sulfate) suggested that the sugars were linked to

6 Karl Meyer was a delightful person with a keen sense of humor. His exchanges with Albert Dorfman at themeetings were the highlight for many of us. For instance, at one meeting Al gave a talk, and in the questioning periodKarl asked Al, “How did you quantitate the keratosulfate?” Al responded that he had not. In a stage whisper, Karlsaid: “I thought as much.” Al, with whom I did my postdoctoral work, was a principal figure in the field. He held bothM.D. and Ph.D. degrees but what made him really unusual was his expertise in both fundamental biochemicalresearch and in clinical practice (pediatrics). He was a leader in the University of Chicago Medical School and laterbecame Chair of Pediatrics. Al came around to see me every day, and we would get into the most vigorous discussionson how to interpret results, the next experiments, etc. He had to be the most tolerant person, considering that I wasfresh out of graduate school and was convinced that I knew everything there was to know (it has been downhill eversince). My paying job was to direct the pediatric blood chemistry laboratory, which was actually very interestingbecause one had to develop ultramicroanalytical methods, especially for samples from the newborn, which were oftenobtained by heel puncture. Most of my research was conducted late in the afternoon and evening. Al lived across theMidway and could see the laboratory window (top floor of Bobs Roberts Hospital) from his bedroom. I always left thelights on when I went home.

7 The abbreviations used are: GAG, glycosaminoglycan; PAPS, 3�-phosphoadenosine-5�-phosphosulfate; MGT, mul-tiglycosyltransferase; GT, glycosyltransferase; ST, sialyltransferase.

8 At the University of Chicago we were fortunate to have the large meat packing houses close by, which were sourcesof necessary tissues, such as bovine eyes (for vitreous humor), testis (for hyaluronidase), etc. The isolation ofchondroitin sulfate started with bovine nasal septa, which were obtained by working on the line and cutting them outof the skulls as they came by on a belt (very hard on the hands). The cartilage was ground and extracted with about0.1 N NaOH for several days in the cold with constant stirring. The alkaline extract was then deproteinized and thepolysaccharide isolated. By hindsight we know now that the alkaline extraction procedure split the polysaccharidefrom its O-serine (or threonine) linkage in the protein by �-elimination.


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serine.9 In 1964, Lindahl and Roden found that the “linkage fragment” in heparin wasO-�-D-xylopyranosyl-L-serine (reviewed in Ref. 10). They later showed that the sequence at thelinkage region in these polymers (chondroitin sulfates, dermatan sulfate, and heparan sulfate)to which the polysaccharide is attached is GlcUA-Gal-Gal-Xyl-Ser. In skeletal keratan sulfate,the O-linkage is to �-GalNAc in place of the Xyl.

At the same time, a different class of complex carbohydrates, now call glycoproteins, was thesubject of intensive study. Neuberger’s laboratory in England showed by isolation and syn-thesis that the linkage region in ovalbumin is �-GlcNAc3Asn, i.e. to the amide N of aspara-gine. There are, of course, a wide variety of N-linked glycoproteins, particularly the glycopro-teins in serum. Since the overriding question in these early studies was purity, the isolationand characterization of the major serum glycoprotein, �1-acid glycoprotein (orosomucoid), byKarl Schmid was a key breakthrough. The protein (44 kDa) contained 17% hexose and 12%hexosamine.

A characteristic of carbohydrate polymers is that they are polydisperse or microheteroge-neous. The template mechanisms of protein and nucleic acid synthesis do not apply to thecarbohydrate polymers, thereby resulting in polydispersity. Human orosomucoid, for instance,contains 6 oligosaccharide chains per molecule, but the chains are different from each other.In the collection of molecules called orosomucoid, at least 8 oligosaccharides have beenidentified (11). Each oligosaccharide can contain up to 5 different kinds of sugars, a givensugar can occur several times in the chain, and the number of possible combinations isoverwhelming (see below).

Aggrecan Aggregate—The major components of cartilage are collagen and a huge macromo-lecular complex called the aggrecan aggregate. An electron micrograph of one such aggregateis shown in Fig. 2A, and Fig. 2B presents a schematic view of 6 aggrecan monomers bound tohyaluronan. Determining the details of these structures is an extraordinary achievement inthis field, equivalent (at least) to delineating the structure of collagen. The structure wasdeveloped through work in the laboratories of Hascall, Muir, and Heinegard and has recentlybeen reviewed (12, 13). This unusually complex “molecule” can have an apparent mass of �6 �109 Da and is a composite of all of the structural units described above. The relationshipbetween the structure of the aggregate and its function is briefly discussed below.

The Erythrocyte Surface, Human Blood Group Activity, and Erythroglycan (Poly-N-acetyllactosamine)—The frequent incompatibility of the blood of a donor and recipient wasrecognized in the 17th century. Starting with the work of Landsteiner (1900), who defined theABO group, we now know that there are at least 27 such families of human blood groupsubstances expressed on the surfaces of erythroid cells and often other cells as well. Thegeneral characteristic of these antigens is that they comprise integral membrane glycopro-teins, both O- and N-linked, and in some cases, glycolipid. Thus far, it has been shown that theglycan units are the epitopes in four of the systems, ABO, Lewis, P, and H/h.10 Some aspectsof the ABO system will be discussed here.

Work on the ABO family was greatly aided by finding these activities in water-soluble formin various secretions and mucins, such as ovarian cysts. The major antigenic determinantswere established by Morgan and his co-workers (particularly Watkins and Aminoff) and byKabat and his co-workers (reviewed in Ref. 14). These determinants were sugars at thenon-reducing termini of oligosaccharide chains linked via Ser and Thr to polypeptides, similarto the mucins. Blood group O chains were terminated by a trisaccharide Gal(�,1–4)[Fuc-(�,1–2)]GlcNAc–X. Blood group A activity was expressed by linking an �-GalNAc to C-3 of the Gal,whereas in B activity a Gal is substituted for the GalNAc.

The erythrocyte membrane was quite another problem. Although Yamakawa showed thatred blood cell glycolipids exhibited such activity (1953), this conclusion was disputed as late as1956 (14). It is now clear that the antigens are carried on the erythroid surface by both lipidsand polypeptides (see review by Hakomori (15)).

These structures are closely related to the glycosaminoglycan keratan (desulfated keratansulfate). The repeating unit in this GAG is N-acetyllactosamine: Gal-(�,1–4)-GlcNAc-(�,1–3)

9 In the alkaline �-elimination step, the oligo- or polysaccharides glycosidically linked to serine or threonine are firstreleased from the protein and then degraded by the alkali at the reducing end of the chain, a reaction called “peeling.”An important advance in the field was Carlson’s alkaline borohydride procedure, which reduced the aldehyde groupas the glycosidic bond was cleaved and protected the oligomer from alkaline degradation (9).

10 I am very grateful to Dr. Olga Blumenfeld (Department of Biochemistry, Albert Einstein Medical School) forhelpful discussions on the blood group substances.


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linked to the next Gal in the chain. The same structural unit but in shorter chains than thepolysaccharide, called poly-N-acetyllactosamine or polylactosamine, is found both O- andN-linked to integral membrane proteins on many cell surfaces and is also found linked toceramide. Polylactosamine can be straight chain or branched and can be “decorated” with Fucor sialic acid residues. Apparently the first references to Gal-, GlcNAc-, and Fuc-rich glyco-peptides in cell membranes came from work by the eminent geneticist/molecular biologistFrancois Jacob and his group on the cell surface antigens found in early embryonic differen-tiation (reviewed in Ref. 16). At about the same time (17, 18), Laine and co-workers isolated“erythroglycan” by extensive Pronase digestion of lipid-free red blood cell stroma and charac-terized the large branched oligosaccharides (7,000–10,000 Da) by methylation, etc. “Band 3,”the major red blood cell integral membrane protein and the anion transporter, is the source ofthe polylactosamine, and it accounts for more than 30% of the total Gal and GlcNAc in the redblood cell membrane. Further, at its non-reducing termini the polymer can carry Fuc and �Galor �GalNAc, thereby becoming an antigenic determinant for A, B, or O activity. The largequantity of polylactosamine peptide derived from the red blood cell membrane corresponds tomost of the antigenic sites in the intact erythrocyte (about 2 � 106). There is now an extensiveliterature on polylactosamine, its enzymatic synthesis (19), and how branching occurs duringdevelopment, tumorigenesis, etc.

Blood group activity is also carried by glycolipids, which are present in small quantities inthe red blood cell membrane (reviewed by Hakomori (15). They consist of a large number ofcompounds derived from N-acetyllactosamine oligomers. This family comprises oligosaccha-

FIG. 2. Electron micrograph of a proteoglycan aggregate purified from calf epiphyseal cartilage. A, theaggregate was spread as a monolayer in a cytochrome c film. Under these conditions the chondroitin sulfate chains ofthe proteoglycan collapse onto the core protein so that each monomer (aggrecan) of the aggregate is distinct. Thenearly uniform length of the monomers is characteristic of proteoglycans from young cartilages, with each �3.5 millionDa in molecular mass. This aggregate contains �180 aggrecan monomers, and the overall molecular mass of theaggregate is �6.5 billion Da. The central strand of hyaluronan is �5500 nm in length. The boxed area encloses 6monomers, the number depicted in the model in B. (Micrograph kindly provided by Joseph Buckwalter, University ofIowa.) B, model of a portion of the proteoglycan aggregate showing 6 aggrecan monomers (see box in A). Each of thesix monomers is depicted with a central core protein strand to which the glycosaminoglycans are covalently linked,giving the appearance of bristles. The core protein (�250,000 Da) contains a midregion with �100 chondroitin sulfatechains (blue) of �30,000 average molecular weight and a nearly equal number of keratan sulfate chains (red) of�10,000 average molecular weight. The monomers are anchored (non-covalently) to the central hyaluronan strand(orange) by: (a) a hyaluronan-binding site in the N-terminal globular-1 (Gl) domain (pink sphere) of the core protein,and (b) a link protein (crystal sphere) that binds to both hyaluronan and to the G1 domain of aggrecan. The core proteinof aggrecan contains two other globular domains, G2 and the C-terminal G3, shown as green and lavender spheres,respectively. The functions of G2 and G3 are not known. The globular domains of aggrecan also contain N-linkedoligosaccharides (red Y symbols). The model is fully extended as it would appear in dilute solution. In the tissue thestructure would be compressed to approximately one-tenth its extended size. See Ref. 13 for additional details. (Modeland supplemental three-dimensional, rotational view kindly provided by Mark Sabo (Art Department) and VincentHascall, Cleveland Clinic Foundation.)


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rides, both straight and branched chain, linked to glucosylceramide and terminated by one ofthe antigenic determinant sugars. The glycolipids change, especially with respect to branch-ing, during the development of erythroid cells.

Glycobiology in the 20th Century: BiosynthesisIsotope Experiments—The complex carbohydrates contain up to 8 different monosaccha-

rides, including D-xylose, hexoses, hexosamines, and hexuronic acids, in addition to varioussialic acids, such N-acetylneuraminic acid (NAN or NeuAc). Until 1950, we did not know howmost of these monosaccharides were biosynthesized. For the 6-carbon sugars, the theoriesranged from (a) direct conversion of the 6-carbon skeleton of D-glucose to the sugar to (b)fragmentation of glucose through glycolysis and other catabolic cycles and recombination ofsuitable fragments. It was suggested, for example, that the GlcNH2-6-P carbon skeleton wasformed by condensing glyceraldehyde-3-P (G3P) and serine, with subsequent reduction of thecarboxyl to the aldehyde. And how could L-fucose possibly arise directly from D-glucose withoutinversion of the carbon skeleton by 180o, which would give the L- from a D-sugar?

These problems were addressed by treating an appropriate biological system with specifi-cally labeled glucose, such as 1-[14C]- and 6-[14C]glucose in companion experiments. The purepolymer was isolated and hydrolyzed, and the monosaccharides were isolated and dissectedcarbon by carbon to determine the specific activity at each C-atom in the skeleton.11 If theorigin of the 6-carbon hexoses was via fragmentation of the Glc 6-carbon chain, followed byrecombination, isotope scrambling would result.12 The results were conclusive, showing thatthe 6-carbon skeleton of Glc was converted intact to GlcNH2, glucuronic acid, galactose, andmannose. Surprisingly, D-Glc was converted to L-Fuc without inversion (20).

Enzyme Experiments—The next step, of course, was to determine the pathways of synthesisand degradation using appropriate “ferments.” A review (21) published in 1959 shows howrapidly the field grew in 10 years. Many of the anabolic/catabolic pathways were established,and although they have since been added to and somewhat modified, the essential elementsremain the same today.

Sialic acid was a separate problem in that enzymatic studies could not proceed until after itscorrect structure was established (22), and N-acetyl-D-mannosamine was found to occurnaturally and to be a precursor of NeuAc. Glucosamine-6-P is the precursor of all nitrogen-containing sugars and is formed from Fru-6-P and glutamine (23), although the catabolicenzyme, GlcN-6-P deaminase (24), which gives Fru-6-P and NH3 as products, is reversible andcan be utilized anabolically when the synthase is mutated in bacteria.

Sugar Nucleotides, Dolichol, and PAPS—Aside from establishing the intermediary metab-olism of the monosaccharides, there were five major developments in the field over the courseof the next 20 years (listed in order of the discussion): (a) isolation of “active sulfate” or PAPS(1958); (b) recognition of lectins (sugar-binding proteins) in animal tissues; (c) identification ofdolichol-linked oligosaccharides as intermediates in the synthesis of the Man-rich core oligo-saccharides of N-linked glycoproteins (1976); (d) isolation of the sugar nucleotides (1950); (e)elucidation of the pathways of synthesis and degradation of the complex carbohydrates and ofthe number and specificities of the glycosyltransferases.

The precursor of the sulfated glycoconjugates, such as chondroitin sulfate, is 3�-phosphoad-enosine-5�-phosphosulfate (or PAPS) characterized and enzymatically synthesized by Robbinsand Lipmann (25). PAPS is, of course, “high energy” or “active” sulfate.

Ricin was apparently the first lectin (proteins that bind carbohydrates) recognized morethan a century ago. Early in their history, lectins were found to agglutinate erythrocytesdepending on blood type. Lectins by now have become a field unto themselves, and the workof I. J. Goldstein, who developed quantitative methods for accurately defining specificity, as

11 My major postdoctoral project was to determine the modes of synthesis of the glucosamine and glucuronic acidmoieties in hyaluronic acid. The biological system was a strain of Group A streptococcus that secreted the polysac-charide, and the organism was grown (in a rich medium) on 1-[14C]- and 6-[14C]glucose. One of the many problems wasthe cost of the labeled sugars (far too expensive for these experiments). [14C]NaCN was more reasonable, and thelabeled sugars were synthesized from this starting material. In the experiments, because of the rich medium, thelabeled glucose, acetate, and lactate were isolated from the medium, as well as the polysaccharide, and were dissectedas well. Konrad Bloch, who was a Professor in the Department of Biochemistry, was of enormous help to me duringthis phase of the work.

12 For instance, at the triose-P level, because of triose-P isomerase 1-[14C]glucose would become 1,6-[14C]hexose, andthe specific activity at C-1 would be half that of the 1-[14C]glucose used for the experiment.


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well as in isolating new lectins, is especially significant. The plant lectins are not only powerfultools for analyzing macromolecules and cell surfaces, but the field became particularly inter-esting to cell biologists when it was realized that animal cells express lectins.

In 1968, Ashwell and co-workers (26) discovered that liver hepatocytes bind and take upasialoglycoproteins (the asialoglycoprotein endocytosis receptor). This receptor is a Gal-spe-cific lectin in mammals and GlcNAc-specific in birds. It is called the “Ashwell protein” in whatfollows. Animal lectins, such as the Siglecs (bind to sialic acids), Ig superfamily lectins,selectins, the integrins, CAMs (cell adhesion molecules), collectins, CD44, and others, havenow become major areas of research.13

Lipid-linked intermediates were discovered around 1964–1965 by three groups (reviewed byOsborn (27)). These studies were conducted in the laboratories of Horecker, Robbins, andStrominger, who were working on the enzymatic synthesis of bacterial lipopolysaccharides andthe peptidoglycan cell wall. This work led to similar studies in a number of laboratories onlipid-linked intermediates in the biosynthesis of complex carbohydrates in eukaryotic organ-isms, including yeast, plants, and higher animals (for review, see Ref. 28). A polyisoprenoid,dolichol, was known to occur in animal tissues and was identified as the lipid carrier ofthe carbohydrate groups. This early work led to the well established dolichol pathway for thesynthesis of the N-linked glycoproteins (29). The dolichol pathway does not apply to theO-linked glycoproteins or to the glycolipids.

The isolation and characterization of sugar nucleotides is one of the most important achieve-ments in the field of carbohydrate metabolism in the 20th century. They were discovered intwo laboratories at about the same time, those of Luis Leloir in Argentina and of James Parkin this country. Leloir’s group (Caputto, Cardini, Paladini, and Cabib) was working on theenzymatic synthesis of Glc-6-P from Gal-1-P using yeast extracts and found that a heat-stablecofactor (“cozymase”) was required. One of the factors was isolated and fully characterized asUDP-Glc (30). This was followed by isolation of UDP-Gal and recognition that the “galacto-waldenase” reaction (epimerization at C-4) occurred at the level of the sugar nucleotides. In anindependent discovery, Park found that Staphylococcus aureus treated with penicillin (whichinhibits cell wall synthesis) accumulated considerable quantities of UDP derivatives andshowed that they contained the cell wall sugar muramic acid and amino acids (31). The Parkcompounds are intermediates in cell wall biosynthesis.

The list of sugar nucleotides is huge (32). It includes virtually every naturally occurringmonosaccharide, purines, pyrimidines, and in some cases, 2-deoxyribose in place of ribose.They are most frequently formed by the action of pyrophosphorylases, which catalyze thereaction (N indicates nucleoside): NTP � glycose-1-P 3 PPi � N-P-P-glycose. As usual, thesialic acids are exceptions in that the nucleotide is CMP (not the diphosphate). CMP-sialicacid, originally isolated from E. coli (33), is formed by condensation of NeuAc or N-glycolyl-neuraminic acid and CTP (34, 35). A similar nucleotide (36) was obtained with 2-keto-3-deoxyoctanoic acid (KDO), a component of bacterial lipopolysaccharides.

Functions of the Sugar Nucleotides—Some sugars in glycoconjugates (Man, GlcNAc, NeuAc)are synthesized from Fru-6-P, whereas others (Gal, GlcUA, Xyl) are synthesized as nucleotidesugars from UDP-Glc or, in the case of L-Fuc, from GDP-Man. Aside from their participationin the biosynthesis of monosaccharides, the sugar nucleotides serve as glycose donors in thebiosynthesis of oligo- and polysaccharides. Enzymes that utilize sugar nucleotides as donorsare designated glycosyltransferases and are the major catalysts for generating the glycosidicbond (also formed by transglycosidases and phosphorylases).

Glycosyltransferases were first reported by the Leloir group (37, 38). The enzymes catalyzedthe synthesis of disaccharides (trehalose, sucrose) and of the �,134 linkage in glycogen. At thetime, it was generally believed that the �,134 linkage in glycogen was synthesized fromGlc-1-P by reversing the glycogen phosphorylase-catalyzed reaction.

The following general glycosyltransferase catalyzed reaction occurs in animal tissues.

13 In the 2-year period 1999–2000, SciFinder lists 1400 papers on selectins and 5900 on the integrins. Early in myservice on National Institutes of Health Study Sections, our section, which comprised a distinguished group ofbiochemists, reviewed what I think may have been the first National Institutes of Health application for funds to studya plant lectin (concanavalin A). A vigorous debate ensued with those opposed asking why a plant protein that bindscarbohydrates should be of any interest to the National Institutes of Health. It should be funded by the NationalScience Foundation! Fortunately, the application was funded. In this connection it was this same group that reviewedapplications by Fritz Lipmann and by Luis Leloir, which were of course funded; these applications basically consistedof describing what the applicants planned to do with very little detail or particular focus. How would they fare today?


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Glycose—PP(U or G) � HO-acceptor ¡ (U or G)DP � Glycoside—O-acceptor

REACTION 1

In the case of the sialic acids, such as NeuAc, the donor is CMP-NeuAc.Specificity of the Glycosyltransferases, Biosynthetic Pathways, and Multiglycosyltransferase

Systems—At the time we began to study macromolecular glycans (around 1960), nothing wasknown about the biosynthetic pathways leading to glycoproteins, mucins, and glycolipids. Toundertake this work, we required substrate quantities of the sugar nucleotides. FortunatelyMoffatt and Khorana (39) had just published a method for the synthesis of UDP-Glc. A veryfruitful and enjoyable summer in Vancouver led to a modified, general method for thesynthesis of sugar nucleotides (40),14 giving us the tools for studying glycosyltransferases.

Some 15–20 glycosyltransferases were characterized, and it soon became obvious that theyformed families such as sialyl-, Gal-, GlcNAc-, GalNAc-, Glc-, and Fuc-transferases. Theenzymes we studied are involved in the synthesis of the mucins, glycolipids, and terminaltrisaccharides of N-linked glycoproteins, and the results can be summarized as follows (seeRef. 2 for review). 1) Each glycosyltransferase is specific for both the donor sugar nucleotideand the acceptor. 2) A different transferase usually catalyzes each step in a pathway. When asugar occurs twice in a molecule, such as NeuAc in disialoganglioside, two different trans-ferases are required.15 3) Chain elongation can be at the non-reducing end or can form branchpoints. 4) The product of one reaction is the substrate for the next, which leads to the conceptof multiglycosyltransferase (MGT) systems, namely that the transferases required for synthe-sis of one glycoconjugate are associated. A different MGT system is required for mucins,glycoprotein trisaccharide termini, and glycolipids (e.g. gangliosides). 5) Polydispersity inglycoconjugates is explained by the MGT hypothesis. For instance, Svennerholm (41) showedthat there is a particular array of human brain gangliosides of different chain length andcomplexity. This array exactly fits the pathway that is predicted by the specificities of theenzymes in the corresponding MGT. One would expect to find only the final products of thepathway (e.g. tetrasialoganglioside) if all conditions were optimum for each enzyme and theyare expressed at high enough levels. It should be noted that many of the transferases requireMn2� for activity and not necessarily at the same concentrations,16 and this may be animportant means of regulating these activities. 6) There is some evidence to support the ideathat glycosyltransferases in an MGT complex bind to one another. In the original work, wefound that all of the transferases in a given MGT were found in the same particulate fraction,ultimately identified as the Golgi apparatus (42). The Gal-transferase involved in synthesizingthe Gal-O-Xyl-O-Ser (protein) linkage region in chondroitin sulfate was purified by binding tothe immobilized xylosyltransferase, and it coprecipitates with antibody directed at the xylo-syltransferase (43). Recent papers present evidence for binding of a glycolipid GalNAc-trans-ferase to a Gal-transferase (44, 45).

The Structure of the Acceptor Can Determine Glycosyltransferase Activity—The activity of aGT is not only determined by whether the acceptor is a glycolipid, mucin, or an N-linkedglycoprotein but can also depend on the fine structure of the termini in these potentialacceptors. One example will be given. Enzymatically synthesized, labeled CMP-NeuAc andCMP-N-glycolylneuraminic acid (34) were used to detect and characterize sialyltransferases(STs), first from rat mammary gland and then in colostrum (goat, bovine, human), bacterialextracts (for synthesizing colominic or polysialic acid), submaxillary gland, and embryonicchicken brain (summarized in Ref. 2). Bovine colostrum and human milk contain mixtures of

14 I arrived with the sugar 1-phosphates and was given space on John Moffatt’s bench. He measured my waist andmarked off the corresponding width on the bench top. Fortunately, my waist was substantially greater than his.

15 The glycosyltransferases that synthesize the GAGs have exceptional characteristics. (a) Elongation of thepolysaccharide chains in chondroitin sulfate, heparan sulfate, and in one hyaluronan (produced by Pasteurella) takesplace in a stepwise manner at the non-reducing ends of the polymers. In these cases, a single glycosyltransferasetransfers first one and then the other glycose unit from their respective sugar nucleotides to the ends of the chain. (b)Single glycosyltransferases also catalyze hyaluronan synthesis by eukaryotic and Streptococcal cells, but in thesecases elongation occurs at the reducing ends of the chain by mechanisms that are not entirely clear. One phenomenonthat has always intrigued this reviewer is how the enzymes or cells “know” when to stop the process of polysaccharideelongation (see Ref. 12 for review).

16 One mechanism for regulating glycosyltransferase activity could well be via local Mn2� concentrations. A briefliterature search found references to analyses of tissues, mostly autopsy material, for trace metals including Mn2� butlittle on its transport. The relevant analyses will require that they be conducted on actively metabolizing cells andorganelles (such as the Golgi) to preserve the in vivo ion gradients.


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3�-sialyllactose (NeuAc-�2,3-Gal�1,4Glc) and 6�-sialyllactose (NeuAc-�2,6-Gal�1,4Glc). Therat mammary gland ST synthesizes 3�-sialyllactose when incubated with CMP-NeuAc andlactose, whereas the colostrum enzyme yields the 6�-isomer. The colostrum enzyme showsgreat specificity for its acceptors (46). In quantitative terms, when the efficiency of the enzymeis expressed as Vmax/Km the following values were obtained (% relative to N-acetyllac-tosamine): Gal(�,1–4)GlcNAc or N-acetyllactosamine, 100; Gal(�,1–4)Glc or lactose, 2;Gal(�,1–3)GlcNAc, 13; Gal(�,1–6)GlcNAc, 0.4; and asialoorosomucoid, 1000.

Thus, the nature of the penultimate sugar in an acceptor, the precise linkage of the terminalto the penultimate sugar, and the size of the acceptor can all play major roles in determining theactivity of a given GT.

The total number of GTs thus far identified exceeds many hundreds (reviewed in Ref. 47).Many of the structural genes have been cloned, and the enzymes were overexpressed, purifiedto homogeneity, and characterized kinetically. At least two have been crystallized and theirthree-dimensional structures determined. Insofar as the topics covered here are concerned, theGlcNAc transferases that act on polylactosamine ((Gal-(�1,4)-GlcNAc-(�1,3))n), a constituentof many cell membranes, are of considerable interest (see review by Renkonen (19)). OneGlcNAc transferase is required for increasing the chain length at the non-reducing terminalGal. Two others add GlcNAc to internal Gal residues, thereby starting the branching process.One of the branching enzymes works at the distal end of the chain, and the other acts“centrally.” Both are greatly influenced by the presence of Fuc residues on the chains. Thus,the combination and interplay of the GalT, the three GlcNAc transferases, the FucT, andpossibly the sialyltransferases determine the final structure on the cell surface, but how theseare regulated with respect to each other remains to be determined.

Speculations on Cell-Cell AdhesionThe human brain contains approximately a trillion neurons, and each averages around 103

connections with other cells or about 1015 specific connections. How can this happen given atotal of about 40,000 genes in the human genome? The data banks list 72,000 publications on“cell adhesion,” and they report CAMs (cell adhesion molecules), cadherins, catenins, ephrins,Eph receptor tyrosine kinases, laminins, selectins, integrins, their relationships to the extra-cellular matrix and the cytoskeleton, to cytokines, and much, much more.

In some instances, the role of carbohydrates is well documented. (a) Leukocyte extravasation(recruiting leukocytes from the blood to the site of infection, injury, or lymphatic circulation)involves a sequence of complicated interactions between the leukocytes and the blood capillaryendothelium comprising selectins, other proteins, and carbohydrates (reviewed in Ref. 48). (b)CD44, a cell surface receptor, binds to hyaluronan (12). (c) Myelin-associated glycoprotein is aSiglec (sialic acid-specific lectin) that binds to complex gangliosides, an interaction essentialfor maintaining normal myelin structure (49, 50).

As indicated below, there is now a rapidly developing interest in the role of glycans indevelopment and in cell recognition. However, in surveying the literature, it appears that someold ideas bear repeating. The discussion will be limited to cell-cell recognition.

Specific Intercellular Adhesion—The crucial importance of cell recognition in developmentwas well established in the late 1800s. In normal embryos, cells exhibit exquisite adhesivespecificity. They “know” where they are, and they “know” where they are going. Under in vitroconditions, cells adhere nonspecifically to many substances, including tissue culture plastic,glass, serum proteins, etc. Nevertheless, adhesive specificity can be demonstrated in vitro andwas shown in 1907 in a classic case of serendipity. Wilson (51) found that when single-cellsuspensions from two species of marine sponges were mixed they first aggregated to form aheterologous chimera, but with time they sorted out to yield aggregates of homotypic cells.Holtfreter (1930s) obtained the same results with cell suspensions from different embryonictissues. Although cadherins are thought to be involved in cell sorting, the underlying biochem-ical basis is very complex, and yet to be fully explained.17 It was subsequently demonstratedthat adult cells, such as hepatocytes and mycocytes (55, 56), exhibit adhesive specificity and

17 Humphreys (52) showed that dissociation of the sponges to single cells released species-specific, heat-labile, largemolecular weight “aggregation factors.” These observations were followed by a series of studies from many laborato-ries, particularly by Burger’s group. Polysaccharides, sulfated polysaccharides, proteoglycans, and lectins have beeninvoked as participants in a multistep process, some of which require Ca2�. In a recent paper (53), a uniquesupramolecular circular proteoglycan complex is described as one of the components involved in the process. One ofthe N-linked glycans contains glucuronic acid, fucose, mannose, galactose, N-acetylglucosamine, and sulfate (54).


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that in liver homogenates, the specific factor was localized to the plasma membranes (56). Theactive factor(s) in the chick membranes is a trace glycolipid (Fig. 3).

A quantitative assay (57) was used to study the kinetics of homologous adhesion (58) andshowed that the process is multistep. The first step does not require metabolic energy; the cellsform a loose association that dissociates even by simple dilution of the suspension. In thesecond energy-requiring step, the aggregate is stabilized and can only be dissociated byvigorous treatment, e.g. proteases. In the third step, the stable aggregates synthesize collagenand sulfated GAGs. All of this takes minutes at 37 °C.

Insofar as the underlying biochemical mechanisms are concerned, there are two obviousquestions. (a) What cell surface molecules participate in the process? (b) How is the informa-tion transmitted to the interior of the cell? Two hypotheses were suggested to answer thesequestions, as indicated in Figs. 4 and 5.

Hypothesis I: Carbohydrates Are Involved in Specific Intercellular Adhesion—Two mecha-nisms were proposed (2) for carbohydrate participation as indicated in Fig. 4. 1) Cell adhesionis mediated by hydrogen bonds between carbohydrates on neighboring cells. That hydrogenbonds can be important in maintaining carbohydrate structures is exemplified by polysaccha-rides such as cellulose and chitin. 2) Cell adhesion is mediated by the binding of carbohydratesto cell surface proteins and enzymes. There were two reasons for extrapolating from proteinsto enzymes and in particular to the glycosyltransferases. (a) The glycosyltransferases as aclass appeared to be much more specific than the lectins, a critical requirement for specificintercellular adhesion. (b) If glycosyltransferases are involved, then one cell could also modifythe surface of its neighbor. However, extracellular modification requires an extracellular cellsurface or soluble enzyme and a source of sugar nucleotides and/or PAPS, either from thecytoplasm or extracellularly. Is any of this possible? 1) Enzymatically active, soluble extracel-lular glycosyltransferases do occur in the fluid surrounding intact embryonic chicken brain andin embryonic and adult chicken serum, vitreous humor, and human spinal fluid (59). 2) Cellsurface glycosyltransferases may occur. Chick embryonic neural retina cells transferred Galfrom UDP-Gal to soluble high molecular weight acceptors (60), suggesting that the reaction

FIG. 3. Scanning electron micrographs of chicken hepatocytes and immobilized chicken plasma mem-brane glycolipid. A quantitative procedure was devised for assaying the effects of immobilized glycolipids andsimilar substances on the rate of adhesion of chicken hepatocytes (81). A glycolipid, present in trace quantities inchicken liver, was the only substance of many pure and mixed lipids and glycolipids tested that stimulated theadhesion of these (but not rat) hepatocytes. The scanning electron micrographs are as follows. A, mixture of chickenhepatocytes and polystyrene beads (red balls) previously immersed in a dilute solution of the specific glycolipid. Nosuch structures were seen with beads treated with the inactive lipids. B and C, cell and bead. The filopodia-likestructures were observed in all cases of cell-bead adhesion. D, cell-cell adhesion. Filopodia-like structures are evidentin the regions of contact. These experiments were conducted with purified but not homogenous preparations of theglycolipid. The specific glycolipid has recently been purified to apparent homogeneity by Dr. Ming Chuan Shao andBarbara Rauch. (The photographs were kindly prepared by Michael McCaffery of the Integrated Imaging Center,Department of Biology, Johns Hopkins University.)


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was catalyzed by a cell surface Gal-transferase. Evidence for and against this conclusion hasbeen presented by other laboratories, and at this time, it remains controversial. However, Fig.3 suggests that only a vanishingly small percent of the cell surface appears to be involved earlyin specific cell-cell interactions. If cell surface glycosyltransferases participate in these inter-actions, they may be present in traces and difficult to detect by any method, includingimmunological procedures. 3) Sugar nucleotides may be secreted. In a recent paper (61), a Gprotein-coupled plasma membrane receptor for UDP-Glc was identified in a wide variety ofhuman tissues, including many regions of the brain. Thus, extracellular sugar nucleotides mayindeed occur.

Hypothesis II: Membrane Messengers—In 1958–1962, a series of studies by Sutherland andco-workers (62, 63) led to the characterization of cAMP, adenylate cyclase, and the effects ofcertain hormones on this enzyme. Sutherland designated cAMP as the “second messenger”(hormones were the first messenger). This seminal work surely ranks as one of the mostimportant biochemical findings of the past century. Somewhat later, Rasmussen invoked Ca2�

as another “second messenger.” These hypotheses were obviously correct, but it seemed to us

FIG. 4. Hypothesis I: carbohydrates are involved in specific intercellular adhesion. The suggested mech-anisms are as follows. (a) Hydrogen bonding between oligosaccharide chains on adjacent cell surfaces. The scheme isnot meant to imply that hydrogen bonds can only form between identical monosaccharides. (b) Enzyme-substratecomplex. The model is meant to suggest that cells can bind to and/or modify adjacent cells or extracellular matrixthrough the action of cell surface glycosyltransferases. See text for discussion.

FIG. 5. Hypothesis II: membrane messengers. An extrapolation of the Sutherland second messenger idea. Avariety of extracellular signals are received by cell membrane receptors, which in turn send specific messages to thecytoplasm or nucleus. The membrane is a transducer, and the membrane messengers were suggested (82, 83) tocomprise both low and high molecular weight substances, such as proteins. Additionally, it was suggested that in somecases the messenger molecules would act stoichiometrically (e.g. repressors of operons), whereas in others, they couldbe enzymes.


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that they were insufficient. Could the diverse stimuli received by a cell and the manyresponses that these signals elicited be explained by only two second messengers? A “mem-brane messenger” hypothesis was therefore devised in 1974 as illustrated in Fig. 5. Themembrane acts as a “transducer” containing multiple receptors that respond to externalsignals by releasing specific intracellular messengers. Signal transduction by the plasmamembrane is now well established, and a section of each issue of this Journal is devoted topapers in this field.

Quantitative Changes in Carbohydrate Ligands Can Have Global Effects on Cellular Phe-notypic Behavior—Qualitative changes in carbohydrate composition of the cell surface or thesubstrata to which the cell adheres can have far reaching effects on cell behavior, but what ofquantitative changes? Although the Ashwell protein catalyzes receptor-mediated endocytosisof glycoproteins in hepatocytes, it does not participate in intercellular adhesion. Nevertheless,it served as a useful model to answer this question.

We have often tried to mimic cell surfaces by adsorbing or covalently linking potentialcarbohydrate ligands to solid matrices (e.g. Fig. 3). This approach was used to test hepatocytes(64, 65) with sugar derivatives covalently linked to polyacrylamide gels. Chicken hepatocytesspecifically adhered to GlcNAc-derivatized gels and rat hepatocytes to Gal-derivatized gels, inaccord with the known specificities of the Ashwell receptors in the two cell types. However,there was a remarkable threshold or critical concentration effect of the sugars as shown in Fig.6. Below this concentration of sugar in the gel, the cells did not bind to the gels. At thethreshold, �15% increases in GlcNAc and Gal concentrations, respectively, in the gels resultedin 100% cell binding to the gels.18

18 An interaction between a protein and its monovalent ligand may be weak, but if the ligand is polyvalent such thatmany protein molecules can interact, the binding affinity for the polyvalent ligand greatly increases. An excellentexample of this is CD44, a cell surface receptor that binds to hyaluronan (12). Hyaluronan oligosaccharides with 6–10sugars are sufficient to interact with CD44 monovalently, and relatively high concentrations of these oligosaccharidescan prevent binding of macromolecular hyaluronan, which otherwise binds with high affinity. However, the interac-tion of the monovalent oligosaccharides with CD44 is sufficiently weak that they do not remain bound through a

FIG. 6. Effect of quantitative changes in carbohydrate concentration on cell binding in a model system.A variety of carbohydrates were covalently linked to polyacrylamide gels and tested with chicken and rat hepatocytes.As expected, the cells bound to the gels in accord with the known specificities of the Ashwell receptor, chicken toGlcNAc, and rat to Gal. In this model system, the concentrations of the sugars in the gels were varied, as indicated.A threshold or critical concentration effect is observed. Below this concentration, there is no binding, and above it, allthe cells bind to the gels.


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The physiological implications are plain if this model represents what can happen in celladhesion. A non-adherent cell can become adherent by a slight change in the cell surfaceconcentration of the appropriate ligand and/or its receptor or in the extracellular matrix. Evenmore likely, the “grouping” of receptors or ligands into microdomains in the plasma membraneresults in binding, and the size of these domains is apparently affected (regulated?) by otherfactors, such as cholesterol.

The Future: Glycans as Informational MacromoleculesThe particular advantage of carbohydrates is that they have enormous potential for serving

as informational macromolecules, starting with their de novo biosynthesis. Laine (67), forinstance, has calculated that a hexasaccharide has 1012 isomeric permutations. Second, theglycans are readily modified after synthesis of the core structure. A few such modifications aresulfation (thought to be essential for leukocyte extravasation), O-acetylation of individualsugars such as sialic acid, addition of a few sugar residues that can convert blood group O toA or B, or initiating a branch point by the action of the branching GlcNAc transferases onpolylactosamine. For instance, in the neural retinotectal system where neuronal pathfindingis essential, immunological methods have shown a dorsoventral gradient in a cell surfaceantigen of the rat embryonic neural retina (68). The antigen was identified as 9-O-acetyl-GD3.At the same time, there was no apparent gradient of the parent ganglioside GD3. Thus,relatively few enzymes can create a large number of molecular variants.

There is no doubt that the molecular events underlying embryogenesis, especially of thenervous system, will be the major goal of biology well into the foreseeable future. Experimentsin a number of laboratories are now in progress to elucidate the roles of glycans in theseprocesses, and some of these are cited above. However, other examples can be given. (a) Byconstructing specific glycosyltransferase mutants in mice and other organisms, the synthesisof specific glycans or classes of glycans can be eliminated. This approach has shown thatgangliosides and glycoproteins of the N-glycan type are essential for the survival of the embryoand/or its normal development in the mouse (69) and in the nematode Caenorhabditis elegans(70).19 (b) A number of papers have reported that proteoglycans and glycosaminoglycans,especially heparan, are essential for normal development of Drosophila and C. elegans. Theaffected genes include Wingless, tout-velu, sugarless, sulfateless, dally, and sqv 3,7,8 (71–77).(c) Notch receptors are highly conserved intercellular signaling pathways that direct embry-onic cell-fate decisions. The activities of these receptors are regulated by Fringe proteins, andrecent evidence (78, 79) shows that Fringe is a fucose-specific GlcNAc-transferase.

To summarize, the huge gap between the 1015 specific connections in the brain and thenumber of genes in the human genome can readily be filled by the glycans.

It is presumptuous to try to predict the future. Who, in the 1960s, could have predicted whathappened to the field we called genetics? At the moment, primary interest seems to be shiftingfrom genomics to proteomics and functional proteomics. But as others have said, glycobiologyis the field of the future. However, the problems are formidable, as I have tried to indicate inthis brief overview.

One “problem” is nothing more than a false perception. On several occasions I have heardstructural biologist colleagues state that the glycan units in a glycoprotein, for instance,cannot be important because they are too flexible to be seen in an x-ray crystal structure or byNMR. In other words, if they don’t have structure, how can they have function? That thisconclusion is gratuitous requires no more than a moment’s reflection.

For instance, one important physiological property of cartilage is that it is reversiblycompressible, acting like a spring to the application of a force. This feature emanates from theflexibility of the aggrecan aggregate, which can be compressed to one-tenth its volume.Hyaluronan provides another example. It is essential as a lubricant in joint fluids where it hashigh viscosity and an extended helical or possibly random coil structure. It is more restrictedin the aggregcan aggregate but must still be flexible, and it forms a gel in cumulus cell-oocytecomplexes (12).

routine washing step (66). The cytoplasmic tail of CD44 interacts with anchorin in the cytoskeleton. Therefore,interaction of CD44 with its polyvalent, linear ligand can contribute to alignment and stabilization of the cytoskeletonand consequently influence cell behavior.

19 Unpublished data: on the N-type glycoproteins by Schachter et al; on the gangliosides by Sandhoff, Proia et al.


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These should be sufficient to make the point. Glycans are different because frequently theyare flexible and adjust to physiological need. In other words, in these substances, functiondefines structure, not vice versa. Certain glycans clearly have highly favored conformations,and lectins may have evolved to reflect those particular three-dimensional structures. Fur-thermore, whereas energy minimization methods can yield the thermodynamically favoredconformers, the less favored conformers may be the biologically active structures that bind totheir ligands.

It would not be a big surprise if different conformers of a single oligosaccharide interactedwith different ligands or receptors or enzymes or possibly even other carbohydrates underdifferent physiological conditions. It is this interplay between proteins and different conform-ers that likely allows a single carbohydrate structure, such as hyaluronan, to be used in manydifferent ways. In the excellent book by Cantor and Schimmel on the conformation of macro-molecules (80), they raise a number of questions about carbohydrate polymers similar to thosediscussed above and then say: “These are all interesting questions, but it will probably takemuch hard work to answer most of them.” Amen to that! What is lacking is adequatebiophysical methodology.

The problem is much more complicated when we deal with membranes. Trying to assignstructure or even distribution (if it is not random) of a particular glycolipid on the surfaceseems impossible at this point because of fluidity of the external monolayer of the lipid bilayer.If glycolipids do exist primarily in “rafts” or domains, these domains are in a constant state offlux and motion within the monolayer, and their sizes, frequency of formation, etc., depend onthe lipid composition of the remainder of the monolayer and whether they are or are notassociated with membrane-bound signaling proteins, such as the Src family of kinases. Thesame problem exists with cell surface glycoproteins, except possibly for those tethered tocytoplasmic components, such as the cytoskeleton. Even in the latter case, publicationssuggest that perturbation of the cell can rapidly result in drastic reorganization of thecytoskeleton.

Thus, it appears that present methods will permit us only to obtain “snapshots” of limitedareas of the cell surface. There is no doubt that the task ahead of us is difficult, but if cells“talk” to other cells via cell surface substances such as the glycans, the problem cannot beavoided. I am optimistic. Breakthrough technological advances are produced at an astonishingrate these days.

Who could have predicted the development of polymerase chain reaction and itsconsequences?

Acknowledgments—I am especially grateful to Drs. Ronald Schnaar and Mark Roseman for critical reading of thismanuscript and for many helpful suggestions. The sections on aggrecan and the aggrecan aggregate and Fig. 2 couldnot have been written without the help of Dr. Vincent Hascall, who also provided numerous other insightfulcomments.

REFERENCES1. (2001) Science 291, 2263–25022. Roseman, S. (1970) Chem. Phys. Lipids 5, 270–2973. Roseman, S. (1989) FEMS Microbiol. Rev. 63, 3–124. Keyhani, N. O., and Roseman, S. (1999) Biochim. Biophys. Acta 1473, 108–1225. Conant, J. B. (1952) Pasteur’s Study of Fermentation, Harvard University Press, Cambridge, MA6. Fruton, J. S., and Simmonds, S. (1953) General Biochemistry, John Wiley & Sons, Inc., New York7. Gottschalk, A. (1972) Glycoproteins: Their Composition, Structure and Function, Elsevier Science Publishers B.V.,

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The Pentose Phosphate Pathway

Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.X200007200

Bernard L. Horecker

From the Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021

I received my basic training in enzymology as a graduate student in the laboratory ofProfessor T. R. Hogness at the University of Chicago from 1936 to 1939. Hogness had con-structed a photoelectric spectrophotometer modeled after the one in Otto Warburg’s laboratoryin Berlin-Dahlem. I was assigned a problem on succinic dehydrogenase from beef heart, usingthe Warburg manometric apparatus, and did not get to use the spectrophotometer until ErwinHaas arrived from Warburg’s laboratory in 1939. Haas asked me to join him in the search foran enzyme that would catalyze the reduction of cytochrome c by reduced TPN (now NADP).This reaction was thought to be the missing link in the electron transport chain from substrateto oxygen and marked the beginning of my interest in what was then thought to function asa direct oxidative pathway for the metabolism of carbohydrate but is now known as the pentosephosphate pathway.

After I left Chicago during the Second World War, my experience with the spectrophotom-eter landed me a job at the National Institute of Health (NIH) in Frederick S. Brackett’s groupin the Division of Industrial Hygiene. Brackett had assembled an automatic recording spec-trophotometer in the basement of Building 2 that I was assigned to use to develop a methodfor the determination of carbon monoxide hemoglobin in the blood of Navy pilots returningfrom combat missions. That and a number of other war-related projects kept me occupied forthe next 4 years.

In 1945, after the end of the war with Japan, I was advised by the Director of the Laboratory,Dr. Paul Neal, that I was free to return to research in enzymology. I began studies on thereduction of cytochrome c by the succinic dehydrogenase system, using what was now my ownBeckman spectrophotometer. One day, which I consider to be a turning point in my career,Arthur Kornberg, who had been working in Building 4 on the biological role of folic acid,appeared in my laboratory. Arthur had become convinced that enzymes were the key to anunderstanding of intracellular biochemical processes and suggested that we work together. Webegan with studies on the effect of cyanide on the succinic dehydrogenase system, becausecyanide was known to bind to and be a general inhibitor of enzymes containing the hemegroup. An exception was cytochrome c, which had been reported to be resistant to the actionof cyanide. Contrary to these early reports, we found that cyanide did react with cytochromec and in 1946 published our first paper together, in the Journal of Biological Chemistry,entitled “The Cytochrome c Cyanide Complex.”

Making History in Building 3Two years later in 1948 when Arthur returned from a study leave in the laboratories of

Severo Ochoa in New York and Carl Cori in St. Louis, he invited Leon Heppel and me to joinhim in setting up a new Section on Enzymes in the Laboratory of Physiology to be housed inBuilding 3, which was being completely renovated. Leon and I were about to be transferred, ithaving been discovered that the Industrial Hygiene Research Laboratory in Building 2 had

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 50, Issue of December 13, pp. 47965–47971, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.




This paper is available on line at http://www.jbc.org

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never been officially part of the NIH but was in the Bureau of State Services, which wasmoving to new headquarters in Cincinnati.

In the fall of 1948 while we waited for the of the renovation of laboratories in Building 3 tobe completed, we all three worked in Building 2. Arthur and I, both of whose planned researchprojects would depend heavily on assays using the “pyridine nucleotide” coenzymes DPN andTPN, collaborated in their isolation. In those early years of American biochemistry there wereno vendors that supplied these materials. We isolated them from sheep liver, using theunpublished procedure from Warburg’s laboratory that Erwin Haas and I had used in Chicago.In 1948 Arthur and I possessed the world’s supply of TPN, and when Warburg visited ourlaboratory in 1948, we were able to present the discoverer of TPN with a gift of 25 mg of thatcoenzyme.

The new Enzyme Section in the Division of Physiology in Building 3 provided an excitingand stimulating atmosphere. Together with Herbert Tabor from the Laboratory of Pharma-cology in Building 4, we organized a daily lunch hour journal club, during which we reviewedthe literature on every facet of enzymology and intermediary metabolism. This was thebeginning of a great history for Building 3, continuously occupied by scientists who were tomake notable contributions in biomedical science. The first NIH recipients of the Paul LewisLaboratories Award in Enzyme Chemistry, then one of the most prestigious awards inbiological research, were all from Building 3: Arthur Kornberg in 1951, myself in 1952, andEarl Stadtman in 1953. Later, our Section on Enzymes became part of the new ExperimentalBiology and Medicine Institute, which Henry Sebrell, then the NIH Director, proposed tofunction as the basic research arm of the NIH. It was later renamed the National Institute ofArthritis and Metabolic Diseases, a change that had no effect on the nature of our research butresulted in increased funding by Congress. We continued to work in the laboratories inBuilding 3.

Use of the pyridine nucleotides in enzyme assays with the Beckman spectrophotometerrequired knowledge of the exact extinction coefficients of the 340 nm peaks of the reducedforms. The published values for DPNH showed considerable variation, and there was scantinformation for TPNH. In the new laboratories in Building 3, Arthur and I designed experi-ments to determine the true extinction coefficients at 340 nm of both coenzymes, which provedto be identical. That work, published in 1948, made possible quantitative spectrophotometricmeasurements in reactions involving the pyridine nucleotides and became one of the mostfrequently cited papers in the biochemical literature.

In the new laboratories in Building 3 Leon Heppel and I also collaborated in thepurification of xanthine oxidase from milk after we found that this enzyme could reduce notonly methylene blue, a reaction that I had studied in Chicago, but also cytochrome c.However, this reduction occurred only if oxygen was present, a curious observation that wasquickly picked up by Fridovich and Handler, who were working at Duke University on theformation of the superoxide anion. The reduction of cytochrome c by superoxide anionbecame a widely used assay for this species of “active oxygen.” I also returned to the studyof cytochrome c reductase, which Haas and I had isolated from yeast, and accomplished thefirst isolation of this flavoprotein from mammalian liver. By then it had become apparentthat these cytochrome c reductases did not function in mitochondrial respiration but ratheras components of the cytochrome P-450 system for the metabolism and detoxification ofdrugs and other xenobiotics.

The Pentose Phosphate PathwayThe Oxidation of Glucose 6-Phosphate—When Otto Warburg discovered TPN as the coen-

zyme required for the oxidation of glucose 6-phosphate to 6-phosphogluconate, the role of theother pyridine nucleotide, DPN, as the coenzyme required for the fermentation of glucose toethanol in yeast or the glycolysis of glucose to lactic acid in muscle had been well established.The finding that the new coenzyme was required for the oxidation of glucose 6-phosphate andalso for the further oxidation of the product, 6-phosphogluconate, led Warburg and also FrankDickens in England and Fritz Lipmann, then working in Denmark, to propose that thereexisted an alternate pathway that functioned as a “direct oxidative pathway.” They hadobtained evidence that the products formed in the oxidation of 6-phosphogluconate by TPNwere carbon dioxide and an unidentified pentose phosphate. Because carbon dioxide was oneof the products, it seemed reasonable to regard this alternate pathway as the one responsible

Reflections: The Pentose Phosphate Pathway

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for the oxidation of carbohydrate. Haas and I had already shown that TPN could serve as anelectron transport link to the cytochromes and therefore to molecular oxygen.

Twenty years after the pioneering work of Warburg, Dickens, and Lipmann, I began studies(with a new laboratory technician, Pauline (Polly) Smyrniotis) on the enzymes involved in theoxidation of 6-phosphogluconate and the metabolic intermediates formed in this pathway. Wewere joined by J. E. Seegmiller, my first postdoctoral student, and he and I worked out a newmethod for the preparation of glucose 6-phosphate and 6-phosphogluconate, which were notyet commercially available. We purified the enzyme, 6-phosphogluconate dehydrogenase, frombrewers’ yeast, the richest source we could find, and by coupling the reduction of TPN to itsre-oxidation by pyruvate in the presence of lactic dehydrogenase showed that the first productof the oxidation of 6-phosphogloconate, in addition to carbon dioxide, was a new pentose ester,ribulose 5-phosphate, which was then converted to ribose 5-phosphate by a pentose-phosphateisomerase present in our purified dehydrogenase preparations. The separation of ribulosephosphate from ribose phosphate and the demonstration that their interconversion wascatalyzed by a pentose-phosphate isomerase were made possible by the recent development atthe Oak Ridge National Laboratory of a separation technique for nucleotides called ion-exchange chromatography.

The identification of the sugar in the new pentose ester as ribulose was based on a numberof criteria, including comparison with the authentic sugar, prepared by the method ofGlatthaar and Reichstein, using a number of chemical and physical criteria, which includedx-ray diffraction of the crystalline nitrophenyl hydrazones. In those days discoveries of newsugar phosphate esters were rare events, and I felt that it was necessary to establish itsidentity beyond a shadow of doubt. The results were first presented at the American ChemicalSociety meeting in Boston in 1951 at a symposium honoring Arthur Kornberg’s Paul LewisLaboratories Award, and I recall the warm reaction at that meeting to the work reported fromour laboratories in Building 3.

During the following year Jay Seegmiller and I showed that the same products were formedin the metabolism of 6-phosphogluconate by enzymes from mammalian tissues.

The Further Metabolism of the Pentose Phosphates—An important clue to the further stepsin what was later to become known as the “pentose phosphate pathway” was already in theliterature. In 1938 Zaccharias Dische had demonstrated that red cell lysates catalyzed theconversion of the 5-carbon sugar, ribose 5-phosphate to hexose monophosphate, an observationthat Seegmiller and I confirmed in 1952 with rabbit bone marrow extracts. These observationsgave rise to the hypothesis that the oxidative pathway was really a cyclic mechanism for thedirect oxidation of carbohydrate. With each turn of the cycle one molecule of carbon dioxidewould be produced, and the pentose phosphates formed would be metabolized back to hexosephosphates to start another cycle. Six turns of the cycle would result in the complete oxidationof one molecule of glucose.

However, the reactions involved in the conversion of the 5-carbon pentose phosphates to the6-carbon hexose phosphates were completely unknown. What ensued was a race involving anumber of laboratories, including ours at the NIH and those of Ephraim Racker at the NewYork City Research Laboratories, later at Cornell University in Ithaca, Seymour Cohen at theUniversity of Pennsylvania in Philadelphia, Oliver Lampen at Washington University in St.Louis, and Frank Dickens in England, to identify the reactions and metabolic intermediatesinvolved.

It had already been established from the work of Dische and others that one of the productsof pentose phosphate metabolism was the 3-carbon sugar, glyceraldehyde 3-phosphate, whichsuggested cleavage of a 5-carbon sugar, probably ribulose 5-phosphate, between carbon atoms2 and 3. The 3-carbon fragment, glyceraldehyde 3-phosphate, was a known intermediate inglycolysis, but what was the fate of the remaining 2-carbon fragment? Polly Smyrniotis and I,now joined by my first foreign postdoctoral fellow, Hans Klenow from Copenhagen, set out topurify the enzyme(s) involved in the cleavage of pentose phosphate, using rat liver as theenzyme source and an assay that measured the appearance of glyceraldehyde 3-phosphate.When we also followed the disappearance of the 5-carbon sugar, using Dische’s “orcinol”reaction, we detected the formation of a new product that also reacted with orcinol butproduced a different color and a visible absorption spectrum distinctly different from thatproduced in the reaction with pentoses. Our clue to the identity of this product came fromMelvin Calvin’s laboratory in Berkeley, where, using radioactive carbon dioxide, they had


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identified both ribulose diphosphate and a 7-carbon sugar, sedoheptulose monophosphate, asearly intermediates in the fixation of carbon dioxide in photosynthesis. Authentic sedoheptu-lose was available from Nelson Richtmyer’s laboratory in Building 4, and on Christmas Eve,1951, when everybody else had gone home, I sprayed a paper chromatogram with orcinol andup came the blue spot characteristic of sedoheptulose. I rushed up and down the laboratoryhallway clutching the paper chromatogram, but there was nobody there to show it to, so I tookit home and hung it on the Christmas tree, singing the little ditty: “It’s sedoheptulose, it’ssedoheptulose, tra la la boom deay, tra la la boom deay,” much to the amusement of my youngdaughters.

We adopted the name “transketolase,” first suggested by Racker and his co-workers, becauseit catalyzed the transfer of a 2-carbon fragment from the ketopentose, ribulose 5-phosphate, tothe other 5-carbon sugar, ribose 5-phosphate, to generate the new ketol linkage in the 7-carbonsugar, sedoheptulose 7-phosphate. Thus the unknown 2-carbon fragment never occurred as afree entity. Later, simultaneously with Racker, we showed that the coenzyme carrier for the2-carbon fragment by transketolase was thiamine pyrophosphate.

Two puzzling observations remained to be explained. One was that the configuration of thehydroxyl group on the third carbon atom of the new product, sedoheptulose 7-phosphate, wasopposite that on the third carbon atom of the presumed substrate, ribulose 5-phosphate. Thislack of stereospecificity, particularly because we had demonstrated that the reaction wasreadily reversible, was highly improbable for an enzyme-catalyzed reaction. The other unex-plained observation was that the cleavage of pentose phosphate by our purified transketolasepreparations from rat liver required the presence of aldolase, a crystalline and supposedlypure enzyme from rabbit muscle that catalyzed the condensation of two triose phosphates toform the hexose, fructose 1,6-bisphosphate. The answer to both of these puzzling observationscame from the discovery by Paul Stumpf, while on sabbatical in my laboratory, and reportedalmost simultaneously from the laboratories of Dickens, Racker, and Ashwell, of anotherenzyme, an “epimerase,” that catalyzed the conversion of ribulose 5-phosphate to its “3-epimer,” xylulose 5-phosphate, which had the same stereo-configuratuion at the 3-carbon atomas sedoheptulose phosphate. With this substrate, we confirmed the earlier report by Rackerand his co-workers that xylulose phosphate, rather that ribulose phosphate, was the truesubstrate for cleavage by transketolase and the true donor of the 2-carbon fragment. Therequirement for aldolase was explained when we found that crystalline preparations of thisenzyme from rabbit muscle contained the epimerase as a contaminant, which could only beremoved by many re-crystallizations.

Thus three different pentose phosphates were now shown to be involved in the new pathway:ribulose 5-phosphate, the first product of the oxidation of 6-phosphate gluconate, and xylulose5-phosphate and ribose 5-phosphate, both formed from ribulose 5-phosphate, one serving asthe 2-carbon donor and the other as the acceptor in the reaction catalyzed by transketolase.The addition of any one of these pentose phosphates to crude tissue extracts would result in theformation of an equilibrium mixture of all three.

Completion of the Cycle—Still to be discovered, however, was a mechanism that wouldconvert the products of the transketolase reaction, sedoheptulose phosphate and glyceralde-hyde phosphate, to the 6-carbon sugars, fructose 6-phosphate and glucose 6-phosphate, andcomplete the cycle. In particular, what was the fate of sedoheptulose phosphate? We found thatpurified enzyme preparations from liver or yeast would catalyze the formation of hexosemonophosphate from sedoheptulose monophosphate but only if triose phosphate was alsopresent. When I described this finding at one of our luncheon journal club meetings, Horace(“Nook”) Barker, who was visiting from the University of California at Berkeley and workingin Kornberg’s laboratory, suggested that we consider the possibility of another transfer, thistime of a 3-carbon fragment from sedoheptulose phosphate to triose phosphate to generatefructose 6-phosphate. When we carried out an experiment with carbon-14-labeled triosephosphate, we found that, as predicted, the fructose 6-phosphate formed had radioactivity inthe last three carbon atoms with the first three unlabeled. We named the enzyme “transal-dolase” because it catalyzed the transfer of an aldol linkage rather than the hydrolytic cleavagecatalyzed by aldolase.

What remained was to account for the fate of the remaining 4 carbon atoms of sedoheptulose7-phosphate. For this work Polly Smyrniotis and I were joined by Paul Marks and HowardHiatt, two young M.D.s working as Clinical Associates in the new Clinical Center in Building


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10, who had asked to join my group to learn enzymology in their “spare time,” which turnedout to be from 5 p.m. to midnight. We identified the missing fragment as another new sugarester, the 4-carbon sugar erythrose 4-phosphate, in a number of tests, including its conversionto the 7-carbon sugar sedoheptulose 1,7-diphosphate in a condensation with dihydroxyacetonephosphate, catalyzed by fructose bisphosphate aldolase. It was also converted to fructose6-phosphate in the reaction catalyzed by transketolase.

The elucidation of the pentose phosphate pathway had now been accomplished. It consistedof two branches, an oxidative branch in which the hexose, glucose 6-phosphate, was convertedto pentose phosphate and carbon dioxide with the reduction of two molecules of TPN, and anon-oxidative branch, in which three molecules of pentose phosphate (15 carbon atoms) werereconverted to two and one-half molecules of hexose phosphate (15 carbon atoms) in a seriesof fully reversible reactions. Our contributions included the discovery of three new sugarphosphate esters, ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose 4-phos-phate, and three new enzymes, transketolase, transaldolase, and pentose-phosphate 3-epime-rase. We shared with Racker the discovery of transketolase and confirmed his finding thatxylulose 5-phosphate, rather than ribulose 5-phosphate, was the 2-carbon donor in the reac-tion catalyzed by that enzyme. We also shared with McLean and Dickens, working in England,the discovery that fructose 6-phosphate was also a substrate for transketolase. If the pathwayoperated as originally envisioned, six turns of the “cycle” would result in the oxidation of onemolecule of 6-carbon sugar to six molecules of carbon dioxide.

Functions of the Pentose Phosphate Pathway—The function(s) of the new pathway, however,turned out to be quite different from the pathway for the direct oxidation of carbohydrate thatwe had expected. It provides two mechanisms for the production of ribose 5-phosphate. One isthe “oxidative branch” of the pathway, which also generates 2 eq of TPNH (NADPH). Ribose5-phosphate can also be formed directly from hexose and triose phosphates by the non-oxidative rearrangements catalyzed by transketolase and transaldolase. Where large quanti-ties of NADPH are required, as in the synthesis of fatty acids or sterols, the excess pentosephosphates produced would be recycled back to hexose monophosphates.

To assist medical students in memorizing the reactions, someone composed the followingsong.

THE PENTOSE PHOSPHATE SHUNT(Tune: “MacNamara’s Band”)

If you’re converting carbohydrate into triglyceride,If you need pentose moieties to make nucleotide,You’ll find that Embden-Meyerhof is not the game to playAnd you’ll do your biosynthesis the pentose phosphate way.

Chorus: With transaldolase, transketolase, G6PDH too,Six times six gives five times six plus six of CO2Carbons passing to and fro, the back becomes the front,Did you ever see a pathway like the pentose phosphate shunt?

First G6P is oxidized, NADP reducedTo give gluconolactone (as might have been deduced).The lactone is then hydrolyzed to make the gluconateAnd decarboxylated to its metabolic fate.

There ends the oxidative phase, now multiply by three,An intermediate balance sheet by way of summary,Six NADPH are formed, three CO2 set free,Three ribulose 5-phosphate formed from three of G6P.

One isomerization from ketose to aldoseTurns ribulose 5-phosphate to the phosphate of riboseThe other two epimerized, inverted at C3,Two xylulose 5-phosphates formed (hence called Xu5P).


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Two carbons from Xu5P transferred from the ketose to aldose(Transketolase needs TPP as everybody knows),Thus three plus seven made to meet transaldolase attack,Three Cs from sedoheptulose the GAP gets back,

Glyceraldehyde 3-phosphate thus becoming F6PLeaves erythrose 4-phosphate looking for some company,But Xu5P number two has two top Cs to spare,Transketolase negotiates their transfer as a pair.

So we’ve made another F6P, a triose phosphate too,To see what we have now achieved let’s multiply by two,Four F6Ps, two GAPs, by glycolytic tricks,Give five glucose 6-phosphates, when we started out with six!

(Author unknown)

The first discovery relevant to the new pentose phosphate pathway, namely the formation ofribulose and ribose phosphates as products of the oxidation of 6-phosphogluconate, wasannounced in the spring of 1952 at the annual meeting of the American Chemical Society inChicago. The outline of the complete pentose phosphate cycle, including the reactions cata-lyzed by the new enzymes transketolase and transaldolase, was published in 1955 in a reviewwritten in collaboration with I. C. (Gunny) Gunsalus and W. A. (Woody) Wood for Bacterio-logical Reviews entitled “Pathways of Carbohydrate Metabolism in Microorganisms.” Theexistence of the cycle in mammalian liver and in plant leaves was confirmed in experimentswith carbon-labeled ribose 5-phosphate in two papers published with Martin Gibbs of theBrookhaven National Laboratory, describing work carried out there during the summer of1953.

The Path of Carbon in PhotosynthesisWhen, in 1952, Calvin’s group at the University of California at Berkeley reported evidence

for ribulose 1,5-diphosphate as the CO2 acceptor for the formation of 3-phosphoglyceric acid,the first CO2 fixation product in photosynthesis, we were excited by the possibility that thepentose phosphate pathway might serve as the mechanism for regenerating this key inter-mediate from hexose monophosphates. Art Weissbach, a newly arrived postdoctoral studentfrom Columbia, and Polly Smyrniotis carried out the first experiments to identify the enzy-matic mechanisms involved. They were able to show that with crude extracts from spinachleaves ribose 5-phosphate was a unique substrate for the formation of phosphoglyceric acid,and they purified a kinase from spinach leaves that they used to prepare the barium salt ofribulose 1,5-bisphosphate (RUDP).

In the fall of 1954 we moved from Building 3 to new laboratories on the 9th floor of the NIHClinical Center, where, joined by Jerry Hurwitz, we isolated the enzyme phosphoribulokinase,responsible for that reaction, as well as the enzyme ribulose-bisphosphate carboxylase, whichcatalyzed the formation of 2 mol of phosphoglyceric acid from ribulose bisphosphate and CO2.Working in laboratories across the hall from each other, Art, Jerry, and I divided responsibil-ities. Jerry was charged with the purification of phosphoribulokinase, I took on the task ofpreparing pure ribulose 1,5-bisphosphate, and Art went after the most important enzyme, theribulose-bisphosphate carboxylase. The last effort deserves a special comment. Although theenzyme was purified only 10-fold from the crude spinach leaf extracts, by all the criteria thatwe could apply it appeared to be a pure protein, which meant that it constituted about 10% ofthe soluble protein in the spinach leaf. Later work in other laboratories around the worldconfirmed this finding, and this enzyme, now known as “Rubisco” for ribulose-bisphosphatecarboxylase/oxygenase is now considered to be the most abundant protein on earth.

Our work was published in three back-to-back papers in the February 1956 issue of theJournal of Biological Chemistry, entitled: “Spinach Phosphoribulokinase,” “The EnzymaticSynthesis and Properties of Ribulose 1,5-Diphosphate,” and “The Enzymatic Formation ofPhosphoglyceric from Ribulose Diphosphate and Carbon Dioxide.” With this work and ourearlier demonstration of the reversible reactions for the interconversion of pentose and hexose


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phosphates, with sedoheptulose phosphate as a prominent intermediate, all of the enzymes forthe reactions of the Calvin Cycle were identified.

ConclusionThe pentose phosphate pathway in animals, as discussed earlier, fulfills two important cell

requirements: 1) for ribose 5-phosphate for the synthesis of nucleotides and nucleic acids; and2) for reducing power in the form of NADPH. In photosynthesis, it functions to regenerate theprimary CO2 acceptor, ribulose bisphosphate, from the hexose phosphates produced. Chloro-plasts utilize radiant energy to produce ATP, required for the production of ribulose 1,5-bisphosphate from ribulose 5-phosphate and also for the reduction of 3-phosphoglyceric acid toglyceraldehyde 3-phosphate. The reducing agent for the latter reaction, NADPH, is alsogenerated by the action of light in the chloroplasts. In both animals and plants, NADP ratherthan NAD appears to function as the coenzyme for reductive synthesis.

CommentBecause these were personal “reflections” they have mainly described work from my labo-

ratory. Calvin and his co-workers provided the first clues leading to the development of thephotosynthetic cycle and also the conclusive evidence for its function as the path of carbon inintact photosynthesizing cells. These pioneering experiments, as well as important contribu-tions from many other laboratories, are cited in a review that I published in 1957 with WolfVishniac and Severo Ochoa in Advances in Enzymology (see last entry of the Bibliography).

Address correspondence to: [email protected].

BIBLIOGRAPHYHorecker, B. L., and Kornberg, A. (1948) The extinction coefficients of the reduced band of the pyridine nucleotides.

J. Biol. Chem. 175, 385–390Horecker, B. L., Smyrniotis, P. Z., and Seegmiller, J. E. (1951) The enzymatic conversion of 6-phosphogluconate to

ribulose 5-phosphate and ribose 5-phosphate. J. Biol. Chem. 193, 383–396Horecker, B. L., and Smyrniotis, P. Z. (1953) The coenzyme function of thiamine pyrophosphate in pentose phosphate

metabolism. J. Am. Chem. Soc. 75, 1009–1010Horecker, B. L., and Smyrniotis, P. Z. (1953) Transaldolase: the formation of fructose 6-phosphate from sedoheptulose

7-phosphate. J. Am. Chem. Soc. 75, 2021Horecker, B. L., Smyrniotis, P. Z., and Klenow, H. (1955) The formation of sedoheptulose phosphate from pentose

phosphate. J. Biol. Chem. 205, 661–682Weissbach, A., Smyrniotis, P. Z., and Horecker, B. L. (1954) Pentose phosphate and CO2 fixation with spinach

extracts. J. Am. Chem. Soc. 76, 3611Horecker, B. L., and Smyrniotis, P. Z. (1955) The purification and properties of yeast transaldolase. J. Biol. Chem. 212,

811–825Horecker, B. L., Smyrniotis, P. Z., Hiatt, H., and Marks, P. (1955) Tetrose phosphate and the formation of sedohep-

tulose diphosphate. J. Biol. Chem. 218, 827–836Hurwitz, J., Weissbach, H., Horecker, B. L., and Smyrniotis, P. Z. (1956) Spinach phosphoribulokinase. J. Biol. Chem.

218, 769–783Horecker, B. L., Hurwitz, J., and Horecker, B. L. (1956) The enzymatic synthesis and properties of ribulose 1,5-

diphosphate. J. Biol. Chem. 218, 785–794Weissbach, A., Horecker, B. L., and Hurwitz, J. (1956) The enzymatic formation of phosphoglyceric acid from ribulose

diphosphate and carbon dioxide. J. Biol. Chem. 218, 795–810Horecker, B. L., Smyrniotis, P. Z., and Hurwitz, J. (1956) The role of xylulose 5-phosphate in the transketolase

reaction. J. Biol. Chem. 223, 1009–1019Vishniac, W., Horecker, B. L., and Ochoa, S. (1957) Enzymatic aspects of photosynthesis. Adv. Enzymol. XIX, 1–77


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My Brief Encounter with the Phosphoinositides and IP3

Published, JBC Papers in Press, October 8, 2004, DOI 10.1074/jbc.X400010200Clinton E. BallouFrom the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

For my first independent research project after my appointment to the Berkeley faculty, Ichose to work on the structures of myoinositol-containing phospholipids, a study that led useventually to the discovery of D-myoinositol 1,4,5-trisphosphate or IP3. Before describing thisresearch, however, I should say how that choice came about. While in graduate school at theUniversity of Wisconsin, I had had the good fortune to study under Karl Paul Link, who waswidely renowned for his discovery of dicumarol and the synthesis of related blood anticoagu-lants such as warfarin, work that was recognized with two Lasker Awards (1). On the side,however, Link remained a carbohydrate chemist at heart, a hobby that had grown out of hisstudies on plant polysaccharides and uronic acids while a student and then a young facultymember. In fact, Stanford Moore had completed his doctoral dissertation with Link on amethod for characterizing aldo-monosaccharides as benzimidazole derivatives (2).

I arrived at Madison in the fall of 1946, fresh from a stint in the United States Navy, andI found Link’s laboratory bursting at the seams with about 15 ex-GIs, all hard at work tryingto make up for lost time. During earlier investigations on the structure-function relationshipof coumarin anticoagulants, an attempt to synthesize the glucoside of dicumarol had beenfrustrated because the acetylated intermediate was degraded in alkali under conditions usedfor deacetylation (3). Because glycosides are acetals, which are typically acid-labile andalkali-stable, I found the anomaly intriguing and decided to study a variety of syntheticcompounds in an effort to understand the structural basis for alkali sensitivity (4). Thisresearch formed the core of my doctoral dissertation, and although I failed to recognize it at thetime, the chance exposure to carbohydrate chemistry was to have a lasting influence on thedirection my career would take.

I continued my indoctrination in sugar chemistry during a postdoctoral year in Edinburgh,Scotland, with E. G. V. Percival in the new Department of Chemistry at Kings Buildingsheaded by Edwin Hirst. This was a time of economic depression in Britain, which was stillsuffering the aftermath of the war, and I discovered that I had left a well equipped laboratoryin Madison to engage an unexpectedly primitive research environment. Wisely I did not letthis change in fortunes discourage me. Instead I undertook a project dealing with the structureof maple sapwood starch and did the best that I could with the available facilities (5). Myefforts were well rewarded because, in the process, I became adept at the uses of analytical andpreparative filter paper and cellulose column chromatography, skills that were to be extremelyvaluable in my later research. The greatest challenge to my ingenuity, however, was toconstruct an electric stirring device from a small board-mounted motor, a couple of woodenpulleys, a piece of string, and a glass rod. The speed of the motor was regulated by adjustinglight bulbs that were wired in series with the power cord to draw off electricity, a crude buteffective method of control. I have always enjoyed working with my hands, so this mundaneproject even took on a certain appeal.

Living in a new environment always has its fringe benefits. While in Edinburgh, I developeda special affection for the Scots and a better understanding for the lingering resentment that

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 53, Issue of December 31, pp. 54975–54982, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.





reflects a long history of conflict within the British Isles. Thus, I could understand why one ofmy graduate student colleagues was proud to proclaim, at every opportunity, that he hadnever been south of the border! It was also during this year that some Scottish separatistssneaked into Westminster Abbey and made off with the Stone of Scone. This symbol of Scottishnationalism, which was taken from Scotland to England by Edward I, had long rested beneaththe chair on which British monarchs were crowned. The incident created quite a stir amongthe local patriots, but after its recovery the stone was returned to the Abbey. (I was recentlyinformed that the Stone of Scone has since been returned to Scotland.)

Although I enjoyed the time, when the year ended I was ready to move on to Berkeley, whereI had arranged to study with Hermann O. L. Fischer. Nicknamed “Hermannol,” probably byhis friend Claude Hudson as a play on the term “polyol,” Fischer was an expatriate Germanscientist who had experienced a turbulent career that eventually led him to the University ofToronto. Then, when the new Biochemistry and Virus Laboratory was set up in 1948, WendellStanley had recruited him to Berkeley. I was attracted to Fischer in part because of hisresearch on phosphorylated sugars but also because during graduate school I had drawnheavily on the published works of his father, Emil Fischer (6). I guess the idea of beingassociated with the son of Emil Fischer just seemed “real cool” to me. As it turned out, it alsoproved beneficial that I happened to go to Berkeley just as the University was entering aperiod of rapid postwar expansion.

This was a time of active research on biosynthetic pathways that involved short chainphosphorylated sugars, as exemplified by the studies of Melvin Calvin on photosynthesis, ofP. R. Srinivasan and David Sprinson on shikimic acid biosynthesis, and of Bernard Horeckeron transaldolase. With Fischer and his colleague, Donald MacDonald, I undertook the syn-theses of several such metabolic intermediates, including D-glyceric acid 2-phosphate, D-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid 3-phosphate,and D-erythrose 4-phosphate (7). The novelty of our approach was to prepare stable dimethylacetal derivatives of the inherently unstable phosphorylated compounds with aldehydo or ketogroups. These could be stored indefinitely and then be converted by mild acid hydrolysis ofthe acetal to the active metabolites as needed. Our success is documented by the fact thattoday, 50 years later, samples of the preparations have survived in pure crystalline usableform. During these first years in Berkeley, I also became interested in inositol chemistry asa result of studies on the cyclitols in sugar pine heartwood (8). Then, when Elvin Kabatcame to Berkeley from Columbia University to spend a sabbatical with Fischer and learnsome carbohydrate chemistry we all collaborated on the methylation analysis of galactinol,an �-D-galactoside of myoinositol. This study established that the galactose was linked tothe L-l-position on the inositol ring (9), a fact that I was to put to good use in my laterstudies.

After my appointment to the faculty in 1955, I was in a position to set up an independentprogram, and this background led me to undertake a project concerned with the character-

FIG. 1. Alkaline degradation of soybean monophosphoinositide (15). The products are D-myoinositol 1-phosphate(I-1-P) and myoinositol 2-phosphate (I-2-P). R is a fatty alkyl chain.

Reflections: Reflections on Phosphoinositides and IP3

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ization of inositol-containing phospholipids. In so doing, I was fortunate to have Finn Wold,Lewis Pizer, and Francis Lane Pizer as my first graduate students. At the time, there wasconvincing evidence from a number of studies that the lipid known as “phosphoinositide” wasa phosphatidylmyoinositol (10), and as expected for such a structure, acid or alkaline hydrol-ysis of the phosphodiester bond had yielded myoinositol phosphate as one of the degradationproducts (11–13). Because the chemical hydrolysis of phosphate diesters with neighboring freehydroxyl groups can lead to phosphate migration, however, the position of attachment of thephosphatidic acid unit to the myoinositol ring was uncertain. Important studies at CambridgeUniversity by Brown and Todd (14), showing that the alkaline hydrolysis of the phosphatediester linkage in nucleic acids proceeds via a cyclic phosphate intermediate, suggested to usa strategy to resolve this uncertainty. We subjected pure soybean phosphoinositide to alkalinehydrolysis and isolated the inositol phosphate fraction. It consisted mainly of myoinositol1-phosphate along with some myoinositol 2-phosphate and other minor products (15). Thisresult indicated that the putative myoinositol cyclic phosphate intermediate had involvedpositions 1 and 2 on the ring.

Because position 2 of the myoinositol ring lies between two adjacent cis-hydroxyls, called D-1and L-1, the phosphatidyl group in the lipid could have been attached to position 2 or to one ofthe adjacent enantiomeric 1-positions.1 The choice between these alternatives was suggestedby the fact that the myoinositol 1-phosphate we isolated was optically active, [�]D �9.8° (water,pH 2). This would be expected from the cyclization and reopening of a phosphate diester grouporiginally on the D-1- or L-1-position, because the intermediate cyclic phosphate would beasymmetric if the myoinositol in the starting diester were asymmetrically substituted (Fig. 1).This would not be the result if the original diester involved position 2, which has a plane ofsymmetry, unless the asymmetry of the glycerol portion were able to exert a directive influenceduring the reaction.

To complete the characterization, we carried out a definitive synthesis of L-myoinositoll-phosphate, starting from galactinol (9). For this synthesis, we perbenzylated galactinol,removed the benzylated galactose moiety by acidic methanolysis, and phosphorylated the freeL-1-position of the recovered penta-O-benzylmyoinositol. Deblocking of the product by hydrog-enolysis yielded L-myoinositol 1-phosphate (Fig. 2), which showed [�]D �9.3° (water, pH 2) (16,17). Because this synthetic L-isomer, which later was found to occur naturally (18), showeda rotation equal to that of the lipid-derived product, but of opposite sign, it must be theenantiomer; and consequently, the 1-phosphatidylmyoinositol (15) must have had the

1 For these assignments, the three adjacent cis-hydroxyls of myoinositol are numbered one to three, and thedirection of numbering is selected to give substituted positions the lowest possible number. When the ring isrepresented with these three hydroxyls projecting downward and the direction of numbering is clockwise, themyoinositol configuration is D and if counterclockwise it is L. Note that myoinositol 2-phosphate and 5-phosphate havea plane of symmetry and are meso compounds.

FIG. 2. Synthesis of L-myoinositol 1-phosphate (16, 17). The starting material for this synthesis was galactinol,1-O-�-D-galactopyranosyl-L-myoinositol (9).


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D-configuration.2 In an important parallel study, Brown et al. (19) degraded horse liverphosphoinositide by the periodate/phenylhydrazine procedure, which avoided the cyclic phos-phate intermediate, and they recovered a single myoinositol 1-phosphate with the same opticalactivity as the isomer we had obtained from the soybean lipid. Together, these studies firmlyestablished that the myoinositol ring was substituted on the D-1-position in phosphatidylmy-oinositol from both plants and animals. This was an important result, although it was notsurprising because most myoinositol derivatives show chirality.

At the time, I was aware of the important work of Jordi Folch at Harvard Medical School,who had isolated a complex phosphoinositide from beef brain (20, 21). This isolation was basedon the facts that phospholipids have low solubility in acetone, but they can be extracted froman acetone powder of brain tissue with chloroform, and the inositides can then be precipitatedselectively by adding ethanol or methanol. Because strong acid hydrolysis of the material hadyielded an “inositol metadiphosphate,” Folch concluded that the original lipid was a polypho-sphoinositide. Because it was known, however, that acid treatment could cause phosphategroups to migrate around the inositol ring (15), we decided to reexamine this characterization.A new graduate student from the University of Chile, Carmen Grado, had just joined mygroup, and I suggested that she should repeat the brain phosphoinositide preparation accord-ing to Folch. When Grado subjected this material to strong acid hydrolysis, she observed thatthe resulting inositol phosphate fraction gave a very diffuse unresolved streak on paperchromatography (22). She then repeated the study, using alkaline degradation of the brainlipid, and found that chromatography of the inositol phosphate fraction gave a well resolvedpattern of five components, one mono-, two bis-, and two trisphosphates of myoinositol. Thissuggested that the Folch brain inositide preparation was a mixture of related substances, andbecause the myoinositol trisphosphates predominated, we proposed that “the lipid might moreaccurately be called a triphosphoinositide” (22). At about the same time, Dittmer and Dawson,at Cambridge University, reported the isolation from ox brain of a lipid fraction with thecomposition expected for a triphosphoinositide (23).

Grado then went on to characterize each inositol polyphosphate in the mixture, using asequence of periodate oxidation to cleave the inositol ring between free glycol groups, borohy-dride reduction of the resulting dialdehyde, and dephosphorylation to yield a free polyalcohol.From an inositol bisphosphate, one could expect a tetritol if the phosphate groups were nextto each other. If they were in a 1,3-position, a pentitol would result; and if they were in a1,4-position, the ring would be cleaved in two places to give two molecules of malondialdehydephosphate, which would be oxidized further by excess periodate to yield inorganic phosphate,formate, and carbon dioxide. From an inositol trisphosphate with the phosphates adjacent toeach other, a pentitol would be formed; if in a 1,2,4 arrangement, a hexitol would result; andif in 1,3,5 arrangement, the inositol ring would survive the treatment. Besides indicating thephosphate positions, the identity and optical activity of the resulting polyol would also revealthe chirality of the inositol derivative. Using these methods, Grado characterized the twobisphosphates as D-myoinositol 1,4- and 4,5-bisphosphate and the major trisphosphate fractionas either the D-1,4,5-isomer or the D-1,4,6-isomer, the uncertainty arising because both of thesetrisphosphates would yield the same D-iditol in the above analytical procedure (22). The

2 The convention for assigning configurations to substituted myoinositols was changed during the 1970s, so thatwhat we designated L-myoinositol 1-phosphate in 1959 (16, 17) was later renamed D-myoinositol 1-phosphate. In thisarticle, I have assigned all configurations in agreement with the convention now used.

FIG. 3. Reactions used to characterize D-myoinositol 1,4,5-trisphosphate (22, 25). P is –PO3H2.


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uncertainty was resolved by Raymond Tomlinson, a graduate student who had made adetailed study of the dephosphorylation of phytic acid (myoinositol hexaphosphate) by wheatbran phytase (24). He observed that alkaline phosphomonoesterase selectively removed phos-phate groups flanked by unsubstituted hydroxyls, and he found that the myoinositol trisphos-phate isolated by Grado was converted to D-myoinositol 4,5-bisphosphate by this treatment(25). Thus, the complete characterization of D-myoinositol 1,4,5-trisphosphate can be summa-rized as shown in Fig. 3.

These studies still left open the question of the true nature of the apparently heterogeneousbrain phosphoinositide. From the composition of his preparation, Folch (21) had postulatedthat it could be represented as a meta-diphosphatidylmyoinositol, whereas Hawthorne (26)proposed a cyclic structure of myoinositol meta-diphosphate with monoacylglycerol. I wasfortunate at the time to be joined by a postdoctoral co-worker, Hans Brockerhoff, who hadstudied with Donald Hanahan at the University of Washington. To obtain the water-solublecomponent(s) of the brain lipid complex with intact phosphodiester linkages, Brockerhoffdeacylated the phosphoinositide preparation with hydroxylamine and separated the productson an ion exchange column (27). This yielded three fractions with compositions correspondingto glycerol myoinositol phosphate (20%), glycerol myoinositol diphosphate (22%), and glycerolmyoinositol triphosphate (58%) (Fig. 4). Further analysis suggested that these products couldbe derived from three lipids: l-phosphatidyl-D-myoinositol, 1-phosphatidyl-D-myoinositol 4-phos-phate, and l-phosphatidyl-D-myoinositol 4,5-bisphosphate. This conclusion was confirmed whenStewart Hendrickson, a postdoctoral fellow who had studied with Herbert Carter at the Univer-sity of Illinois, developed an ion exchange procedure using a homogeneous chloroform/methanol/water solvent. This solvent dissolved the intact brain phosphoinositide preparation and allowedits separation into three homologs (28), analysis of which agreed with Brockerhoff’s assignments(27). Hendrickson also found that the three lipids were closely related in that each was predom-inantly acylated by the same mixture of stearic, oleic, and arachidonic acids. Later, Brown andStewart (29) also characterized purified triphosphoinositide, using the selective degradationprocedure Brown and co-workers had exploited so effectively earlier (19).

During the 1960s when we were conducting the above studies, very little was known aboutthe cellular function(s) of the inositol phospholipids. Mabel and Lowell Hokin at the Universityof Wisconsin had investigated the possible role of these lipids in cellular secretion (30), andthey, along with others, had studied the incorporation of 32Pi into the brain lipids (31, 32).These studies had yielded only limited information owing, in part, to uncertainty about theactual structure of the brain inositide. From the insight we had gained by our structural work,it appeared to us likely that the three components would be interconvertible in cells by anenzyme-catalyzed process of cyclic phosphorylation-dephosphorylation. When Brockerhoff in-vestigated the incorporation of [32P]phosphate, [3H]myoinositol, and [14C]glycerol into theindividual inositides in brain tissue slices, the results proved to be consistent with such apathway (33, 34). Thus, the monoester phosphate groups turned over rapidly, whereas theglycerol, myoinositol, and phosphodiester groups were much more stable. Moreover, turnoverof the monoester phosphate groups was not random, because partial enzymic dephosphoryl-ation of 1-phosphatidylmyoinositol 4,5-bisphosphate to the next lower homolog occurred by theselective removal of the 5-phosphate group, indicative of a specific 5-phosphomonoesterase inbrain tissue (35).

I became eligible for a sabbatical leave in 1961, and because our work on the brainpolyphosphoinositides was going well, I asked Edgar Lederer if I could spend a year with himto study the glycophosphoinositides of mycobacteria. He welcomed me in his very gracious

FIG. 4. Products isolated from deacylated brain polyphosphoinositide (27). The intact lipids were found to beacylated by a mixture of stearic, oleic, and arachidonic acids (28).


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manner, and he even arranged the rental of a spacious apartment in Paris on rue Pierre Curie(later renamed rue Pierre et Marie Curie). Thus, I had only a short stroll to catch the Ligne deSceaux at Luxembourg Station for the daily ride to his CNRS laboratory at Gif-sur-Yvette.Myoinositol, as a lipid constituent, was first reported by R. J. Anderson to occur in the phospho-lipids of mycobacteria (36), and Lederer subsequently described a dimannosyl phosphoinositidefrom the same source (37). While at Gif, I collaborated with his colleague, Erna Vilkas, onexperiments to establish the linkages of both the phosphatidyl and the mannosyl groups to themyoinositol ring (38). In later investigations by Yuan Chuan Lee, a postdoctoral student from theUniversity of Iowa, we determined the structures of the family of mannosyl phosphoinositides inMycobacterium smegmatis (39, 40). Like the other phosphoinositides, the phosphatidyl group wasfound attached to the D-1-position of myoinositol, whereas a single mannose was linked to position2 and one to four mannoses were linked to the D-6-position (Fig. 5). This phospholipid was laterfound to serve as an anchor for the lipoarabinomannan in mycobacteria (41), and it is interestingthat the glycophosphoinositide protein anchor has the analogous structure in which a carbohy-drate chain is also attached to position 6 of myoinositol (42).

In a report to the International Congress of Biochemistry on the “Structure of MyoinositolPhospholipids” (43), I summarized the results of our studies and observed that: “In attemptingto assess the role of phospholipids in cellular metabolism, one can place primary emphasis onthe lipid end of the molecule and its modification according to the type of fatty acid thereesterified. Or, one can direct attention to the hydrophilic end. In the case of the inositolphospholipids, we find, in the great structural variability of the inositol part, evidence thatherein may lie the prime functional center of these molecules.” I have never considered myselfa clairvoyant, and as it turned out, both the polar and nonpolar ends of the inositides haveimportant regulatory functions. Today, we can look back and see that our earlier studies weresignificant mainly in helping to prepare the groundwork for the explosive developmentsconcerning the cellular functions of the phosphoinositides that followed upon the importantdiscoveries described in the review by Berridge and Irvine (44).

In these Reflections, I have limited myself to that early period of the 1960s in which I wasdirectly involved, and I have referred only peripherally to the many subsequent importantdevelopments. I can’t avoid reference, however, to the role that has been discovered for1-phosphatidylmyoinositol 3-phosphate and its derivatives (45), substances we never encoun-tered in our investigations. I should also admit that I am a little disappointed that I neverencouraged my co-workers to pursue a detailed study of the enzymes involved in the metab-olism of the polyphosphoinositides. My only excuse is that we were drawn in other directionsby our discovery of the mycobacterial polymethylpolysaccharides (46, 47), which were foundlater to act as regulators of fatty acid synthesis in this microorganism (48), and to investiga-tions on the genetic control of yeast mannoprotein structure (49). Both of these developmentsare traceable to the sabbatical leave I spent at Gif in 1961, a testament to the unpredictableinfluence such an experience can have. Despite the minor doubt expressed above, I must saythat I enjoyed a wonderful ride with the phosphoinositides, and it was all great fun! I amespecially grateful to the many co-workers who shared this journey with me.


FIG. 5. Structures of mycobacterial mannophosphoinositides (40). M is �-D-mannopyranosyl.


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methanol. II. The identification of seven aldo-monosaccharides as benzimidazole derivatives. J. Biol. Chem.133, 293–311

3. Huebner, C. F., Karjala, S. A., Sullivan, W. R., and Link, K. P. (1944) Studies on the hemorrhagic sweet cloverdisease. VI. Glucosides of 4-hydroxycoumarins. J. Am. Chem. Soc. 66, 906–909

4. Ballou, C. E. (1954) Alkali-sensitive glycosides. Adv. Carbohydr. Chem. 9, 59–955. Ballou, C. E., and Percival, E. G. V. (1952) Wood starches: the structure of the sapwood starch of the maple (Acer

spp.). J. Chem. Soc. 1054–10566. Fischer, E., and Beensch, L. (1894) Uber einige synthetische glucoside. Ber. Dtsch. Chem. Ges. 27, 2478–24867. Ballou, C. E., Fischer, H. O. L., and MacDonald, D. L. (1955) The synthesis and properties of D-erythrose

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H86

Lectins: Carbohydrate-specificReagents and BiologicalRecognition Molecules

PUBLISHED, JBC PAPERS IN PRESS, DECEMBER 4, 2006, DOI 10.1074/JBC.X600004200

Nathan Sharon

From the Department of Biological Chemistry, The Weizmann Institute of Science,

Rehovot 76100, Israel

The occurrence in nature of proteins with hemagglutinating activity that in later years

were shown to be sugar-specific and eventually named lectins has been known since

the turn of the 19th century, but until about two decades ago they aroused little interest

(for a historical survey, see Ref. 1). My own involvement with these proteins began

inadvertently and initially on a part-time basis in the early 1960s after my return to theWeizmann

Institute from two and a half years of exciting and educational postdoctoral studies in the United

States. During the first of these I worked in the laboratory of Fritz Lipmann at the Massachusetts

General Hospital, Boston. Lipmann, one of the most influential biochemists of the last century,

was then interested in the mechanism of protein biosynthesis. I was assigned to study the amino

acid activation reaction (the first step in this process), work that resulted in two publications (2, 3).

Concurrently, I greatly enriched my knowledge of biochemistry, mainly from my fellow postdoc-

toral students, and especially from the guest seminars in which ongoing biochemical discoveries

and developments were reported. I spent the second postdoctoral year at the Massachusetts

General Hospital with Roger Jeanloz, a leading carbohydrate chemist, where I got my training in

the subject and also succeeded in isolating an unusual diamino sugar from a Bacillus polysaccha-

ride I had brought with me from Rehovot (4) (see below); the remaining time I worked with Dan

Koshland at Brookhaven National Laboratory on the mechanism of action of myosin ATPase (5,

6). Dan was then starting to make his mark on enzymology with his “induced fit” concept of

enzyme action, originally greeted with much skepticism (7).

Back at Rehovot my original aim was to establish the structure of that diamino sugar; I was

fortunate to receive for this purpose my first National Institutes of Health (NIH) grant, a modest

one of some $25,000 for 3 years. (This would have been unheard of at the present time because

nothing was known then about the function of the compound.) The task tookme (with a couple of

graduate students) over a decade; eventually we were able to prove by degradation and synthesis

that the compound in question, which we named bacillosamine, is 2,4-diamino-2,4,6-trideoxy-D-

glucose (8, 9). To my delight, the di-N-acetyl derivative of bacillosamine has recently been found

attached glycosidically to the amide of asparagine or the hydroxyl of serine in the carbohydrate-

peptide linkage region of several interesting glycoproteins of pathogenic bacteria (10). By a strange

twist of fate, most of these glycoproteins were originally isolated in 2002 byMartin Young and his

colleagues at the National Research Laboratories, Ottawa, from Campylobacter jejuni by affinity

chromatography on immobilized soybean agglutinin (SBA) (11), the first lectin I got involved with

40 years earlier.

From Soy Proteins for Nutrition to Glycoproteins and Lectins

My studies of SBA began together with Halina Lis with whom it has been my good fortune to

collaborate to this very day. It aroused our curiosity not because of its ability to bind sugars

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 5, pp. 2753–2764, February 2, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

FEBRUARY 2, 2007 • VOLUME 282 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY

REFLECTIONS This paper is available online at www.jbc.org

H87

specifically and reversibly and to agglutinate cells, the hall-

marks of proteins of this class, but because of other rea-

sons that I shall presently mention. We did not have the

slightest idea that lectins would become extremely useful

carbohydrate-specific reagents, that they would be found

to function as mediators of cell recognition, or that they

would make a major contribution to glycobiology (12). In

fact, for a time we were not even aware of the term lectin,

which was originally proposed in 1956 byWilliamC. Boyd

from Boston University for blood type-specific hemagglu-

tinins. Because SBA, like the majority of the hemaggluti-

nins, is not blood group-specific, we began referring to it as

a lectin only in 1970, when it occurred to us that the orig-

inal definition should be broadened to include all cell-ag-

glutinating and sugar-specific proteins (13).

Our interest in SBA developed in the course of investi-

gations on soybean proteins carried out within the frame-

work of a generous and long term grant from the United

States Department of Agriculture that I received in 1961

jointly with Katchalski-Katzir (14). Katchalski was the

founding Head of the Department of Biophysics at the

fledglingWeizmann Institute, whichwas officially inaugu-

rated in 1949. I came to the department in 1954 after hav-

ing receivedmy Ph.D. degree from theHebrewUniversity,

Jerusalem; Halina, with a Ph.D. degree from Uppsala Uni-

versity, joined the department 5 years later. The purpose

of the above grant was to carry out a fundamental study of

the soy proteins with the aim of providing information for

their improved utilization for human nutrition. Katchalski

and I were persuaded to embark on this project by Tim

(M. L.) Anson and Aaron Altschul, close friends, noted

protein chemists, and enthusiastic believers in these pro-

teins as the best solution to world hunger. After some

time, Katchalski became immersed in his pioneering stud-

ies of polyamino acids as protein models and on enzyme

immobilization and turned over the whole project to me,

for which I am extremely grateful.

Halina and I set out by trying to obtain pure proteins

from soybeans by chromatographic techniques, but this

proved to be a difficult task as most of them lack biological

activity, are poorly soluble, and undergo complex associa-

tion-dissociation reactions. We therefore chose to focus

on SBA, originally isolated and characterized in the 1950s

by Irvin E. Liener at the University of Minnesota, St. Paul.

The main reason for our choice was the evidence pre-

sented by Liener that it contained glucosamine, raising the

likelihood that it may be a glycoprotein (15). In those days,

research on glycoproteins was in its infancy, but I became

intrigued by these compounds because of my interest in

carbohydrates, as described elsewhere (16).

Soybean Agglutinin, a Plant Glycoprotein

Working on SBA, Halina and I soon found that it con-

tains not only glucosamine but also mannose. We then

isolated from a proteolytic digest of SBA an asparaginyl-

oligosaccharide that contained all the N-acetylglucosa-

mine and mannose of the lectin (17). Eventually we also

isolated from the lectin N-acetylglucosaminylasparagine

(18), the carbohydrate-peptide linking group, that was

identical with the one originally obtained in 1963 byAlbert

Neuberger, the founding father of modern glycoprotein

research, in his pioneering studies of ovalbumin.

As pointed out recently by Liener (19), “The fact that

SBA was shown to be a glycoprotein may not be particu-

larly surprising to the modern day biochemists, but at the

time the finding of a sugar moiety in a plant protein was

accepted with reservation. It was thought that glycopro-

teins were strictly of animal origin and that the finding of a

sugar with a plant protein was most likely because of non-

covalent contamination.”

In 1981, jointly with Hans (J. F. G.) Vliegenthart from

the University of Utrecht, the complete structure of the

carbohydrate of SBA was established by NMR as the

branched oligomannoside Man9(GlcNAc)2, found in ani-

mal glycoproteins too, demonstrating that protein N-gly-

cosylation is a process conserved in plants and animals

(20). A unique feature of SBA is that all its molecules carry

the same oligosaccharide (21) in contrast to essentially all

other glycoproteins, which bear a variety of glycans at each

attachment site, i.e. consist of mixtures of distinct glyco-

forms. SBA serves therefore as an excellent source of this

oligosaccharide (for an example, see Ref. 22).

Emerging from Obscurity

Our few 1960s publications on SBA attracted little

attention, and we sometimes felt like wanderers in a

desert. Although the studies of lectins were in their eighth

decade and several hundreds of these proteins (almost all

from plants) had already been identified, the handful of

other scientists active in the field at the time did not fare

better. Irwin J. Goldstein from the University of Michigan

at Ann Arbor, still a leading lectin researcher, tells that

when he sent a note in 1963 to Biochemical and Biophys-

ical Research Communications describing the purification

of concanavalin A by affinity chromatography, it was

rejected forthright because “this represents a modest

advance in an obscure area.” The note was eventually pub-

lished in the Biochemical Journal (23), and affinity chro-

matography soon became the method of choice for lectin

isolation.

REFLECTIONS: Lectins

JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 5 • FEBRUARY 2, 2007H88

However, as the 1960s were folding, the attitude toward

lectins began to change, and a number of leading biochem-

ists and immunologists, among them Gerald Edelman at

Rockefeller University,MelGreaves at LondonUniversity,

Elvin Kabat at Columbia University, Jerker Porath at Upp-

sala, and Jon Singer at University of California, San Diego,

became involvedwith them.The reasons for this change in

attitude were summarized by Kabat, who had become

intrigued with lectins primarily because their combining

sites seemed similar to those of antibodies andwho in 1977

stated: “During the past 10 years there has been an extraor-

dinary burst of activity in the study of plant and animal

lectins, stimulated largely by the findings that they have

specific receptor sites for carbohydrates and react with

glycoproteins in solution or on cell membranes . . . ” (24).

In 1970, affinity chromatography of glycoproteins on

immobilized lectins was introduced (among others) by

Donnely and Goldstein (25). It became a must at one step

or another for the isolation of membrane proteins, all of

which are glycosylated, a classical case being that of the

insulin receptor with the aid of wheat germ agglutinin

(WGA) (26). Lectins proved also to be useful for the sep-

aration of purified glycoproteins into their glycoforms, i.e.

differently glycosylated forms of the same protein. A very

recent telling example is of different glycoforms of IgG

with different degrees of sialylation, obtained by fraction-

ation on the sialic acid-specific Sambucus nigra agglutinin

and shown to differ in their anti-inflammatory activity

(27).

Tools for Study of Membranes and Cells

Interest in lectins intensified with the realization that

they are extremely valuable reagents for the investigation

of cell surface sugars, for the assessment of the role of the

latter in cell growth and differentiation, in interactions of

cells with their environment, and also in a variety of path-

ological processes. In this connection it is instructive to

refer to two classical studies with lectins that provided

very early evidence for the presence of sugars on cell sur-

faces and their potential role as cell identity markers, a

common theme in modern glycobiology. One came from

the laboratory of James Sumner at Cornell University,

Ithaca, who in 1919 isolated concanavalin A in crystalline

form but only in 1936, together withHowell, reported that

it agglutinates cells such as erythrocytes and yeasts and

that this agglutination is inhibited by sucrose, thus dem-

onstrating for the first time the sugar specificity of lectins

(28). Moreover, with much foresight they suggested that

the hemagglutination induced by the lectin might be a

consequence of its reactionwith carbohydrates on the sur-

face of the red cells. The other study was by Walter Mor-

gan andWinifredWatkins at the Lister Institute, London,

who in the early 1950s used blood type-specific hemagglu-

tinins to show that the blood type A immunodeterminant

is �-linked N-acetylgalactosamine and that the H(O)

determinant is �-L-fucose (reviewed in Ref. 29). This was

the first demonstration that cell surface carbohydrates can

serve as carriers of biological information.

Much excitement was created in the following decade

by the reports of Joseph C. Aub from the Massachusetts

General Hospital (30) and Max Burger from Princeton

University (31), who were both working with WGA (spe-

cific for N-acetylglucosamine and N-acetylneuraminic

acid), and of Leo Sachs with Michael Inbar from the

Department of Genetics of our Institute, who used con-

canavalin A (specific for mannose and glucose) (32), that

these lectins agglutinated malignantly transformed cells

but not their normal parental cells. The reports provided

compelling evidence that cancer might be associated with

a change in cell surface sugars, an idea that only a few years

before had been considered completely unfounded. In col-

laboration with Leo Sachs and Ben-Ami Sela, we found

soon thereafter that SBA (specific for galactose and

N-acetylgalactosamine) also possesses the remarkable

ability to distinguish between normal and malignant cells

(33). Numerous subsequent studies have demonstrated

that high susceptibility to agglutination by lectins is a

property shared by many, albeit not all, malignant cells.

Several basic features of membranes were revealed, or

their existence confirmed, with the aid of lectins. Thus,

using ferritin-conjugated concanavalin A and ricin as an

electron microscopic probe, Garth Nicolson and Jon

Singer at University of California, San Diego, found that

the lectin derivatives bind specifically to the outer surface

of the human and rabbit erythrocyte membrane and con-

cluded that the oligosaccharides of the plasma membrane

of eukaryotic cells are asymmetrically distributed (34).

Further support for such distribution was obtained by

Vincent Marchesi at Yale University, who used ferritin-

labeled phytohemagglutinin (PHA) and showed that gly-

cophorin, themajor sialoglycoprotein of the human eryth-

rocyte membrane, is oriented so that its carbohydrate-

carrying segment is exposed to the external medium,

whereas the other segments of the same molecule are

embedded in the lipid bilayer or protrude into the cyto-

plasm (35).

Other ultrastructural studies with lectins provided

some of themost convincing evidence for the fluidmosaic

membrane model of Singer and Nicolson, according to

which the membrane consists of proteins and glycopro-

teins floating in a lipid bilayer (reviewed in Ref. 36). Prom-


FEBRUARY 2, 2007 • VOLUME 282 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRYH89

inent among these was the finding of the lectin-induced

clustering and patching of the corresponding membrane

receptors on lymphocytes and other kinds of cell, as illus-

trated for example by the treatment with fluorescein-la-

beled concanavalin A of rat or mouse lymphocytes (37).

Reorganization of cell surface carbohydrates was later

shown to be required for various activities of lectins on

cells such as mitogenic stimulation and induction of

apoptosis.

The toxicity for animals of certain plant lectins has been

recognized since the earliest days of lectin research, at the

end of the 19th century. However, research on the toxic

action of lectins on cells started only many decades later

with special attention being paid to mammalian cell lines

(e.g. CHO and BHK) resistant to different lectins, primar-

ily the highly toxic ricin and the less toxic PHA andWGA

(reviewed in Ref. 38). Leading the field was one cell phe-

notype independently isolated in 1974 by Stuart Kornfeld

at Washington University, Colin Hughes at the National

Institute for Medical Research, Mill Hill, London, and

Pamela Stanley at Toronto University, Canada. This phe-

notype lackedGlcNAc transferase I, the key enzyme in the

biosynthesis of complex and hybrid N-linked carbohy-

drate units of glycoproteins. Soon thereafter many other

lectin-resistant cell lines with different enzymatic glycosy-

lation defects became available. They proved extremely

valuable for the investigation of the biosynthesis of glyco-

proteins and glycolipids and of the function of their car-

bohydrates, especially those expressed on the cell surface.

Currently they also serve for the large-scale production

of pharmacologically useful glycoproteins such as

erythropoietin.

Review Articles

In the fall of 1970 I arrived at the Department of Bio-

chemistry, University of California at Berkeley, for a sab-

batical year as Visiting Professor.My host was Clint Ballou

with whom I discussed at length the possibility of using

lectins to examine the ideas on the roles of carbohydrates

as information and recognition molecules. Such ideas had

been entertained by Saul Roseman from Johns Hopkins

University (39) and Victor Ginsburg at the NIH (40).

Although there existed a few books and several reviews on

lectins, none of them dealt with their molecular properties

nor did they indicate their enormous potential as tools for

biological research. Because Dan Koshland from the same

department at Berkeleywas then amember of the editorial

board of Science, I approached him with the suggestion

that I write a review on lectins for that journal. This sug-

gestionwas readily accepted by PhilipAbelson, then editor

of Science.

Writing was started by me in the fall of that year in the

laboratory of Albert Neuberger at St. Mary’s Hospital in

London, where I arrived for a few months to study

lysozymes, on which Neuberger and I were at that time

working. However, I ended up purifying WGA by ion

exchange chromatography from commercial wheat germ

together with Tony (A. K.) Allen, separating it into three

isolectins and showing that its specificity is similar to that

of lysozyme (41), because it too exhibited a pronounced

affinity not only for oligosaccharides derived from chitin,

as originally demonstrated by Burger and Goldberg (31),

but also of peptidoglycan. In addition we also proved that,

contrary to suggestions in the literature, WGA was not a

glycoprotein. This work further stimulated the interest of

Neuberger in lectins with which he continued to be

engaged for several years into his eighties.

The Science review was completed jointly with Halina

upon my return to Rehovot early in 1972 (13). It summa-

rized the history of the research on lectins since their dis-

covery, their specificity for monosaccharides and cells,

and the properties of concanavalin A and the few other

lectins that had been purified at the time. The changes that

occur on cell surfaces upon malignant transformation, as

revealed by lectins, were discussed, although their signifi-

cance was not clear and doubts were raised by us, amply

supported later, as to whether they are a distinctive char-

acteristic of malignant cells. Regardless of this, we con-

cluded that lectins, both native and modified, provide a

new and useful tool for the study of the chemical architec-

ture of cell surfaces. Finally, we dealt in brief with the spec-

ulations on the role of lectins in nature, about which noth-

ing was known with certainty. Another review on lectins

was published by us in the following year in the Annual

Review of Biochemistry (42) and a third appeared in the

same series in 1986 (43). In these reviews we tried to con-

vey to the readers our fascination and enthusiasm for the

subject.

In 1989 we prepared a monograph on lectins and in

2003 a second edition of the same (44), both of which have

been translated into Japanese. Some 20 years ago I co-

edited a treatise on lectins to which Halina and I contrib-

uted several chapters (45). A related activity of mine was

the publication in 1975 of a book entitled “Complex Car-

bohydrates” in which lectins are featured and where I

expressed my firm belief “that the specificity of many nat-

ural polymers is written in terms of sugar residues, not of

amino acids or nucleotides” (46). The book was based on

notes that I prepared for the graduate students taking my

course on the same subject and remained in use for a long



time. I still continue teaching this course, now under the

title “Molecular and Cellular Glycobiology.”

Structural Diversity of Lectins

The 1970s witnessed the intensification of the study of

the molecular properties of individual lectins, a prerequi-

site for a deep understanding of their activities at the

molecular level. In 1972 concanavalinAbecame the first of

these proteins for which the primary and three-dimen-

sional structures have been established, the latter by x-ray

crystallography. This was thanks to the efforts of Gerald

Edelman’s group at the Rockefeller University (47) and of

the efforts of Karl Hardman and Clinton F. Ainsworth

at Argonne National Laboratories (48). The fold first

observed in this structure, an elaborate arrangement of

extended beta strands into two sheets, became known as

the jelly roll or lectin fold (126). The publication of the

concanavalin A structure was soon followed by the deter-

mination by Christine Schubert Wright at Virginia Com-

monwealth University of the three-dimensional structure

of WGA as well as of its complexes with ligands even

before the complete amino acid sequence of this lectin had

become available (49). It is worth noting that at present the

structures of close to 100 lectins have been solved, almost

all also in complexes with ligands.

In my laboratory, we continued to be occupied with

SBA,mainlywithReubenLotan, a talented andhardwork-

ing graduate student. Among others we demonstrated

that lectin is a tetramer made up of four nearly identical

subunits (50) (all legume lectins consist of two or four

subunits) and that the carbohydrate of SBA is not essential

for its biological activity (51). Final proof for the latter

conclusion came when we obtained the carbohydrate-free

SBA in a bacterial expression system in a fully active form

(52); still, why SBA, like most lectins, is glycosylated

remains an enigma.

Analysis of the amino acid composition of SBA showed

that it is similar to that of other legume lectins, the com-

position of which was known at the time. In particular, it

was devoid of sulfur-containing amino acids, in striking

contrast to WGA that is rich in such residues. We have

therefore proposed that although “lectins have many bio-

logical properties in common, they represent a diversified

group of proteins with respect to size, composition, and

structure,” which is indeed the case (53).

The primary sequence of SBA was determined in the

early 1980s at the Rockefeller University by conventional

methods (reviewed in Ref. 54). Although homologouswith

the sequences of the other two legume lectins known at

the time (from lentil and fava bean), homology with con-

canavalin A (also a leguminous lectin) could only be

obtained by circular permutation of the latter. This means

by aligning residue 119 of concanavalin A with the amino-

terminal residue of the SBA, proceeding to the carboxyl

end of concanavalin A (residue 237), and continuing with

its amino-terminal region along the sequence of SBA. This

kind of circular homology, never observed before, was

shown by Diana Bowles and her co-workers at York Uni-

versity, United Kingdom, to be the result of a unique rear-

rangement of the peptide chain that occurs in the last step

of the biosynthesis of concanavalin A (55).

Crystals of SBA suitable for x-ray diffraction studies

were obtained by Boaz Shaanan and colleagues from our

Department of Structural Chemistry in 1984 (56), but the

high resolution structure of the lectin, in complex with a

ligand, was solved only a decade later by James Sacchettini

and co-workers at Albert Einstein College of Medicine,

New York (57).

Biological Activities and Functions of Plant Lectins

In 1960 Peter Nowell at the University of Pennsylvania

discovered that PHA, the lectin of the red kidney bean, acts

as amitogen for lymphocytes, namely that it has the ability

to stimulate these cells to grow and divide (58). This find-

ing shattered the belief, held until then, that lymphocytes

are dead-end cells that could neither divide nor differen-

tiate further.Within a short time several other lectinswere

proven to be mitogenic. Of special significance was the

finding, first reported byWerner G. Jaffe at the University

of Venezuela, Caracas, that concanavalin A acts as a mito-

gen (59) because its binding to the lymphocytes could be

inhibited and reversed by low concentrations of mannose,

in contrast to PHA for which no effective inhibitor was

available at the time. It was thus concluded that mitogenic

stimulation is the result of binding of lectins to cell surface

sugars, providing another early demonstration of a biolog-

ical function of the latter compounds. Not all lectins that

bind to cells are, however, mitogenic, indicating that

attachment to selected carbohydrates is required for cell

stimulation.

In subsequent years there was an explosive growth in

the use of mitogenic lectins in biological research. Until

the advent of monoclonal antibodies they served as a pop-

ular tool in attempts to clarify the mechanism of signal

transmission through the cell membrane and of cell acti-

vation. Mitogen-stimulated lymphocytes were found,

among others, to produce many growth factors collec-

tively known as lymphokines or cytokines, the first of

which was discovered by Robert C. Gallo and co-workers

at the National Institutes of Health, Bethesda, as T-cell

growth factor in 1976 and later named interleukin-2 (60).

When the immune system is malfunctioning, the mito-



genic response of lymphocytes is defective. Stimulation by

PHA, and to some extent also concanavalin A and

pokeweed mitogen, is routinely employed in clinical labo-

ratories as a simple means to assess the immunocompe-

tence of patients suffering from different diseases and to

monitor the effects of various immunosuppressive and

immunotherapeutic treatments.

Studies with mitogenic lectins have provided informa-

tion on the cell surface sugars and other factors involved in

cell activation. Thus, using SBA, Abraham Novogrodsky

and Ephraim Katchalski found that the lectin stimulated

mouse lymphocytes only after the cells had been treated

with sialidase, which unmasked the subterminal galactose

and N-acetylgalactosamine residues of the surface glyco-

proteins and glycolipids to which the lectin bound (61),

and we have subsequently demonstrated that peanut

agglutinin (PNA, see below) exhibits the same property

(62). We then found that SBA was mitogenic only in poly-

merized form (63). It was an early demonstration of the

requirement of receptor cross-linking for cell activation.

The latter findings were published in the European Jour-

nal of Immunology over the objection of one of the referees

in whose opinion papers on lectins had no place in an

immunological journal. Luckily, this view was then not

generally held; indeed, at the same time Michael Sela

invited Halina and me to contribute a chapter on lectins

forThe Antigens, a treatise he was editing (64). A few years

later I wrote a review for Advances in Immunology on the

application of lectins for lymphocyte identification and

separation (65). At present lectins form an integral part of

immunology because of their pivotal role in innate immu-

nity (see later).

Because lectins do not stimulate plant cells, it is

highly unlikely that they have been selected by evolu-

tion for this purpose. Based on findings inmy laboratory

that SBA (as well as PNA) inhibits the sporulation and

growth of fungi such as Trichoderma viride, Penicilium

notatum, and Aspergillus niger, we raised the possibility

that lectins may protect plants against pathogenic

microorganisms (66). Work in other laboratories has

subsequently extended this proposal to include the

defense of plants against predatory animals and phyto-

pathogens (reviewed in Ref. 67). According to another

suggestion, plant lectins may be responsible for the spe-

cific association between leguminous plants and nitro-

gen-fixing rhizobia that provide the plants with the

needed nitrogen (for review, see Ref. 68). However, this

suggestion can account for the role of lectins in only one

plant family, the Leguminosae.

For Cell Separation and Bone Marrow

Transplantation

Toward the end of his doctoral research, Reuben Lotan,

withYehudaMarikovsky andDavidDanon fromour Insti-

tute, purified PNA by affinity chromatography (69). The

lectin was also purified at the same time by Toshiaki

Osawa from Tokyo University, friend and pioneer lectin

researcher (70). The detailed specificity of PNA was then

established in collaboration with Miercio Pereira and

Elvin Kabat at Columbia University, confirming among

others that the lectin has a high affinity for Gal�3GalNAc

(known also as T antigen), a characteristic glycan ofO-gly-

coproteins (71).

Just as PNA had become available in my laboratory, it

was my good fortune that Yair Reisner joined me as a

doctoral student. Bright and imaginative, he set his mind

to find out whether lectins can serve as markers for lym-

phocyte subpopulations. He soon found that immature,

cortical mouse thymocytes were agglutinated by PNA but

that themature,medullar cells were not. This served as the

basis for the development by him of a facile method

(sometimes referred to by us as “poor man’s cell sorter”)

for separation, by selective agglutination with PNA, of the

two thymocyte subpopulations in good yield and with full

viability. A manuscript we sent early in 1976 to Nature

magazine describing themethodwas promptly rejected by

the editor on the grounds that it was not of general inter-

est. Time proved Nature wrong because soon after it was

published in Cellular Immunology (72) the method

became very popular. This was primarily because it

afforded access to the immature thymocyte subpopulation

needed for the investigation of T lymphocyte maturation

and because it became “a classic technique for defining the

cortical and medullar regions of the thymus” (73). In our

study, we have found a marked difference in the level of

PNA binding to the thymocyte subpopulations, which is

abolished upon treatment of the mature subpopulation

with sialidase.We have therefore proposed that sialylation

of the PNA receptor may be an important step in the mat-

uration of the thymic cells. Nearly 20 years later evidence

was indeed presented by Linda Baum and her co-workers

from UCLA and UCSD that regulated expression of a sin-

gle glycosyltransferase, a Gal�3GalNAc �2,3-sialyltrans-

ferase, can account for this glycosylation change (73). This

enzyme sialylates Gal�3GalNAc, the preferred ligand of

PNA, forming the sequence NeuAc�2,3Gal�3GalNAc

and thusmasking the PNA-binding sites; expression of the

enzyme is inversely proportional to that of the PNA recep-

tor. The PNA receptor continues to serve as a differentia-

tion marker for lymphocytes (74). It has also been



employed as a similar marker in other systems, for exam-

ple in embryonal carcinoma cells ofmice, as shown byYair

in a joint study with Francois Jacob and Gabriel Gachelin

of the Pasteur Institute, Paris (75).

What proved to be highly significant was Yair’s demon-

stration, together with Asher Meshorer and Lea Itzico-

vitch from our Experimental Animal Center, that sequen-

tial agglutination of mouse bonemarrow or spleen cells by

SBA and PNA afforded a cell fraction suitable for trans-

plantation across histocompatibility barriers. In the paper

reporting on these results, we stated that the same

approach “may prove useful for bone marrow transplan-

tation in humans” (76). For his postdoctoral research, Yair

joined Robert A. Good, then President of the Sloan Ket-

tering Institute, New York, and Richard O’Reilly, Chief of

Bone Marrow Transplantation at that Institute, with the

express aim of adapting the lectin separation method to

humans. By 1981 he had found that treatment of human

bonemarrowwith SBA alone removed the bulk of the cells

responsible for the lethal graft-versus-host disease and

that, after additional processing, such bone marrow, even

from haploidentical donors, could be safely transplanted

into children born with severe combined immune defi-

ciency (SCIDs or “bubble children”) (77). It is a matter of

great pride and satisfaction to Yair, and to me, that over

75% of the hundreds of “bubble children” who received

since then transplants of bone marrow that had been

purged with SBA have been cured and lead a normal life.

For several years SBA-purged bone marrow was also used

on an experimental basis for treatment of end stage leuke-

mia patients (78).

Legume Lectins, a Large Family of Homologous

Proteins

I owe the last turning point in my research on plant

lectins to Jose Luis Iglesias, a youngmedical student at the

University of Montevideo, Uruguay, who had become fas-

cinated by these proteins and found one in the seeds of

Erythrina cristagalli, an ornamental leguminous tree com-

mon in Uruguay. Because of a lack of facilities in his coun-

try he was unable to isolate the lectin so he came to my

laboratory for a short stay early in 1981, bringing with him

6 kg of the flour of E. cristagalli seeds. In no time he had

ascertained that his lectin, which we had designated ECL,

is galactose-specific and purified it by affinity chromatog-

raphy on the immobilized sugar (79). He also demon-

strated that ECL is a glycoprotein containing fucose and

xylose in addition to mannose and N-acetylglucosamine

present in SBA. The structure of the carbohydrate of ECL

was established in the laboratory of Raymond Dwek at

Oxford University, United Kingdom, as the branched

Asn-linked heptasaccharide Man�3(Man�6)(Xyl�2)-

Man�4GlcNAc�4(Fuc�3)GlcNAc (80). It was one of the

first examples of this plant-specific oligosaccharide

reported in the literature. The oligosaccharide is allergenic

for humans and presents a major obstacle that needs to be

overcome before plants can be employed for the produc-

tion of pharmacologically useful glycoproteins such as

monoclonal antibodies.

When we exhausted the supply of E. cristagalli seed

flour that Jose had brought with him from Uruguay, I

turned my attention to Erythrina corallodendron, the

coral tree that grows commonly in Israel, the seed lectin of

which (ECorL) was originally isolated in 1980 byNechama

Gilboa-Garber at Bar Ilan University, Ramat Gan (81).

Working on ECorL proved to be highly rewarding. Its pri-

mary sequence was established by Rivka Adar using con-

ventional methods and by her (jointly with Rafael Arango,

a graduate student fromMedeillin, Colombia, and Shmuel

Rozenblatt from Tel Aviv University) by recombinant

techniques (82). The sequence was homologous to that of

other legume lectins, providing further evidence for the

proposal that I made in 1977 with Donny Strosberg, then

at the Free University of Brussels, that despite their dis-

tinct sugar specificities, legume lectins are members of a

single protein family and that the genes coding for them

have a common ancestry (83). According to a recent

count, 210 sequences of legume lectins are known, all

homologous (84). In the 1980s sequence similarities were

found for lectins from unrelated taxonomic families,

including lectins from animal sources, starting with the

galectins and C-type lectins (85).

Identical Tertiary Structures, Different Quaternary

Structures

In collaboration with Boaz Shaanan, then at the Weiz-

mann Institute, the three-dimensional structure of ECorL

in complex with lactose was established by high resolution

x-ray crystallography (86). Although the tertiary structure

observed was superimposable on that of other legume lec-

tins, the quaternary structure wasmarkedly different from

the canonical one, such as that of concanavalin A (which is

devoid of carbohydrate); the same was found also for the

structure of ECL recently solved in collaboration with Ute

Krengel and colleagues from Goteborg University, Swe-

den (87). We originally assumed that the bulky N-linked

carbohydrate of ECorL interfered with the formation of

the canonical structure by the lectin. This interpretation

has been proven to be incorrect, because in a recent joint

study with Avadesha Surolia and colleagues from the

Indian Institute of Scientific Research, Bangalore (88) as

well as by K. Ravi Acharya and colleagues at the University



of Bath, United Kingdom (89), the quaternary structure of

the bacterially expressed ECorL, devoid of carbohydrate,

was identical with that of the native one. The non-canon-

ical mode of dimerization of the Erythrina lectins is there-

fore most likely because of factors intrinsic to the protein.

Indeed, according to Surolia and his co-workers, legume

lectins are a family of proteins inwhich small alterations in

essentially the same tertiary structure lead to large varia-

tions in quaternary association (90).

An exceptional feature of the three-dimensional struc-

ture of the Erythrina lectins is that all the seven protein-

linked monosaccharide residues are seen with extraordi-

nary clarity, whereas in almost all other such structures of

glycoproteins at most 3 or 4 of the protein-proximal mon-

osaccharides have been observed (86–89). The structures

also show that in the crystal lattice the glycosylation site

and the carbohydrate-binding site are involved in inter-

molecular contacts through water-mediated interactions.

How Lectins Combine with Carbohydrates

To obtain detailed information on the combining site of

ECorL, RivkaAdar togetherwithHansjorg Streicher, post-

doctoral fellow from the University of Konstanz, carried

out extensive site-directed mutagenesis of the lectin. In

addition they made a thorough examination of its speci-

ficity in collaboration with Jonas Ångstrom and co-work-

ers from theUniversity ofGoteborg andwith Ray Lemieux

from the University of Alberta, Edmonton. Taking also in

consideration the three-dimensional structure of the

ECorL-ligand complex (87), we have concluded that a

constellation of three key amino acid residues, an aspartic

acid, an asparagine, and an aromatic one, is essential for

galactose binding (91–94). The first two residues form

hydrogen bonds with the hydroxyls of the ligand, whereas

the third interacts with it hydrophobically. Moreover, an

identical constellation of amino acid residues is involved

also in binding of mannose by other legume lectins, for

example by concanavalin A. We suggested therefore that

homologous lectins with distinct specificities might bind

different monosaccharides primarily by the same set of

invariant residues that are identically positioned in their

tertiary structures although with the ligand in a different

orientation (95). Quite surprisingly, the constellation

present in the combining sites of legume lectins is also

found in certain animal lectins, such as the mannose-spe-

cific ERGIC-53 that serves as a carrier of a specific subset

of nascent glycoproteins between the ER and Golgi com-

partments (96). Comparison with the combining sites of

lectins from other sources, including of animals, led to an

additional conclusion, namely that structurally different

lectinswith similar specificitiesmay bind the same saccha-

ride by different sets of combining site residues (95). A

survey of the literature showed that lectins bind their

ligands most commonly by hydrogen bonds (some medi-

ated by water) and hydrophobic interactions and that in

rare cases electrostatic interactions (ion pairing) and coor-

dination with metal ions also play a role. On the whole,

most of the side chains of the protein amino acids, as well

asmain chain groups, can participate in ligand binding. All

the above seems to indicate that lectins are products of

convergent evolution.

Cell-Cell Recognition Molecules in Microbial

Infection

In the foregoing, I dealt almost exclusively with plant

lectins. However, for the last 30 years I have also been

interested in microbial lectins, which I wish to describe

now. This interest was motivated by the arrival in my lab-

oratory in 1975 of Itzhak Ofek as a postdoctoral fellow.

Through him I learned that primarily thanks to the efforts

of J. P. Duguid of Ninewells Hospital Medical School in

Dundee it had been known that many strains of Esche-

richia coli possess the ability to agglutinate erythrocytes

and that this activity is inhibited by mannose and methyl

�-mannoside (97), but little attention was paid to these

findings. Moreover, the idea that sugar-specific adhesion

to host cells might be a prerequisite for bacterial coloniza-

tion and infection was not considered at all. In retrospect,

this is all the more surprising because it had already been

established that initiation of infection by influenza virus

required its attachment via its hemagglutinin to a sugar

(sialic acid) on cells (for an early review, see Ref. 98).

TogetherwithDavidMirelman, a former graduate student

ofmine, andOfek (who latermoved to the SacklerMedical

School, Tel Aviv University) we found that E. coli adheres

readily to buccal epithelial cells and that this adhesion was

inhibited specifically by mannose and methyl �-manno-

side. The adhesion was also inhibited by pretreatment of

the epithelial cells with concanavalin A but not with other

lectins such as SBA. Extraction of the bacteria afforded a

lectin-like constituent specific for mannose. We con-

cluded that mannose, a common constituent of most

mammalian cell surfaces, acts as a receptor for E. coli (99).

The same conclusion was reached independently and at

the same time by Irving Salit and Emil Gotschlich from the

Rockefeller University, who demonstrated that binding to

monkey cells of purified fimbriae obtained from a man-

nose-specific strain of E. coliwas inhibited by analogues of

mannose or by preincubation of the cells with mannose-

specific plant lectins (100). These studies provided a clear

example (I believe the first of its kind) that lectins function

in cell-cell recognition.



WithOfek andNurit Firon, a graduate student, we have

mapped the combining site of the type 1 fimbriae, mainly

by the ability of a variety of mannose-containing oligosac-

charides and �-mannosides to inhibit the agglutination of

yeasts by the bacteria or by the isolated fimbriae (101, 102).

Hydrophobic mannosides in particular were found to be

powerful inhibitors of the agglutination, up to three orders

of magnitude more effective than methyl �-mannoside.

Based on our results, we proposed that the site is extended

with an adjoining hydrophobic region. A very recent study

by Julie Bouckaert and colleagues at the Free University of

Brussels of FimH, the carbohydrate-binding subunit of the

fimbriae, has confirmed our results and extended them

(103). By x-ray crystallography and modeling of FimH-

ligand complexes a molecular explanation was obtained

for the high affinity of hydrophobic mannosides to the

fimbriae, which is based on the interaction of their agly-

coneswith aromatic side chains (referred to as “hydropho-

bic gateway”) close to the mannose-binding site of the

subunit.

That blocking of the bacterial lectinsmay prevent infec-

tion was proven by us in a study carried out in collabora-

tion with Ofek andMirelman together with Moshe Aron-

son from Sackler Medical School, Tel Aviv University.

Infection ofmouse bladder with amannose-specific E. coli

strain was markedly diminished by pre-suspension of the

organism in a solution ofmethyl�-mannoside but was not

affected by glucose, a sugar to which the bacteria do not

bind (104). The prophylactic effects of adhesion-inhibi-

tory saccharides have been demonstrated in many other

animal models, such as pneumococcal pneumonia in rats

(105) andHelicobacter pylori gastric infection in monkeys

(106). In addition to demonstrating unequivocally that

recognition of cell surface carbohydrates is a prerequisite

for infection by lectin-carrying bacteria, the above data

serve as a definitive proof for the validity of the concept of

anti-adhesion therapy of microbial diseases. However,

success of such treatment in humans has not yet been

achieved (for review, see Ref. 107).

The specificity of the bacterial surface lectins is a key

determinant in their animal tropism, as first illustrated by

Victor Ginsburg in the case of E. coli K99 (108). The fim-

brial lectin of this organism is specific for glycolipids con-

taining N-glycolylneuraminic acid but not for those con-

taining N-acetylneuraminic acid. The former sialic acid is

present on intestinal cells of newborn piglets but is

replaced by N-acetylneuraminic acid when the animals

develop. It is also not normally formed by humans,

explaining why E. coli K99 can cause lethal diarrhea in

piglets but not in adult pigs or in humans.

In addition to their role in initiation of infection, the

mannose-specific bacterial surface lectins may also func-

tion in protection against infectious agents. This is the

case also for surface lectins of phagocytic cells (such as

granulocytes and macrophages). As we have shown some

time ago, bacteria and yeasts may bind to these cells in the

absence of opsonins, leading to uptake and killing of the

organisms. This phenomenon, named by us “lectinoph-

agocytosis” (109), is an early example of innate immunity,

a phenomenon of great importance, in which lectins are

now known to be major players (reviewed for example in

Refs. 110 and 111).

Concurrent with our studies of the mannose-specific

E. coli, Catharina Svanborg and Hakon Leffler discovered

strains of this organism with galabiose (Gal�4Gal)-spe-

cific fimbriae that also serve as mediators of attachment of

the bacteria to host tissues as a prelude to infection (112).

Two surface lectins were discovered soon thereafter in the

pathogenic protozoan Entamoeba histolytica. One of

these, specific forN-acetylgalactosamine, was reported by

Jonathan Ravdin and Richard Guerrant from the Univer-

sity of Virginia, Charlottesville (113); the other, specific for

N-acetylglucosamine oligomers, was described at the

same time by David Mirelman and David Kobiler in our

department (114). During the years, compelling evidence

has been obtained for the involvement of the former lectin

in host cell binding, as well as in a variety of other proper-

ties of the ameba, that determine the severity of its infec-

tion (for review, see Ref. 115).

Animal Lectins in Cell Recognition

Despite the evidence presented above, wide acceptance

of the concept that lectins function as recognition mole-

cules was slow to come and had to await the isolation and

characterization of mammalian lectins. The first of these

was the galactose-specific liver lectin discovered in 1974

by Gilbert Ashwell at NIHwith Anatol G.Morell at Albert

Einstein College of Medicine in the course of their inves-

tigation of the mechanisms that control the lifetime of

glycoproteins in blood circulation (reviewed in Ref. 116).

This lectin recognizes and binds asialo-glycoproteins and

is responsible for their uptake by the liver and eventual

degradation.

In our department and at about the same time, my for-

mer graduate student Vivian Teichberg and Gad Reshef,

another graduate student of mine who was killed in the

October 1973 war, isolated from the electric eel (Elec-

trophorus electricus) and from chicken muscle the first

members of the family of the soluble �-galactose-specific

lectins (117) designated galectins, several of which are

known to be mediators of cellular interactions (for



reviews, see Refs. 118 and 119). One of these, galectin-3,

that is involved inmetastatic spread of B16melanoma cells

was isolated by Reuben Lotanwhile still in our department

together with Avraham Raz from the Department of

Membrane Research (120, 121). Another member of this

family, galectin-8, has also been isolated at theWeizmann

Institute by Yehiel Zick and co-workers from the Depart-

ment of Molecular Cell Biology (122); by binding to cell

surfaces, this lectinmodulates cell-matrix interactions and

regulates cellular functions in a variety of physiological

and pathological conditions.

What fully convinced the skeptics was the discovery in

about 1990 of the selectins, a class of C-type mammalian

lectins, and the demonstration of their crucial role in the

control of lymphocyte migration (homing) to specific

lymphoid organs and to sites of inflammation (for a recent

review, see Ref. 123). Since then, numerous other mam-

malian lectins have been discovered, many of which are

cell surface constituents, strategically located to serve as

recognitionmolecules in a variety of systems, both normal

and pathological (for reviews, see Refs. 110, 111, 122, 124,

and 125). Indeed, recognition by lectins in animal tissues is

undoubtedly one of the major developments in glycobiol-

ogy during the last part of the 20th century. But that is

another story.

This Reflections article is dedicated to Ephraim Katchalski-Katzir, men-

tor and friend, on the occasion of his 90th birthday, May 16, 2006.

Acknowledgments—I am most grateful to my long time colleague, Dr.

Halina Lis, for her help in the preparation of these reflections. Thanks are

also due to my friend, Dr. Uriel Littauer, for his constructive criticism of

the manuscript.


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the hemagglutinin of jack bean. J. Bacteriol. 32, 227–23729. Morgan, W. T. J., and Watkins, W. M. (2000) Unraveling the biochemical

basis of blood group ABO and Lewis antigenic specificity. Glycoconjugate J.

17, 501–53030. Aub, J. C., Sanford, B. H., and Wang, L. H. (1965) Reactions of normal and

leukemic cell surfaces to a wheat germ agglutinin. Proc. Natl. Acad. Sci.

U. S. A. 54, 400–40231. Burger, M., and Goldberg, A. R. (1967) Identification of a tumor-specific

determinant of neoplastic cell surfaces. Proc. Natl. Acad. Sci. U. S. A. 57,359–366

32. Inbar, M., and Sachs, L. (1969) Interaction of the carbohydrate-binding pro-

tein concanavalin A with normal and transformed cells. Proc. Natl. Acad. Sci.

U. S. A. 63, 1418–142533. Sela, B. A., Lis, H., Sharon, N., and Sachs, L. (1970) Different locations of

carbohydrate-containing sites at the surface membrane of normal and trans-

formed mammalian cells. J. Membr. Biol. 3, 267–27934. Nicolson, G. L., and Singer, S. J. (1971) Ferritin-conjugated plant agglutinins

as specific saccharide stains for electron microscopy: application to sacchar-

ides bound to cell membranes. Proc. Natl. Acad. Sci. U. S. A. 68, 942–94535. Marchesi, V. T., Tillack, T. W., Jackson, R. L., Segrest, J. P., and Scott, R. E.

(1972) Chemical characterization and surface orientation of the major glyco-

protein of the human erythrocyte membrane. Proc. Natl. Acad. Sci. U. S. A.

69, 1445–144936. Nicolson,G. L. (1974) Interactions of lectinswith animal cell surfaces. Int. Rev.

Cytol. 39, 89–19037. Inbar, M., and Sachs, L. (1973) Mobility of carbohydrate-containing sites on



the surface membranes in relation to control of cell growth. FEBS Lett. 32,124–128

38. Stanley, P. (1987) Glycosylation mutants and the functions of mammalian

carbohydrates. Trends Genet. 3, 77–8139. Roseman, S. (1970) The synthesis of complex carbohydrates by multiglyco-

syltransferase systems and their potential function in intercellular adhesion.

Chem. Phys. Lipids 5, 270–29740. Ginsburg, V., and Kobata, A. (1971) in Structure and Function of Surface

Components of Mammalian Cells in Structure and Function of Biological

Membranes (Rothfield, L. I., ed) pp. 439–459, Academic Press, New York

41. Allen, A., Neuberger, A., and Sharon, N. (1973) The purification and specific-

ity of wheat germ agglutinin. Biochem. J. 131, 155–16242. Sharon, N., and Lis, H. (1973) The biochemistry of plant lectins (phytohem-

agglutinins). Annu. Rev. Biochem. 42, 541–57443. Lis, H., and Sharon, N. (1986) Lectins as molecules and tools. Annu. Rev.

Biochem. 55, 35–6744. Sharon, N., and Lis, H. (1989) Lectins. Chapman & Hall, London; (2003)

Lectin’s, 2nd Ed., Kluwer Academic Publishers, Dordrecht, The Netherlands

45. Liener, I. E., Sharon,N., andGoldstein, I. J. (eds) (1986)The Lectins, Properties,

Functions and Applications in Biology and Medicine, Academic Press,

Orlando, FL

46. Sharon, N. (1975)Complex Carbohydrates, Their Properties, Biosynthesis and

Functions, Addison Wesley, Reading, MA

47. Edelman, G. M., Cunningham, B. A., Reeke, G. N., Jr., Becker, J. W., Waxdal,

M. J., andWang, J. L. (1972) The covalent and three-dimensional structure of

concanavalin A. Proc. Natl. Acad. Sci. U. S. A. 69, 2580–258448. Hardman, K. D., and Ainsworth, C. F. (1972) Structure of concanavalin A at

2.4 Å resolution. Biochemistry 11, 4910–491949. Wright, C. S. (1977) The crystal structure of wheat germ agglutinin at 2.2 Å

resolution. J. Mol. Biol. 111, 439–45750. Lotan, R., Siegelman, H. W., Lis, H., and Sharon, N. (1974) Subunit structure

of soybean agglutinin. J. Biol. Chem. 249, 1219–122451. Lotan, R., Debray, H., Cacan,M., Cacan, R., and Sharon, N. (1975) Labeling of

soybean agglutinin by oxidation with sodium periodate followed by reduction

with sodium [3H]borohydride. J. Biol. Chem. 250, 1955–195752. Adar, R., Streicher, H., Rozenblatt, S., and Sharon, N. (1997) Synthesis of

soybean agglutinin in bacterial and mammalian cells. Eur. J. Biochem. 249,684–689

53. Sharon, N., Lis, H., and Lotan, R. (1974) On the structural diversity of lectins.

Colloq. Int. du C.N.R.S. 221, 693–70954. Hemperly, J. J., and Cunnigham, B. A. (1983) Circular permutation of amino

acid sequences among legume lectins. Trends Biochem. Sci. 8, 100–10255. Bowles, D. J., Marcus, S. E., Pappin, D. J., Findlay, J. B., and Eliopoulos, E., et al.

(1986) Posttranslational processing of concanavalin A precursors in jack bean

cotyledons. J. Cell Sci. 102, 1284–129756. Shaanan, B., Shoham, M., Yonath, A., Lis, H., and Sharon, N. (1984) Crystal-

lization and preliminary x-ray diffraction studies of soybean agglutinin. J.Mol.

Biol. 174, 723–72557. Dessen, A., Gupta, D., Sabesan, S., Brewer, C. F., and Sacchettini, J. C. (1995)

X-ray crystal analysis of the soybean agglutinin crosslinkedwith a biantennary

analog of the blood group I carbohydrate antigen. Biochemistry 34,4933–4942

58. Nowell, P. (1960) Phytohemagglutinin: an initiator of mitosis in cultures of

normal human leukocytes. Cancer Res. 20, 462–46659. Wecksler,M., Levy, A., and Jaffe,W. G. (1968)Mitogenic effects of extracts of

Canavalia ensiformis and concanavalin A. Acta Cient. Venez. 19, 154–15660. Mier, J. W., and Gallo, R. C. (1982) The purification and properties of human

T cell growth factor. J. Immunol. 128, 1122–112761. Novogrodsky, A., and Katchalski, E. (1973) Transformation of neuramini-

dase-treated lymphocytes by soybean agglutinin. Proc. Natl. Acad. Sci. U. S. A.

70, 2515–251862. Novogrodsky, A., Lotan, R., Ravid, A., and SharonN. (1975) Peanut agglutinin,

a new mitogen that binds to galactosyl sites exposed after neuraminidase

treatment. J. Immunol. 115, 1243–124863. Schechter, B., Lis, H., Lotan, R., Novogrodsky, A., and Sharon, N. (1976)

Requirement for tetravalency of soybean agglutinin for induction of mito-

genic stimulation of lymphocytes. Eur. J. Immunol. 6, 145–14964. Sharon, N., and Lis, H. (1997) Lectins: their chemistry and applications to

immunology. The Antigens, Vol. 4, pp. 429–529, Academic Press, New York

65. Sharon, N. (1983) Lectin receptors as lymphocyte surface markers. Adv.

Immunol. 34, 213–29866. Barkai-Golan, R., Mirelman, D., and Sharon, N. (1978) Studies on growth

inhibition by lectins of Penicillia and Aspergilli. Arch. Microbiol. 116,

119–124

67. Peumans, W. J., and Van Damme, E. J. M. (1995) Lectins as plant defense

proteins. Plant Physiol. 109, 347–35268. Kijne, J. W. (1997) Root lectins and Rhizobia. Plant Physiol. 115, 869–87369. Lotan, R., Skutelsky, E., Danon, D., and Sharon, N. (1975) The purification,

composition, and specificity of the anti-T lectin from peanut (Arachis

hypogaea). J. Biol. Chem. 250, 8518–852370. Terao, T., Irimura, T., andOsawa, T. (1975) Purification and characterization

of a hemagglutinin from Arachis hypogaea. Hoppe-Seyler’s Z. Physiol. Chem.

356, 1685–169271. Pereira, M. E. A., Kabat, E. A., Lotan, R., and Sharon, N. (1976) Immuno-

chemical studies on the specificity of peanut (Arachis hypogaea) agglutinin.

Carbohydr. Res. 51, 107–11872. Reisner, Y., Linker-Israeli, M., and Sharon, N. (1976) Separation of mouse

thymocytes into two subpopulations by the use of peanut agglutinin. Cell.

Immunol. 25, 129–13473. Gillespie, W., Paulson, J. C., Kelm, S., Pang, M., and Baum. L. G. (1993) Reg-

ulation of �-2,3-sialyltransferase expression correlates with conversion of

peanut agglutinin (PNA)� to PNA� phenotype in developing thymocytes.

J. Biol. Chem. 268, 3802–380474. Daniels, M. A., Hogquist, K. A., and Jameson, C. S. (2002) Sweet ’n sour: the

impact of differential glycosylation on T cell differentiation.Nat. Immunol. 3,903–910

75. Reisner, Y. Gachelin, G., Dubois, P., Nicolas, J. F., Sharon, N., and Jacob, F.

(1977) Interaction of peanut agglutinin, a lectin specific for terminal D-galac-

tosyl residues, with embryonal carcinoma cells. Dev. Biol. 61, 20–2776. Reisner, Y., Itzicovitch, L., Meshorer, A., and Sharon, N. (1978) Hemopoietic

stem cell transplantation usingmouse bonemarrow and spleen cells fraction-

ated by lectins. Proc. Natl. Acad. Sci. U. S. A. 75, 2933–293677. Reisner, Y., Kapoor, N., Kirkpatrick, D., Pollack,M. S., Cunningham-Rundles,

S., Dupont, B., Hodes, M. Z., Good, R. A., andO’Reilly, R. J. (1983) Transplan-

tation for severe combined immunodeficiency with HLA-A,B,D,DR-incom-

patible parental marrow cells fractionated by soybean agglutinin and sheep

red blood cells. Blood 61, 341–34878. Aversa, F., Tabilio, A., Velardi, A., Cunningham, I., Terenzi, A., Falzetti, F.,

and Ruggeri, L., et al. (1998) Treatment of high-risk acute leukemia with

T-cell-depleted stem cells from related donors with one fully mismatched

HLA haplotype. N. Engl. J. Med. 339, 1186–119379. Iglesias, J. L., Lis, H., and Sharon, N. (1982) Purification and properties of a

galactose/ N-acetyl-D-galactosamine-specific lectin from Erythrina crista-

galli. Eur. J. Biochem. 123, 247–25280. Ashford, D., Dwek, R. A.,Welply, J. K., Amatayakul, S., Homans, S.W., Lis, H.,

Taylor, G.N., Sharon,N., andRademacher, T.W. (1987) The�(1–2)-D-xylose

and �(1–3)-L-fucose substituted N-linked oligosaccharides from Erythrina

cristagalli lectin. Isolation, characterization, and comparison with other

legume lectins. Eur. J. Biochem. 166, 311–32081. Gilboa-Garber,N., andMizrachi, L. (1981)Anewmitogenic D-galactosephilic

lectin isolated from seeds of the coral-tree Erythrina corallodendron. Com-

parison with Glycine max (soybean) and Pseudomonas aeruginosa lectins.

Can. J. Biochem. 59, 315–32282. Arango, R., Adar, R., Rozenblatt, S., and Sharon, N. (1992) Expression of

Erythrina corallodendron lectin in Escherichia coli. Eur. J. Biochem. 205,575–581

83. Foriers, A., Wuilmart, C., Sharon, N., and Strosberg, A. D. (1977) Extensive

sequence homologies among lectins from leguminous plants. Biochem.

Biophys. Res. Commun. 75, 980–98584. Chandra, N. R., Kumar, N., Jeyakani, J., Singh, D. D., Gowda, S. B., and Pra-

thima,M.N. (2006) Lectinb: a plant lectin database.Glycobiology16, 938–94685. Drickamer, K. (1988) Two distinct classes of carbohydrate recognition do-

mains in animal lectins. J. Biol. Chem. 263, 9557–956086. Shaanan, B., Lis, H., and Sharon, N. (1991) Structure of a lectin with ordered

carbohydrate in complex with lactose. Science 253, 862–86687. Svensson, C., Teneberg, S., Nilsson, C. L., Schwarz, F. P., Kjellberg, A., Sharon,

N., and Krengel, U. (2002) High-resolution crystal structures of Erythrina

cristagalli lectin in complex with lactose and 2�-�-L-fucosyllactose and their

correlation with thermodynamic binding data. J. Mol. Biol. 321, 69–8388. Kulkarni, K. A., Srivastava, A., Mitra, N., Sharon, N., Surolia, A., Vijayan, M.,

and Suguna, K. (2004) Effect of glycosylation on the structure of Erythrina

corallodendron lectin. Proteins 56, 821–82789. Turton, K., Natesh, R., Thiagarajan, N., Chaddock, J. A., and Acharya, K. K.

(2004) Crystal structure of Erythrina cristagalli lectin with bound N-linked

oligosaccharide and lactose. Glycobiology 14, 923–92990. Brinda, K. V., Mitra, N., Surolia, A., and Wishveshwara, S. (2005) Determi-



nants of quaternary association in legume lectins. Protein Sci. 13, 1735–174991. Adar, R., and Sharon, N. (1996) Mutational studies of the combining site

residues of Erythrina corallodendron lectin. Eur. J. Biochem. 239, 668–67492. Adar, R., Ångstrom, J., Moreno, E., Karlsson, K. A., Streicher, H., and Sharon,

N. (1998) Structural studies of the combining site of Erythrina collarodendron

lectin. Role of tryptophan. Protein Sci. 7, 52–6393. Moreno, E., Teneberg, S., Adar, R., Sharon,N., Karlsson, K.-A., andÅngstrom,

J. (1997) Redefinition of the carbohydrate specificity of Erythrina coralloden-

dron lectin based on solid-phase binding assays and molecular modeling

of native and recombinant forms obtained by site-directed mutagenesis.

Biochemistry 36, 4429–443794. Lemieux, R., Ling, C. C., Sharon, N., and Streicher, H. (2000) The epitope of

the H-type trisaccharide recognized by Erythrina corallodendron lectin. Evi-

dence for both attractive polar and strong hydrophobic interactions for com-

plex formation involving a lectin. Isr. J. Chem. 40, 167–17695. Sharon, N., and Lis, H. (2001) The structural basis for carbohydrate recogni-

tion by lectins. Adv. Exp. Med. Biol. 491, 1–1696. Velloso, L. M., Svensson, K., Schneider, G., Pettersson, R. F., and Lindqvist, Y.

(2002) Crystal structure of the carbohydrate recognition domain of p58/ER-

GIC-53, a protein involved in glycoprotein export from the endoplasmic

reticulum. J. Biol. Chem. 277, 15979–1598497. Duguid, J. P., andOld, D. C. (1980) in Bacterial Adherence (Beachey, E. H., ed)

pp. 185–217, Chapman and Hall, London

98. Schauer, R. (1973) Chemistry and biology of acylneuraminic acids. Angew.

Chem. Int. Ed. Engl. 12, 127–13899. Ofek, I., Mirelman, D., and Sharon, N. (1977) Adherence of Escherichia coli to

human mucosal cells mediated by mannose receptors. Nature 265, 623–625100. Salit, I. E., and Gotschlich, E. C. (1997) Type I Escherichia coli pili: character-

ization of binding to monkey kidney cells. J. Exp. Med. 146, 1182–1194101. Firon, N., Ofek, I., and Sharon, N. (1983) Carbohydrate specificity of the

surface lectins of Escherichia coli, Klebsiella pneumoniae, and Salmonella

typhimurium. Carbohydr. Res. 120, 235–249102. Firon, N., Ashkenazi, S., Mirelman, D., Ofek, I., and Sharon, N. (1987) Aro-

matic alpha-glycosides of mannose are powerful inhibitors of the adherence

of type 1 fimbriated Escherichia coli to yeast and intestinal epithelial cells.

Infect. Immun. 55, 472–476103. Bouckaert, J., Berglund, M., Schembri, E., De Genst, L., Cools, M., and Wu-

hrer, M., et al. (2005) Receptor binding studies disclose a novel class of high-

affinity inhibitors of the Escherichia coli FimH adhesin. Mol. Microbiol. 55,441–455

104. Aronson, M., Medalia, O., Schori, L., Mirelman, D., Sharon, N., and Ofek, I.

(1979) Prevention of colonization of the urinary tract ofmicewith Escherichia

coli by blocking of bacterial adherence with methyl �-D-mannopyranoside. J.

lnfect. Dis. 139, 329–332105. Indapaan-Heikkila, I., Simon, P. M., Zopf, D., Vullo, T., Cahill, P., et al. (1997)

Oligosaccharides interfere with the establishment and progression of exper-

imental pneumococcal pneumonia. J. Infect. Dis. 176, 704–712106. Mysore, J. V., Wigginton, T., Simon, P. M., Zopf, D., Heman-Ackah, L. M.,

and Dubois, A. (1999) Treatment of Helicobacter pylori infection in rhesus

monkeys using a novel anti-adhesion compound. Gastroenterology 117,1316–1325

107. Sharon, N. (2006) Carbohydrates as future anti-adhesion drugs for infectious

diseases. Biochim. Biophys Acta 1760, 527–537108. Kyogashima, M., Ginsburg, V., and Krivan, H. C. (1989) Escherichia coli K99

binds to N-glycolyl-sialoparagloboside and N-glycolyl-GM3 found in piglet

small intestine. Arch. Biochem. Biophys. 270, 391–397109. Ofek, I., and Sharon, N. (1988) Lectinophagocytosis: a molecular mechanism

of recognition between cell surface sugars and lectins in the phagocytosis of

bacteria. Infect. Immun. 56, 539–547110. Crocker, P. R., and Varki, A. (2001) Siglecs, sialic acid and innate immunity.

Trends Immunol. 22, 337–342111. Lu, J., Teh, C., Kishore, U., and Reid, K. B. (2002) Collectins and ficolins: sugar

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genic Escherichia coli on human erythrocytes and uroepithelial cells. Infect.

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mechanisms ofEntamoeba histolytica: studies withmammalian tissue culture

cells and human erythrocytes. J. Clin. Invest. 68, 1305–1313114. Kobiler, D., and Mirelman, D. (1981) Adhesion of Entamoeba histolytica tro-

phozoites to monolayers of human cells. J. Infect. Dis. 144, 534–546115. Frederick, J. R., and Petri, W. A., Jr., (2005) Roles for the galactose-/N-acetyl-

galactosamine-binding lectin of Entamoeba in parasite virulence and differ-

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hepatic recognition and transport of surface glycoproteins. Adv. Enzymol.

Relat. Areas Mol. Biol. 41, 99–128117. Teichberg, V. I., Silman, I., Beitsch, D. D., and Reshef, G. (1975) A �-D-galac-

toside binding protein from electric organ tissue of Electrophorus electricus.

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(Paris) 83, 667–676119. Hernandez, J. D., and Baum, L. G. (2002) Ah, sweet mystery of death! Galec-

tins and control of cell fate. Glycobiology 12, 127R–136R120. Raz, A., and Lotan, R. (1981) Lectin-like activities associated with human and

murine neoplastic cells. Cancer Res. 41, 3642–3647121. Meromsky, L., Lotan, R., and Raz, A. (1986) Implications of endogenous tu-

mor cell surface lectins as mediators of cellular interactions and lung coloni-

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Mol. Biol. 6, 149–164



In Search of the MessagePublished, JBC Papers in Press, March 31, 2008, DOI 10.1074/jbc.X800001200

John H. Exton

From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of

Medicine, Nashville, Tennessee 37232-0615

Iwasborn inAuckland, NewZealand, in 1933. Hitler was on the rise inGermany, but this was

of little concern to most of the inhabitants of this beautiful country so far from the political,

economic, and cultural centers of the world. During my early years, my family lived by the

seaside, andmy sister and I led an idyllic existence playing on the beach, exploring rock pools

teeming with life, and climbing the cliffs to see the nests of ocean birds.My exposure to the unique

flora and fauna of New Zealand engendered an early interest in biology. I left all this behind when

my family moved to the city, where my education began in earnest. This was when atomic bombs

were dropped on Japan to endWorldWar II. I did not recognize the moral issues involved in this

act, but it stimulated my intense interest in nuclear physics. I was probably one of few elementary

school pupils in New Zealand who read about Madame Curie, Niels Bohr, Max Planck, Ernest

Lawrence, Enrico Fermi, Walter Heisenberg, Erwin Schrodinger, and, of course, New Zealand’s

own Ernest Rutherford.

For my secondary education, I was sent to a Church of England preparatory school that was

patterned after an English public school. Discipline was severe, with the cane being liberally

applied and bullying rampant. However, my parents did not pay high fees just for the infliction of

pain on their son, but for an excellent academic program inwhich Latin wasmandatory andGreek

recommended. This classical education was broadened by exposure to history and literature,

almost exclusively British, and to mathematics and the sciences. For sport, we were required to

play cricket and rugby football whatever our competence, which in my case was low. The chem-

istry teacher frequently put on dramatic explosive displays of chemical reactions, causing us to

crouch behind our desks while he dropped solid sodium or potassium into concentrated nitric,

sulfuric, and hydrochloric acids!

InNewZealand, there is no college system, and at that time, relatively few high school graduates

proceeded to university. I was fascinated by both chemistry and biology and had to choose

between a degree inmedicine or the sciences, withmy teachers strongly recommendingmedicine.

So I took the two-day journey from Auckland in the North Island to Dunedin in the South Island,

where the onlymedical school at that timewas located at theUniversity ofOtago. I had led a rather

sheltered life and was initially shocked by the riotous and decidedly unintellectual behavior of my

fellow students. Dunedin was founded by dour Scottish settlers, many of them Calvinists, but

somehow they tolerated the frequent and outrageous student pranks. One of Dunedin’s greatest

assets is its proximity to some of New Zealand’s most beautiful lakes and mountains, and groups

of us would spend weekends and other breaks hiking in the spectacular scenery.

Medical training in New Zealand takes six years, with almost two years devoted to anatomy,

physiology, and biochemistry. Unlike the vast majority of students, I was fascinated by biochem-

istry because it fused my interests in chemistry and biology. Another factor was that it was taught

by Norman Edson, one of the unsung heroes of New Zealand Science. He had worked with Hans

Krebs in Gowland Hopkins’ laboratory in Cambridge, England, where he had studied the regula-

tion of ketone body production in the liver. He had an encyclopedic memory and kept up with the

latest advances in the U. S. and U. K. I decided to take a year out of my medical training to do

biochemical research and studied the breakdown of pyrimidines. At that time, there was contro-

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 22, pp. 14901–14909, May 30, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MAY 30, 2008 • VOLUME 283 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY


H99

versy about the degradation pathway, but I showed that

the scheme proposed by Robert M. Fink and co-workers

was correct. This involved the intermediate formation of

�-ureidopropionic acid from uracil and �-ureidoisobu-

tyric acid from thymine (1). There was an initial reduction

of the pyrimidines to dihydropyrimidines, and I set about

to find the dehydrogenase responsible. This was unsuc-

cessful because the only reduced nicotinamide nucleotide

commercially available at that time was NADH, and the

enzyme was later shown to use NADPH. I continued with

my medical studies, and my social life greatly expanded

when Imetmy future wife, whowas a great companion on

our rugged hikes and sailing adventures on the icy waters

of Otago Harbor. She was also a student at the University

of Otago and came from a sheep farming community in

the North Island with the exotic name of Ongaonga. Her

father often employed me on the farm during vacations.

Being a city slicker, I was of doubtful use to him except

during lambing, when my obstetrical skills learned on the

sturdy Scottish women of Dunedin were useful when the

ewes were in difficulties. I suspect he tested me as a suita-

ble son-in-law by sending me out to the endless task of

draining a large swamp by hand shovel. I must have passed

muster because he later gave his daughter in marriage

when I was a final year student.

During my student years at Otago, I supplemented my

scholarship funds by working in a variety of occupations

during the summer breaks. These included working in an

ice cream factory, as a surveyor’s assistant, in a packing

house, in a wool store, and as a longshoreman. These gave

me the opportunity to interact with a wider socioeco-

nomic group than my middle class family and friends and

encouraged my thinking away from the relentless conser-

vatism of my parents!

At several times during my training, I contemplated

switching to a Ph.D. in Biochemistry, but Norman Edson

advised me strongly to continue in medicine, a course I

have never regretted, particularly because there were

aspects that I really enjoyed, and it gaveme fortitude when

I later had to lecture tomedical students. After completing

medical studies and an internship in Auckland, I returned

to Dunedin to do a Ph.D. in Biochemistry under Norman

Edson. The thesis topic was the metabolism of isolated rat

liver cells. Michael Berry, an energetic student in the lab-

oratory, had discovered how to prepare these cells, andmy

project was to study their carbohydrate and lipid metabo-

lism and to understand why they produced large amounts

of ketone bodies (2, 3). This was traced to a limitation in

the citric acid cycle such that the fatty acids were prefer-

entially oxidized to ketone bodies. At that time, the med-

ical school had only one electron microscope, and when

the cells were examined later, it was found that their

plasma membranes were disrupted and their mitochon-

dria distorted. Nevertheless, the findings were reported in

the Biochemical Journal because studies of isolated mam-

malian cells were unusual at that time. A visitor from the

U. S. at that time was Harland Wood from Western

Reserve, who came to study pyruvate metabolism. I am

sure an additional attraction for him was the proximity of

great deer hunting to Dunedin. Because of the great fear of

radioactivity in the 1960s, Norman Edson built a small

laboratory for himon the roof of themedical school. Imag-

ine his chagrin when Harland loaded a syringe with

[14C]pyruvate and squirted part of it into the air to expel an

air bubble!

When my Ph.D. was nearing completion, I looked

around for possible postdoctoral positions. The popula-

tion of New Zealand was then not much more than three

million, and Ph.D. and medical graduates desiring further

training had to look overseas. Britain was the traditional

place, and relatively few went to America. I sought a fel-

lowship to Trinity College Oxford to work on a D.Phil.

under Hans Krebs, although why I needed an additional

degree escapes me now. In case I was not successful in the

fellowship, I looked into several possibilities in the U. S.

The most attractive of these was the Department of Phys-

iology at Vanderbilt University under Charles R. (Rollo)

Park, whose group had just published an impressive series

of papers in the Journal of Biological Chemistry dealing

with the effects of insulin, diabetes, and other hormones

on glucose transport and phosphorylation in heartmuscle.

The rigor of their data and their in-depth analysis

impressed me greatly.

I did win a fellowship to Oxford, but when the details

arrived from the British Council, I was frankly outraged.

At that time (1963), my family had grown to two children,

and the Council secretary advisedme to leavemy family in

New Zealand for two years because the stipend could sup-

port only a single person. Furthermore, I could travel only

at the lowest level on the ship to Britain. On the other

hand, a letter came from Rollo Park offering to pay the

travel expenses of the whole family, so I outraged the Brit-

ish by refusing their fellowship to Oxford, which may

never have been done before by a mere colonial and cer-

tainly did not please Krebs!

Wewent by ship to theU. S., stopping at such exotic and

erotic places as Tahiti, Panama City, and Jamaica and dis-

embarking at Port Everglades in Florida. We hired two

Cadillac limousines to take our large pile of baggage to the

train station and then took one of themon a tour ofMiami.

REFLECTIONS: In Search of the Message

JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 22 • MAY 30, 2008H100

It seemed that we were living the American dream a few

hours after arrival. We took a train to Nashville, but it had

been rerouted, and no one on the train knewwhich station

was Nashville! We discovered for sure only upon alighting

on the platform! Rollo turned out to be as generous as his

correspondence indicated and told me that the scientific

environment of the department had recently been

enhanced by the arrival of Earl Sutherland’s group from

Western Reserve. Earl had achieved great recognition as

the discoverer of cyclic AMP, the second messenger of

many hormones.

My project was to look at direct effects of insulin and

other hormones on the liver using the isolated perfused rat

liver preparation. However, the existing perfusion system

left a lot to be desired in that the oxygenation system was

immersed in a water bath for temperature control and

frequently leaked. The temperatures and flow rates of con-

trol and experimental livers were often not the same, and

there were other problems.When I arrived, a postdoc was

running the experiments, and he overlooked any perfusion

disasters by ignoring the apparatuswhile readingLifemag-

azine.When it was discovered that he did not actually have

a Ph.D., he was dispatched back to England posthaste,

where he opened a candy store! The first order of business

was to develop a better perfusion apparatus, and this was

done with the assistance of Howard Morgan and Bailey

Moore, the skilled head of the apparatus shop. Howard

had a very pragmatic attitude and later became Chair of

Physiology at Penn State, President of the American Phys-

iological Society, and President of the American Heart

Association. With the problems of the perfusion system

solved, I began a lengthy series of studies of the hormonal

control of gluconeogenesis and glycogen metabolism in

the isolated liver. In an interesting turn of events, this put

us in competition with the laboratory of Hans Krebs.

Gluconeogenesis occurs principally in the liver and

involves the formation of glucose from non-sugar sources

such as lactate, pyruvate, glycerol, and certain amino acids.

It is essential for life during starvation and recycles lactate

formed during exercise back to glucose (the Cori cycle).

My work with Rollo Park showed that physiological

increases in amino acids and other substrates could alone

increase gluconeogenesis, andmeasurements ofmetabolic

intermediates in the pathway revealed that themajor rate-

limiting stepwas the substrate cycle between pyruvate and

phosphoenolpyruvate (4, 5). Later, we showed that hor-

mones such as glucagon and epinephrine could directly

stimulate gluconeogenesis in the liver by acting on this

cycle and that their effects were antagonized by insulin.

Work in several other laboratories subsequently showed

that the enzyme affected was pyruvate kinase. Diabetes

also stimulated the process, as did glucocorticoids, which

also exerted a permissive effect on the actions of glucagon

and epinephrine (5, 6). In collaboration with the Suther-

land group, we determined that cyclicAMPplayed amajor

role in the regulation of gluconeogenesis at the level of the

pyruvate-phosphoenolpyruvate cycle (7, 8). This workwas

conducted with the help of two excellent associates, Leon-

ard (Jim) Jefferson, a graduate student fromKentuckywho

drove a Cadillac and ultimately became Chair of Physiol-

ogy at Penn State Medical School and President of the

American Physiological Society, and Tom Miller, a per-

petually good-natured technician who came from a small

town in West Tennessee and became Professor and Dean

of Graduate Studies at the University of Massachusetts

Medical School. These were superb experimentalists,

although I had to avoid scheduling complex experiments

on mornings after they had enjoyed the bars of Nashville.

Another important contributor was Sandy Harper, who

truly qualified for the title of super-tech. Supervising all

this work was the tall aristocratic Rollo Park, with his

impeccable heritage of clerics, generals, and academics.

He provided invaluable insights and conveyed an urbane

relaxed attitude in which issues such as research funding

and priority in publication were not of much concern.

When I complained that one of my papers was taking over

17 months to be submitted because of constant revising,

his response was, “In the end, the best paper wins.” Rollo

sent me out to look at many departmental chairmanships

despite my relatively young age because he believed that

any candidate older than 40 was over the hill!

A year after my arrival in Nashville, we were joined by

J. G. T. Sneyd, who was also from the Biochemistry

Department at Otago. He was nicknamed Sam after the

golfer Sam Snead, although he never played golf. He was a

brilliant guy but an inveterate prankster. A list of his she-

nanigans would occupy this entire essay, but I will report

only the episodes when he served rat liver pate to his

mother-in-law and staged a cricketmatch insidehis house.

The ultimate Sneyd story was when his wife put an adver-

tisement in the local newspaper in which she tried to sell

him plus his TV (both in working order) because of his

addiction to football during the holiday season. This was

taken up by countless news sources in the U. S. and

throughout the world and occasioned many TV and radio

interviews in which his wife played it straight!

The presence of Earl Sutherland’s group gave a great

boost to my research, but discussions with him frequently

involved mental gymnastics because he would often

change topics mid-sentence. I remember talking about


MAY 30, 2008 • VOLUME 283 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRYH101

glycogen metabolism when he suddenly mentioned TG.

For the life of me I could not see the relevance of triglyc-

eride, but he was referring to transglucosylase, an uncom-

mon name for glycogen synthase! He was quite convinced

that cyclicGMPwas the secondmessenger for insulin, and

this seemed quite logical. However, when Joel Hardman,

subsequently Chair of Pharmacology at Vanderbilt, and I

put this to the test in the perfused liver, the results were

negative (9). Everyone in the department recognized the

fundamental importance of Earl’s discovery of cyclic AMP

and waited each year for the call from Stockholm. How-

ever, when a crew from Swedish TV arrived in late Octo-

ber 1971 to film him and his laboratory, we knew his time

had finally arrived.

In 1968, I was promoted to Associate Professor and also

appointed (anointed) as an Investigator of the Howard

Hughes Medical Institute. At that time, Investigators

received only a stipend and a travel allowance, which

would get me to California and back. Nowadays, it would

get me only a one-way ticket to St. Louis! In 1976, under

pressure from the Internal Revenue Service, which was

perpetually suspicious of the Institute, it was compelled to

disbursemore funds, and a cornucopia of equipment, sup-

plies, and support for postdocs and technicians descended

upon my grateful head! The Institute even renovated the

lab using only first-rate materials. At that time, Hughes

Investigators lived in a form of research paradise that was

even better than it is now. Oversight was not very rigorous

and the annual reviews perfunctory. The Medical Advi-

sory Board believed that once Investigators were

approved, they should be left to their own devices. All this

changed in 1978, when the reclusiveHowardHughes died,

and the Institute was restructured. Now, the review proc-

ess became much more rigorous (terrifying), and they

actually terminated some Investigators. I survived the

review process and retired in 2004 as the longest tenured

Investigator. I felt like a living fossil among all the young

Investigators!

In 1970, I spent a sabbatical in Geneva at the Institut de

Biochimie Clinique, headed by the renowned diabetes

researcher Albert Renold. I worked primarily with his

gifted associate Bernard Jeanrenaud, and I was greatly

impressed by the charming and multilingual Albert and

the handsome and debonair Bernard. I studied the hormo-

nal regulation of glycogenolysis and gluconeogenesis in

mouse liver, working with a superb graduate student,

Francoise Assimacopoulos-Jeannet. I depended on her

skill in adapting our procedures to this small animal. We

rented a nice villa in Geneva and took frequent trips to

France, Italy, and the rest of Switzerland to explore the

countryside and culture. One of my first conferences was

at a Swiss ski resort, where the Nobel Laureate Feodor

Lynen sat at the rear of the room on a large throne-like

chair andmade audible derogatory comments about most

of the talks. I approached him with some trepidation after

my talk, but, thankfully, he was complimentary.

While I was in Geneva and after I returned, studies on

the regulation of gluconeogenesis by glucagon and insulin

continued using the perfused liver system. Some of this

work focused on amino acids as gluconeogenic substrates

and was carried out by Larry Mallette, a brilliant M.D./

Ph.D. student, andMichio Ui from Japan, who would later

become famous through his work on a Bacillus pertussis

toxin that inactivates Gi through ADP-ribosylation. Other

work studied the antagonistic action of insulin on the

effects of glucagon in the liver and revealed the primacy of

cyclic AMP changes in this interaction. A key player in this

workwas Steve Lewis, anM.D.with enormous energywho

would try any experimental approach including perfusing

the livers backwards or using distilled water as the perfu-

sionmedium! It was difficult to restrain him fromGeneva.

Soon aftermy return toNashville, I switched our exper-

imental system from the liver perfusion procedure to the

use of isolated rat liver parenchymal cells. After years of

persistence by my former New Zealand colleagueMichael

Berry, structurally and metabolically intact liver cells

could now be prepared using collagenase. Now the many

perfusion apparatuses were used only to prepare these

hepatocytes. Although the use of hepatocytes greatly

increased the throughput of experiments and permitted

better controls, I was faced with a dilemma as follows.

Whereas all our data obtained using the hepatocytes sup-

ported a role for cyclic AMP in the action of glucagon on

gluconeogenesis and glycogen breakdown, this was not

the case for epinephrine. Try as we could, we were unable

to correlate the changes in these processes induced by this

catecholamine with increases in cyclic AMP (10–12). Our

collaborator was Al Robison, a member of the Sutherland

group who was a nocturnal worker and subsequently

Chair of Pharmacology at the University of TexasMedical

School at Houston. We would leave our samples on the

desk, and in the morning, the cyclic AMP measurements

would appear like magic! Our findings caused some con-

sternation in the Sutherland group because the original

work leading up to the discovery of cyclic AMP involved

the effects of epinephrine on glycogen breakdown in dog

liver. This challenge to the dogma was resolved when it

was realized that there was a species difference. In dog

liver, the effects of epinephrine are mediated by �-adre-

nergic receptors acting via cyclic AMP, whereas in rat



liver, they are mediated by �1-adrenergic receptors acting

via Ca2�. The work with epinephrine had a very long her-

itage starting with Claude Bernard, who found in the 19th

century that stimulation of the brain increased the blood

glucose level (piqure hyperglycemia), and Walter B. Can-

non, whose equally classic work (“fight or flight response”)

showed that increases in blood glucose and other changes

occurred in response to activation of the sympathetic

nervous system. When Mahmoud El Refai, an Egyptian

postdoc, found that the rat liver receptors involved in gly-

cogenolysis were of the �1-type, a competing group tried

to prevent us from publishing because it was contrary to

their incorrect findings! A collaborator in some of this

work was Craig Venter, who had not yet developed his

reputation as the enfant terrible of the sequencing world.

Work by Jim Putney, then at the Medical College of

Virginia, and other investigators utilizing salivary gland,

smoothmuscle, and other tissues indicated that the�1-ad-

renergic mechanism involved a rise in intracellular Ca2�,

and we soon confirmed this for rat liver. However, a prob-

lem arose because it was then believed that the increase in

Ca2� came from an influx through channels in the plasma

membrane, but our data indicated that it came from an

internal pool (12). A key collaborator in this project was

Peter Blackmore from Australia, who was nicknamed

“Quokka” after the small Australian marsupial. He was a

remarkably adept experimentalist and is now a Professor

of Physiological Sciences at the East Virginia Medical

School. Also involved were Nancy Hutson, a smart and

very personable graduate student who is now an executive

at Pfizer, and Francoise Assimacopoulos-Jeannet from

Geneva, who is now a Professor Titulaire at the Centre

Medical Universitaire there. The role of Ca2� in the

actions of �1-adrenergic and related agonists was demon-

strated unequivocally by the use of Quin-2, a reagent

developed by Roger Tsien that measured free cytosolic

Ca2� (13). The postdoc involved in this work was Bob

Charest. He was a chain smoker, and I worried about the

effects of cigarette ash on the Ca2� measurements!

During this period, we also engaged in some experi-

ments dealing with the effects of epinephrine and insulin

on glycogen metabolism and glucose uptake in skeletal

muscle using the isolated perfused rat hind limb prepara-

tion, and also themodulation of thesemetabolic processes

by adrenalectomy and thyroid status (14, 15). These stud-

ies were of value in the interpretation of related in vivo

experiments in man and experimental animals because

they were not confounded by secondary effects. The

importance of studying direct effects of hormones uncom-

plicated by in vivo changes also applied to our rat liver

perfusion studies. Interestingly, later experiments with in

vivomodels largely confirmed our findings in the perfused

liver, thus establishing the validity of our system. Jean-

Louis Chiasson, an energetic researcher from Montreal,

Melissa Dietz (Lojek), a graduate student and ballet

dancer, and Mike Caldwell, a surgeon, were key to these

experiments.

With the ready availability of hepatocytes, we also con-

ducted many studies examining factors that modulated

hormone effects on the liver. In an extension of our earlier

work indicating a species difference in the adrenergic

receptors involved in epinephrine action in the liver, we

found that age, adrenal cortical status, and gender also

influenced the extent to which epinephrine acted through

�- versus �-adrenergic receptors (16). Another finding

was that agonists acting through cyclic AMP enhanced

�1-adrenergic responses, whereas insulin and phorbol

esters inhibited them (17, 18). These observations not only

cleared up some confusion in the literature, but also

involved some rather colorful postdocs. For example,

there was the energetic, likeable, but budget-busting Chris

Lynch, now a Professor of Physiology at theHersheyMed-

ical School. His philosophy was to order what he might

possibly need rather than what he actually needed. Others

were Bernie Hughes, an Australian with an inexhaustible

store of risque stories, the charming Bernard Bouscarel

from Toulouse, who was notable because his girlfriends

sent him flowers, and Jean-Paul Dehaye from Brussels,

who was nicknamed “The Pope” after the pope at that

time, Jean Paul II, although he was not as infallible as the

pontiff! These characters were balanced by Tim Chan,

now a Dean at the University of Southern California, and

Noel Morgan, a steadfast Englishman who is now Head of

Biochemistry at amedical school inCornwall. At this time,

we were visited by Fatima Bosch from Barcelona, whose

energetic pursuit of research and stylish clothing

impressed us greatly.

Studies of the mechanisms involved in the actions of

Ca2�-mobilizing agonists reached an exciting phase in

which the hunt was on for the signal that came from the

receptor in the plasma membrane to the internal calcium

pool. Here we became misguided by our findings and

thought that the pool was in the mitochondria, a well

known source of Ca2�. Other workers deduced correctly

that the pool was in the endoplasmic reticulum, and I had

to endure some abuse for our sins at several meetings. We

were also skeptical of the emerging phosphoinositide

hypothesis as the basis of signaling to the internal Ca2�

store. This posited that the breakdown of phosphatidyl-

inositol 4,5-bisphosphate (PIP2) induced by certain ago-



nists generated a second messenger that released the

Ca2�. Because we found that an increase in Ca2� stimu-

lated the breakdown of this phospholipid in liver cells and

plasmamembranes, we felt that the hypothesis waswrong.

In point of fact, we did not realize that there was a much

earlier phase in the action of the agonists when PIP2 break-

down preceded a rise in Ca2�. Our questioning of the

phosphoinositide hypothesis raised the ire of the English

scientists, who were the main proponents. With the rec-

ognition of inositol 1,4,5-trisphosphate (IP3) as the intra-

cellular messenger by Robin Irvine, Michael Berridge, and

associates, the controversy went away, except for some

diehards with lingering resentment!

Much evidence was accumulating that a G protein was

involved in the actions of many agonists to stimulate the

breakdown of PIP2with the resultantmobilization of Ca2�

from the endoplasmic reticulum (19, 20). This early work

was done by three American postdocs and one from

Croatia with very different personalities and communica-

tion skills: Thom Fitzgerald, who barely said a word, Janet

Atkinson (Colbran), who exuded Southern charm and vol-

ubility, RonUhing, whowas notmuchmore talkative than

Thom, and Vera Prpic, whose accent did not prevent a

marriage with Ron. Because none of the knownG proteins

was found to be involved in the breakdown of PIP2, the

hunt was on for a novel G protein. We were successful in

isolating and purifying aG protein that activated the phos-

pholipase C that hydrolyzed PIP2 (PI-PLC), but could not

identify it (21). At the same time, the group of Paul Stern-

weis at the University of Texas Southwestern Medical

School at Dallas had raised antibodies to two novel G pro-

teins, but could not demonstrate that they activated PI-

PLC. So we joined forces to demonstrate that the novel G

proteins were in fact activators of the phospholipase and

hence the mediators of hormone action on intracellular

Ca2� mobilization (22). The two proteins were named Gq

andG11, and I am often asked why the Gq. It turns out that

a postdoc involved in the research in Paul’s laboratory was

an avid reader of the Gentleman’s Quarterly! A key player

in this research was Steve Taylor, a taciturn English post-

doc who preferred to fly on British Airways because of the

quality of their gin and tonics. Karen Shaw, a spunky Eng-

lish postdoc, was also involved in this work, as was Iro

Georgoussi from Athens, whose vibrant personality and

social skills livened up the social life of the laboratory. In a

later extension of this work, Gita Venkatakrishnan local-

ized the residues in the Gq �-subunit that were specifically

involved in the activation of PI-PLC.

Two interesting side projects arose from this work. The

first was the confirmation by Jonathan Blank, another

English postdoc, of the finding of Peter Gierschik at the

University of Heidelberg that the ��-components of G

proteins could activate some forms of PI-PLC (23), a find-

ing also reached by Ken Harden’s group at the University

of North Carolina. At that time, the idea that ��-subunits

of G proteins could be involved in cell signaling was an

issue of great controversy, sometimes vicious, and Gier-

schik had had great difficulty in publishing his findings.

Another side project involved the discovery by Jonathan

Blank and Gabriel Berstein in Elliott Ross’ laboratory at

UT Southwestern that PI-PLC could stimulate the GTP

hydrolase (GTPase) activity of Gq (24). In other words, the

phospholipase could inactivate its G protein activator,

indicating a novel feedback mechanism. All this and other

work extending to 1999 were supported by the efforts of

Annette Ross, a truly superb technician. Three very com-

petent secretaries, Penny Stelling, Carolyn Sielbeck, and

Judy Childs (Nixon), aided immensely in the preparation

of our publications during my tenure as a Hughes Investi-

gator. After two terms on the Editorial Board of the Jour-

nal of Biological Chemistry, I was appointed an Associate

Editor in 1988. I was again lucky to have the superlative

services of two editorial assistants: Carolyn Sielbeck, doing

double duty as my secretary, and Carolyn McDonald,

whose gentle Southern voice calmed many an agitated

author.

The breakdown of PIP2 generates 1,2-diacylglycerol

(DAG) as well as IP3, and if the theory is correct, these

should be produced in equimolar amounts in cells in

which PI-PLC is activated by agonists. Imagine our cha-

grin when Steve Bocckino, another great postdoc, meas-

ured these compounds chemically and found that, in vaso-

pressin-stimulated liver cells, the production of DAG

greatly exceeded that of IP3 and was much more pro-

longed (25). Now we were at odds with the experts in the

protein kinase C (PKC) area because it was believed that

the DAG that activated this kinase came from the break-

down of PIP2, making a nice story in which second mes-

sengers (IP3 and DAG) were simultaneously released in a

bifunctional signaling system. Further work confirmed

that PIP2 was breaking down but that another phospho-

lipid was being hydrolyzed to a greater extent. Measure-

ments of choline release by Helen Irving, an Australian

postdoc, and analyses of the fatty acid composition of the

DAG by Steve Bocckino and Guy Augert, a dashing post-

doc and fearless skier from the Haute-Savoie region of

France, indicated that the phospholipid was phosphatidyl-

choline (PC) (26, 27). This was a surprise because PC is the

major phospholipid of cell membranes, and its breakdown

would be expected to have deleterious effects on the cell.



However, chemicalmeasurements of PC later showed that

the decrease induced by agonist stimulation was barely

detectable, although the resulting increase in DAG was

sufficient for signaling.

The next surprise came when we set out to determine

the phospholipase responsible for the breakdown of PC.

By analogywith the phosphoinositide signaling system,we

expected the phospholipase to be of the C type. However,

chemical measurements showed that the initial product of

PC breakdown was phosphatidic acid, which was then

converted to DAG. In other words, the phospholipase was

of the D type (PLD) (28). This was somewhat disconcert-

ing because it was generally believed that this enzyme was

present in plants, but not animals! Julian Kanfer’s group in

Winnipeg had earlier obtained evidence of PLD in mam-

malian tissues, but their work had been largely ignored.

Once again, these findings were not welcomed by the afi-

cionados of the phosphoinositide hypothesis of cell signal-

ing. However, further work by others, including Claire

Allan, a Scottish postdoc with a delightful accent, showed

that both systems operated, i.e. agonists caused an initial

hydrolysis of PIP2, followed by a larger and more pro-

longed breakdown of PC.

Reverting back to our studies of PLD, it became clear

that a G protein played a major role in its regulation, but

none of the known heterotrimeric G proteins could be

shown to affect its activity. Then a report from the group

of David Lambeth at Emory appeared showing that

RhoGDI, an inhibitor of the activation of the small G pro-

tein Rho, inhibited the activation of PLD by guanosine

5�-O-(3-thiotriphosphate) (GTP�S) in membranes,

implying a role for members of the Rho family. Ken Mal-

com, an environmentally conscious postdoc, used recom-

binant forms of these proteins to show that they could

activate PLD directly (29). An important collaborator in

these studies wasMarc Symons, then atOnyx Pharmaceu-

ticals. There were also reports from the Sternweis and

Cockcroft laboratories that another small G protein, ADP-

ribosylation factor (ARF), could also activate partially

purified PLD or when tested in permeabilized neutrophils.

We confirmed this and focused on factors involved in the

action of ARF. This led to the discovery of the novel pro-

teins arfaptins 1 and 2 (30) and arfophilin (31). Interest-

ingly, both arfaptins and arfophilin were found to bind

other families of small G proteins, implying the existence

of novel signaling networks. Two gifted postdocs,

Hiroyuki Kanoh from Japan and Ok-Ho Shin from Korea,

were responsible for this work.

Because Rho was a major regulator of PLD1 in vitro, we

were interested in examining its role in vivo. Steve Plonk,

an M.D./Ph.D. student, showed that introduction of the

C3 exoenzyme of Clostridium botulinum, which inhibits

Rho, inhibited the activation of PLD by lysophosphatidic

acid (LPA) and other G protein-linked agonists, but not

platelet-derived growth factor (PDGF) (32). On the other

hand, Jean Hess, a postdoc now living in rural Vermont,

found that Rac mediated the effect of growth factors. It

was also found that LPA induced the membrane translo-

cation of Rho, but this did not occur with PDGF (33). The

G protein involved in the activation of Rho was shown by

ourselves and others to be G13 and not Gq, illustrating a

new signaling paradigm (34). Because heteromeric G pro-

teins do not link directly to monomeric G proteins, we

next searched for the guanine nucleotide exchange factor

(GEF) involved in agonist activation of Rho family pro-

teins. In collaboration with John Collard’s group in

Amsterdam, we showed that Tiam1, a GEF for Rac, was

translocated to membranes and phosphorylated on thre-

onine in cells treated with LPA (35, 36). Both PKC and

Ca2�/calmodulin-dependent kinase II were shown to

phosphorylate Tiam1, but only the calmodulin-dependent

enzyme induced activation (36). These findings did not

explain howG13 activated Rho (another group showed the

involvement of other GEFs), but pointed to a mechanism

for Rac activation. The postdocs principally involved in

thisworkwere Ian Fleming, not related to the author of the

James Bond series, and Greg Buchanan, the ultimate Uni-

versity of Tennessee fan who wore orange almost

continuously!

Many groups had obtained evidence that PKC could

activate PLD in vivo. This included evidence by Eui-JuYeo,

a Korean postdoc in my lab, that PKC mediated the effect

of epidermal growth factor on the enzyme. However, a big

surprise came in in vitro experiments showing that PKC

could directly activate PLD but that this did not involve

phosphorylation, but merely protein-protein interaction

(37). This nontraditional view of how PKC acted was sup-

ported by the findings of several other groups, but was not

accepted by others. The lead postdoc in this work was

Kevin Conricode, who was notable in having seven broth-

ers but no sisters. Our studies also showed that the activa-

tion of PLD1 by PKC involved the conventional �- and

�-isozymes, but not the other isozymes. Later work by two

Chinese postdocs, Tianhui Hu and Jun-Song Chen,

showed that PKC could phosphorylate PLD but that this

caused inactivation. This work on PKC led us to examine

the specific PKC isozymes activated by various agonists in

fibroblasts. Surprisingly, Kwon-SooHa, an energetic post-

doc fromKorea, found that�-thrombin and PDGF caused

a differential translocation/activation of PKC isozymes in



these cells. Thrombin caused a rapid membrane associa-

tion of the �- and �-isozymes, which correlated with rapid

increases in IP3/Ca2� and DAG due to PIP2 breakdown.

On the other hand, PDGF did not cause a translocation of

PKC�, but caused a delayed translocation of PKC� that

correlated with an increase in DAG due to PC breakdown

(38). Our general interest in PKC led us to a study of the

atypical PKC� isozyme, about which very little was known.

This first had to be isolated and purified before its regula-

tion could be studied. Hiroyuki Nakanishi, a postdoc from

Kobe, examined the effects of various lipids and found the

enzyme to be the first known target of phosphatidylinosi-

tol 3,4,5-trisphosphate (39), a lipid now known to be gen-

erated by insulin and other growth factors and an impor-

tant second messenger.

We next devoted a lot of effort to the purification, char-

acterization, and cloning of mammalian PLD. However,

the cloning of the two mammalian (human) PLD1 and

PLD2 isozymes was first accomplished by the group of

Michael Frohman at the State University of New York at

Stony Brook based on their earlier cloning of yeast PLD.

Seung-Kiel Park from Korea, in association with Joe Pro-

vost, a colorful ex-Armyman, cloned the rat liver enzymes

by an analogous approach (40), and this initiated an exten-

sive program to characterize their structure, kinetics, and

regulation (41). Much of what we found paralleled the

findings of the Frohman group, and in contrast to my ear-

lier research, no controversies arose between us! The

PLD1 isozyme was of particular interest because of its

complex regulation by PIP2, PKC, ARF, and members of

the Rho family of small G proteins (Rho, Rac, and Cdc42).

Furthermore, PKC and the Rho proteins were shown to

mediate the actions of certain agonists on the enzyme in

intact cells. Interestingly, in our early efforts to purify PLD

from rat brain, we inadvertently purified phospholipaseA1

to homogeneity, and because little was known about this

enzyme, I encouraged the postdoc to characterize it. This

was Matthew Pete, an ex-Marine who tackled the project

like it was Iwo Jima!

PLD isozymes from most sources are characterized by

the presence of two highly conservedmotifs, termedHKD,

which are required for catalytic activity. In PLD1, they are

separately located in the N and C termini. Zhie (Julie) Xie,

a gifted postdoc, ably assisted by Wan-Ting (Tina) Ho,

found that expression of one-half of the enzyme contain-

ing oneHKDmotif produced no catalytic activity, whereas

expression of both halves did (42, 43). The conclusion that

bothHKDmotifswere required to formadimeric catalytic

center was borne out by subsequent structural studies.

Julie and Tina later found that a short C-terminal

sequence in mammalian PLDs was absolutely required for

activity. The reason for this remains unknown. Seunghyi

Kook and Do SikMin, two postdocs from Korea, localized

the binding site of PKC on PLD1 to certain sequences in

the N terminus (44) while Songmin Cai from China iden-

tified the binding site for Rho in the C terminus (45). We

also did the reciprocal experiments. Thus, Tianhui Hu

identified a residue in PKC� that was critical for PLD1

activation, and Chang Dae Bae from Korea identified res-

idues in the activation loop of RhoA that were specifically

involved in activating the enzyme. Chang was a brilliant

postdoc, but was concerned about his health. He askedme

tomove a freezer nearer to his bench because the distance,

not far, was straining his legmuscles! An interesting aspect

of the regulation of PLD1 is the striking synergism

between certain of its activators. In an analysis of this in

collaboration with Alex Brown and Lee Henage, a brilliant

graduate student, it was found that the synergism

occurred only if a catalytic activator such as ARFwas com-

bined with a binding activator such as one of the Rho pro-

teins or a mixed activator such as PKC (46).

PLD is a ubiquitous enzyme that is amember of a super-

family that has been conserved through evolution. This

implies that it subserves some important functions. We

and others have identified a variety of cellular functions of

the PLD1 and PLD2 isozymes, but no underlying themes

have emerged, suggesting that the fundamental impor-

tance of PLD has yet to be defined. Another area of impor-

tance in PLD research is to determine the crystal structure

of the mammalian enzymes. The structures of the enzyme

from Streptomyces antibioticus and of some lowmolecular

mass members of the PLD superfamily have been defined

and have provided very useful information about the cat-

alytic center and themechanism of catalysis. However, the

reasons why certain isozymes are critically dependent on

PIP2 for activity and the molecular mechanisms by which

PKC, ARF, and Rho activate PLD1 remain unknown.

Thus, much more work needs to be done to define the

structure, regulation, and functions of these enzymes, the

distribution of which is so widespread.


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(1992) Phospholipase C-�1 is a GTPase-activating protein for Gq/11, its physi-

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diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and

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From the �-Glutamyl Cycle tothe Glycan Cycle: A Roadwith Many Turns andPleasant Surprises

Published, JBC Papers in Press, October 19, 2009, DOI 10.1074/jbc.X109.023150

Naoyuki Taniguchi

From the Department of Disease Glycomics, Institute of Scientific and Industrial Research, Osaka

University, Ibaraki, Osaka 567-0047 and the Systems Glycobiology Research Group, Chemical

Biology Department, Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan

As I have written previously about my personal history and research journey since

graduating frommedical school (1), in this article, I wish to focus on the biochemistry

of glutathione and glycosyltransferases, which, on the surface, would appear to be

quite different subjects.

Early Training in Biochemistry and Studies of Glutathione Metabolism during

Hepatocarcinogenesis

After obtaining an M.D. and finishing an internship at the university hospital in 1965, my

early training in research was obtained in the Department of Biochemistry, Hokkaido Uni-

versity Graduate School ofMedicine, where I received a Ph.D. degree in 1972. At that time, the

department was chaired by Professor Hidematsu Hirai, and the focus of the departmental

research effort was primarily on the biological and clinical significance of �-fetoprotein, an

oncofetal protein that is synthesized by embryonic tissues and primary hepatomas but not by

normal tissues (2). I had already developed an interest in enzymology, and I also became

interested in an area that was very different from the mainstream program in the department.

I decided to focus on the enzymes involved in glutathione metabolism during experimental

hepatocarcinogenesis of rats that had been fed 0.06% 3�-methyl-4-dimethylaminoazobenzene

under the guidance of Dr. Yutaka Tsukada. In these studies, rats were killed at weekly inter-

vals, and the glutathione levels and glutathione-related enzymes were assayed spectrophoto-

metrically. The experiments started early in the morning and usually ended at around mid-

night. I found that the activity of �-glutamyl transpeptidase, one of the glutathione-degrading

enzymes, in rats the fed azo dye underwent two biphasic changes, i.e. the activity was

increased at an early stage of hepatocarcinogenesis, then decreased, and finally increased

again at the late stage. Similar changes in the pattern for �-fetoprotein had been reported by

a group in the same department. The actual level of glutathione decreased at the early stage,

then increased, and again decreased at the late stage, mirroring the pattern observed for

�-glutamyl transpeptidase (3).

I hypothesized that �-glutamyl transpeptidase might also be an oncofetal protein, analogous to

�-fetoprotein. In fact, �-glutamyl transpeptidase activity is high in fetal tissues, but the enzyme is

also ubiquitously distributed and abundant in the kidney.

After receiving my Ph.D., I spent one year as a research fellow in the same laboratory and then

moved to the Department of Hygiene and Preventive Medicine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 50, pp. 34469 –34478, December 11, 2009Author’s Choice © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

DECEMBER 11, 2009 • VOLUME 284 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY


H108

�-Glutamyl Cycle and Alton Meister’s Laboratory

in New York

In 1974, Alton Meister, Professor and Chair of the

Department of Biochemistry at the Cornell Medical

School, presented a plenary lecture on the �-glutamyl

cycle at the annual meeting of the Japan Biochemical Soci-

ety held inOkayama, Japan. He was agreeable tomywork-

ing with him in New York, and I joined his group as a

visiting associate professor on September 1, 1976. At that

time, there were many postdoctoral fellows and faculty

members in Meister’s laboratory, and they published

numerous excellent papers on glutathione metabolism,

including the chemical synthesis of buthionine sulfoxi-

mine, a specific inhibitor of �-glutamylcysteine synthe-

tase; the chemical synthesis of glutathione esters that are

able to enter the cell; the feedback inhibition of �-glu-

tamylcysteine synthetase; and the characterization ofmet-

abolic disorders of glutathione via the �-glutamyl cycle,

first thought to play a role in amino acid transport but later

shown to be largely involved in the turnover of glutathione

(Fig. 1) (4, 5). He was a heavy smoker, so I could soon

recognize him by the smell of his cigars and pipes when he

showed up in the lab and when I visited him to discuss

research progress in his office on the first floor.

Glutathione is synthesized via two enzymes, glutathi-

one synthetase and �-glutamylcysteine synthetase, both of

which require ATP. Likewise, the degradation of glutathi-

one is catalyzed by two enzymes, �-glutamyl transpepti-

dase and �-glutamylcyclotransferase. I was involved in the

purification of �-glutamylcyclotransferase from rat kid-

ney, whichwas highly heterogeneous because of sulfhydryl

modifications of the enzyme.

Hokkaido University and Cancer-associated Changes

in Glycoproteins: A Saint’s Maid Quotes Latin

I was supposed to become an associate professor in a

newly launched department, i.e. the Graduate School of

Environmental Science at Hokkaido University. I was

obliged to return to Japan, but the research situation was

not well organized, and I became interested in moving to

another laboratory. Half a year later, Professor Akira

Makita kindly offered me a position of associate professor

in the Cancer Institute of the same university. His group

discovered various cancer-associated changes in various

enzymes such as arylsulfatases (6), �-glucuronidase, hex-

okinases, cathepsinD, and galactosyltransferase-1 and gly-

colipids such as sulfatides. Some of the work on sulfatides

was subsequently carried out by Koichi Honke, who puri-

fied sulfatide synthase, cloned the cDNA and gene, pro-

FIGURE 1. �-Glutamyl cycle proposed by Alton Meister (12). The cycle is involved primarily in the turnover of glutathione, i.e. glutathionesynthesis and degradation. AA, amino acid.

REFLECTIONS: From the �-Glutamyl Cycle to the Glycan Cycle

JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 50 • DECEMBER 11, 2009H109

duced null mice, and discovered many interesting pheno-

types (7). He later joined our group as an associate

professor and was a valuable member of our department.

When I first joined theMakita laboratory, I was not famil-

iar with glycoprotein or glycolipid research, and I was just

like a saint’s maid quoting Latin. Finally, however, I

learned many techniques that subsequently proved to be

useful in the area of glycobiology and published several arti-

cles under his guidance. I enjoyed my tenure as an associate

professor in his group very much, as well as my interactions

with the various graduate students in the group. I also pub-

lished several papers on non-enzymatic glycosylation (glyca-

tion), theMaillard reaction, in relation to free radical research

or diabetes, which subsequently became the other project of

my research journey when I moved to Osaka University (8).

While there, I was able to restart experiments on �-glu-

tamyl transpeptidase. At that time, several glycolytic

enzymes such as aldolase C, hexokinase, and pyruvate

kinase had been reported to be activated in cancer and

fetal and muscle tissues, and isozymic forms such as the

muscle type were present in hepatoma tissues. Even

though my preliminary experiments on �-glutamyl

transpeptidase suggested that isozymic forms were not

present, I nevertheless continued to wonder if a specific

isozymic form of �-glutamyl transpeptidase was also acti-

vated in hepatomas. �-Glutamyl transpeptidase is a glyco-

protein and was first purified from bovine kidney byMeis-

ter’s group. Therefore, I was interested in purifying the

enzymes again from various rat tissues such as fetal, kid-

ney, normal liver, and tumor tissues with the objective of

comparing their biochemical properties as judged by

kinetic properties, isoelectric focusing patterns, antigenic-

ity, and amino acid analysis. The only difference we found

was in the sugar composition, i.e. the levels of neutral sug-

ars and sialic acid, as the isoelectric focusing patterns var-

ied before and after treatment with sialidase (9, 10). Since

then, I decided to focus on the sugar chains of the enzyme.

In collaboration with Professor Akira Kobata (Kobe

University), an eminent glycobiology expert, we found

that the enzyme purified from ascites hepatoma AH-66

cells contained bisecting GlcNAc, whereas the enzyme

purified from normal liver did not (11).

Osaka University Medical School, Department of

Biochemistry: From Protein Chemistry to Glycobiol-

ogy (Purification of Glycosyltransferases) and from

Gene Cloning to Functional Glycomics

In February 6, 1986, I received an unexpected phone call

from the dean of the Osaka University Medical School,

who told me that I was a candidate for the position of

Professor and Chair of the Department of Biochemistry,

one of the oldest and most prestigious biochemistry

departments in Japan. The First Chair of the department

was Professor Yashiro Kotake, who elucidated the chem-

istry of kynurenine and was an eminent biochemist and an

expert in tryptophan metabolism. The Third Chair was

Professor Osamu Hayaishi, a world-renowned and emi-

nent biochemist famous for his pioneering work on oxy-

genase and prostaglandins. I was very happy and honored

to accept this position and subsequently moved to Osaka

on April 1, 1986. I decided to change my project from

protein chemistry to glycobiology in this new department.

Purification of Glycosyltransferases and Gene Cloning—

As 1988 was the 100th anniversary of the discovery of

glutathione, I co-organized a meeting on the “Glutathione

Centennial: Molecular Perspectives and Clinical Implica-

tions” with Dr. Yukiya Sakamoto and his group. Alton

Meister also attended the conference as an organizer. At

the meeting, he comprehensively reviewed the history of

glutathione and its metabolism, designated as the �-glu-

tamyl cycle (12), as shown in Fig. 1. His group had been

involved in studies in this area for the last 10 years or so.

We reported on the purification and cDNA cloning of

human �-glutamyl transpeptidase and also reported on

the significance of highly activated UDP-N-acetylglucos-

amine:�-D-mannoside �1,4-N-acetylglucosaminyltrans-

ferase III (GnT-III), which may modify �-glutamyl

transpeptidase in hepatoma tissues and hyperplastic nod-

ules. In 1988, we also reported on a marked activation of

GnT-III activity in primary hepatoma and ascites hepa-

toma, which could explain the unique changes in the sugar

units attached to �-glutamyl transpeptidase, i.e. the addi-

tion of a bisecting GlcNAc, a product of GnT-III.

In 1992, we organized the Sapporo Cancer Seminar,

which was founded by Dr. Hiroshi Kobayashi, Professor

Emeritus ofHokkaidoUniversity; this seminar is aGordon

Conference-like meeting that focuses on cancer research.

I invited Alton Meister, Owen Griffith, Susumu Nish-

imura, Cecil Pickets, Harold Deutsch, and other distin-

guished speakers who were actively involved in glutathi-

one research, redox regulation, and nitric oxide and

reactive oxygen species (Fig. 2). Immediately after the

meeting, we celebrated Alton’s 70th birthday in Osaka,

andmany Japanese scientists, good friends of his or former

colleagues, attended. Since Yoshitaka Ikeda in our group

joined Alton’s laboratory in 1993, we continued the work

on the catalytic properties of �-glutamyl transpeptidase in

a collaborative effort, which focused mainly on studies of

the catalytic mechanism of the enzyme by site-directed

mutagenesis. Two years later, in 1995, Alton suffered a

stroke during a trip toNewZealand, and after returning to


DECEMBER 11, 2009 • VOLUME 284 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRYH110

New York, he passed away in December at the age of 72.

The final paper that Alton actually handled was ours,

which appeared in the Journal of Biological Chemistry in

September 1995 (13); this article was the culmination of

his work. Alton’s death was a great loss not only for gluta-

thione metabolism but also for biochemistry in the world.

His enormous contributions to our understanding of glu-

tathione metabolism through the concept of the �-glu-

tamyl cycle (Fig. 1) and amino acidmetabolismwill remain

forever as his legacy. Our data on the catalytic mechanism

of �-glutamyl transpeptidase, including the work with

Alton, were published (14).

Starting in 1986, we began to purify the glycosyltrans-

ferase, which catalyzes the biosynthesis of the bisecting

GlcNAc found in �-glutamyl transpeptidase purified from

ascites hepatoma AH-66 cells. At that time, the method

used in assaying glycosyltransferases involved the incor-

poration of 14C- or 3H-labeled nucleotide sugars into the

acceptor substrate, but this was a time-consuming and

rather expensive method. Therefore, we attempted to

develop a simpler, less costly method of analysis. The

groups of Drs. Hase and Ikenaka at Osaka University

developed amethod involving the pyridylamination of the

nonreducing end of glycans to analyze sugar chains.

Atsushi Nishikawa and co-workers (15) successfully em-

ployed this technique to assay the activity of various gly-

cosyltransferases such as GnT-III, -IV, -V, and -VI. Using

this assay method, we measured the GnT-III activity of

various hepatoma cells and azo dye-induced hepatomas

and found that the enzyme activity was substantially ele-

vated in most hepatomas. In some cases, the activity was

100-fold greater than that in a normal rat liver.

To obtain a cDNA clone, we attempted to purify GnT-

III by affinity chromatography using biantennary sugar

chains, the substrate for GnT-III.We first prepared a large

amount of bisected biantennary sugar chains from human

serum transferrin. Because GnT-III activity is very high in

rat kidney, we collected a large number of kidneys from

rats that had been killed during physiological experiments

in other laboratories. We ultimately collected 10 kg of rat

kidneys. The enzyme was purified �153,000-fold in 1.5%

yield from a Triton X-100 extract of rat kidneys by frac-

FIGURE 2. 12th Sapporo Cancer Seminar in 1992. Shown are Alton Meister, to the left of me (front row, fourth from the right), and Hiroshi Kobayashi,to the left of Meister.



tionation procedures utilizing QAE-Sepharose, Cu2�-

chelating Sepharose, and affinity chromatography on

UDP-hexanolamine and substrate-conjugated Sepharose,

and finally, the cDNA encoding GnT-III was successfully

cloned (16). The enzyme is responsible for the formation

of a bisecting glycan, as first published by Dr. Harry

Schachter, who reported the presence of numerous

N-acetylglucosaminyltransferases such as GnT-I, -II, -III,

-IV, -V, and -VI. His group purified GnT-I and GnT-II for

the first time and then cloned their genes (17). His contri-

bution in this field has been enormous, and ImetHarry for

the first time in 1989, when Dr. Makita organized the Sec-

ond Sapporo Cancer Seminar at a time when I was a new

comer in this field. Since then, every time I met Harry, he

gave me valuable suggestions and encouragement in our

work on glycosyltransferases even though his lab has been

our major competitor in this area because very few groups

have attempted to purify and biochemically characterize

the enzyme and then clone the gene (Fig. 3).

We further purified various glycosyltransferases in-

volved in N-glycan branch biosynthesis one by one and

cloned their genes; these included GnT-V (18) and GnT-

IV, GnT-VI, synthetic enzyme for poly-N-acetyllac-

tosamine, and �1,6-fucosyltransferase (FUT8) (19, 20).

Our group also cloned other glycosyltransferases using

molecular cloning techniques as depicted in Fig. 4. In 1989,

I organized the first glycosyltransferase meeting in Osaka,

and numerous eminent scientists in this field, including

Robert Hill, Pamela Stanley, Jamey Marth, Gerald Hart,

Ajit Varki, Jurgen Roth, Minoru Fukuda, Laurence Tabak,

Andre Verbert, John Lowe, and many renowned Japanese

glycobiologists, attended, and the significance of glyco-

syltransferases was discussed. Last year, the sixth meeting

(nowcalledGlycoT)was organized byRichardCummings

and Michael Pierce and held in Atlanta.

Functional Glycomics Approach and Lessons fromGnT-

III Transfectants—Using the cloned glycosyltransferase

genes, we next initiated studies related to functional gly-

comics using sugar-remodeling techniques by gene trans-

fections, knock-out and/or transgenic mice, and small

interfering RNA.Wewere able to show various functional

changes in glycans as the result of aberrant glycosylation

(21–24) using the cloned glycosyltransferases. Moreover,

to utilize a functional glycomics approach, we emphasized

the importance of identifying the target protein(s) of the

glycosyltransferase genes. At that time, some researchers

asked, “What would happen if you were able to identify

one or two target proteins among the numerous glyco-

sylated proteins that could undergo glycosylation? This is

analogous to pointing out one star among the universe.”

However, we now all realize that, even though the knock-

out of a certain glycosyltransferase gene may lead to vari-

ous phenotypic changes due to a lack of glycan in various

glycoproteins, in fact, phenotypic changes accompanied

by functional changes in glycans are very limited. This

indicates that the identification of target protein(s) is very

important in characterizing glycan functions. In 2002, I

co-edited and published a handbook in which most of the

biochemical and molecular properties of glycosyltrans-

ferases and related genes are described (25). Our major

glycosyltransferases of interest are depicted in Fig. 4.

As we observed previously, �-glutamyl transpeptidase

was one of the target proteins for GnT-III. To explore the

other functions of theGnT-III gene and its target proteins,

our group conducted experiments using cells that had

been transfected by the GnT-III gene and found different

types of likely target proteins as follows (21–24). K562 cells

are usually killed by the immune systems in the spleen, but

GnT-III-transfected K562 cells are not. This is due to a

decreased sensitivity to natural killer cell cytotoxicity,

which leads to increased spleen colonization by these cells

in athymic mice. We identified target proteins that recog-

nize bisecting GlcNAc, a product of GnT-III, as annexin V

and apoprotein B.

FIGURE 3. Dr. Schachter’s laboratory in Toronto (from left to right):Jim Dennis, Harry Schachter, Naoyuki Taniguchi, and Jeremy P.Carver.

FIGURE 4. Glycosyltransferases involved in N-glycan branchingcharacterized by our group. The enzymes in boxes indicate the namesof glycosyltransferases purified and their cDNAs cloned by our group.GnT-IX was cloned based on the genome sequence.



Epidermal growth factor receptors (EGFRs) also con-

tain N-glycans, and several researchers have proposed

that they play a role in growth factor signaling. The

deletion of Asn241 from the EGFR permits the molecule

to dimerize spontaneously without phosphorylation

(26). Erb-2 glycans play a key role in growth factor sig-

naling because, without glycans, Erb-2 dimerizes with-

out phosphorylation and enhances tumor development

in vitro (27). In GnT-III-transfected glioma cells, EGF

binding was blocked, and autophosphorylation of the

EGFR occurred. In HeLa cells, GnT-III transfection inhib-

ited the low affinity binding of EGF to the EGFR but

enhanced the high affinity binding and the internalization

rate of the EGFR. The transfection of GnT-III further

resulted in 1) the inhibition of the growth factor response

of tyrosine phosphorylation of the Trk/nerve growth fac-

tor receptor following the addition of nerve growth factor,

2) the inhibition of neurite growth induced by co-stimula-

tion of EGF and integrins, and 3) the inhibition of EGFR-

mediated extracellular signal-regulated kinase activation.

The target proteins ofGnT-III responsible for these effects

were identified as the EGFR and integrin �3�1. CD44

obtained from GnT-III-transfected mouse melanoma

cells exhibited increased adhesion to hyaluronate, which

enhanced CD44-mediated tumor growth and metastasis

in the spleen after subcutaneous inoculation into mice.

The �1–3Gal epitope is a xenotransplantation antigen

absent in humans. Thus, in the case of xenotransplanta-

tion from pigs to apes or humans, subacute rejection will

occur because of the presence of an antibody against the

�Gal epitope in humans and apes. Interestingly, the trans-

fection of GnT-III into pig cells or the generation of GnT-

III transgenic pigs markedly reduced the �Gal epitope by

inhibiting �1–3Gal-transferase action, thus reducing its

antigenicity.

GnT-III and GnT-V Have Opposite Effects on Cancer

Metastasis and Cell Adhesion—GnT-III serves as a STOP

signal for other glycosyltransferases such as GnT-II, -IV,

and -V, and once a bisecting GlcNAc is incorporated,

those enzymes no longer act. Therefore, a high expression

of GnT-III would be expected to inhibit the synthesis of

multiantennary complex carbohydrates and to evoke var-

ious phenotypic alterations reflecting abolished glycan

functions. Gu et al. (18) confirmed this substrate specific-

ity using purified GnT-V and demonstrated that GnT-V

does not act on a bisected biantennary chain. This catalytic

function was originally reported by Schachter et al. (17)

using a crude enzyme preparation. B16-hm cells, in which

GnT-V is highly expressed, therefore leading to increased

�1,6 branching, have a high metastatic potential. In con-

trast, lung metastasis in syngeneic and athymic mice after

an intravenous injection of B16-hm cells transfected with

the gene encoding GnT-III was dramatically suppressed

(28). GnT-V catalyzes the addition of a �1–6 linkage to

the Man�1–6 residue of GlcNAc�1–2Man�1–6Man

between the arm of the N-glycan core. In the GnT-III

transfectants, the level of �1,6 branches is decreased due

to competition for the substrate by intrinsic GnT-V and

ectopically expressed GnT-III (Fig. 5). The underlying

mechanism by which lung metastasis is suppressed is due

to elevated E-cadherin levels on cell-cell borders. Upon

overexpression of GnT-III in melanoma cells, E-cadherin

remains on the cell-cell border and enhances the attach-

ment of cancer cells to protect cancer metastasis. As

FIGURE 5. Competitive reaction between GnT-III and GnT-V. Because GnT-III and GnT-V act on the same substrate but GnT-III acts on the substratefirst and produces a bisecting GlcNAc structure, GnT-V is not able to act further. This leads to a suppression of the metastatic potential in vivo.Computer modeling of these reactions provided support for the this hypothesis.



E-cadherin��-catenin complexes are quite stable, the

increased E-cadherin levels result in the complete down-

regulation of the tyrosine phosphorylation of �-catenin

after EGF treatment (23, 24).

Conversely, the disruption of E-cadherin-mediated cell

adhesion appears to be a central event in the transition

fromnoninvasive to invasive carcinomas. Interestingly, we

recently found that E-cadherin-mediated cell-cell interac-

tion up-regulated GnT-III expression, suggesting that the

regulation of GnT-III and E-cadherin expressionmay rep-

resent a positive feedback loop and may be implicated

in endothelial-mesenchymal transition (29). E-cadherin

contains four N-glycan sites, and the role of glycans in

terms of cell adhesion is now well recognized.

The increased GnT-V levels were attributed to the up-

regulation of the Ets family of transcriptional activators,

which increase GnT-V expression. Knock-out mice of

Mgat5, encoding GnT-V, develop into apparently normal

adults, but the incidences of polyoma virusmiddleT onco-

gene-induced mammary tumor growth and metastases

are considerably lower in knock-out mice than in trans-

genic littermates. N-Glycans bearing the GnT-V branch

amplify oncogene signaling of tumor growth in vivo (30).

Integrins, crucial molecules for cell adhesion, have been

shown to be the major N-glycan-containing molecules on

cell membranes. For example, integrin �5�1 contains 14

and 12 putative N-glycosylation sites on the �5 and �1

subunits, respectively. Introduction of a bisecting GlcNAc

toN-glycans of integrin �5 by the overexpression of GnT-

III diminished ligand binding, cell adhesion, and cell

migration. Contrary to the functions of GnT-III, the over-

expression of GnT-V promoted integrin �5�1-mediated

cellmigration (31). Indeed, the opposing effects ofGnT-III

and GnT-V have been observed for the same target pro-

tein, integrin �3�1. N-Glycosylation of the �-propeller

domain on the �5 subunit (32) and the I-like domain on

the �1 subunit is essential to both the heterodimerization

and biological functions of the subunits. In addition,

among many N-glycosylation sites of the �5 subunit, only

N-glycosylation site-4 on the �-propeller was specifically

modified by GnT-III, thereby regulating integrin �5�1-

mediated cell spreading and cell migration.

Our group identified matriptase as a target protein for

GnT-V and showed that the overexpression of GnT-V

results in the increased expression ofmatriptase due to the

resistance of matriptase to autodegradation. Matriptase is

known to be associated with cancer invasion andmetasta-

sis, and its expression is enhanced in some types of tumor

cells. Moreover, GnT-V may function as an inducer of

angiogenesis, thereby promoting metastasis (22–24).

TIMP-1 was identified as a target protein for GnT-V. The

poly-N-acetyllactosamine chains on GnT-V-dependent

N-glycan antennae serve as ligands for galectin-3, and it

has been suggested that GnT-V strengthens the galectin-

3/glycoprotein lattice on the T-cell receptor, thereby

restricting the recruitment of T-cell receptors to the site of

antigen presentation and impeding antigen-dependent

receptor clustering and signal transduction (33).

Core Fucosylation of Glycoproteins Is Implicated in

Growth, Lung Emphysema, Antibody Therapy, and a Can-

cer Biomarker—L-Fuc:N-acetyl-�-D-glucosaminide �1,6-

fucosyltransferase (FUT8) catalyzes the transfer of a

fucose residue from GDP-Fuc to position 6 of the inner-

most GlcNAc residue (core fucose residue) of anN-linked

sugar hybrid and complex types of N-linked oligosaccha-

rides on glycoproteins. The enzyme was first purified to

homogeneity and cloned by our group from porcine brain

as well as from human gastric cancer cells (19, 20). Dele-

tion of the Fut8 gene resulted in marked phenotypic

changes in mice, namely 60% of the mice died during the

neonatal period, and the remainder died within 3 weeks.

Using Fut8 knock-out mice, we found that themRNA lev-

els of MMP-1, -9, and -12 were specifically up-regulated

among the various matrix metalloproteinases (MMPs)

examined. MMP gene expression occurs specifically due

to the deletion of the core fucose in the transforming

growth factor-� (TGF-�) receptor, which usually sup-

presses MMP expression.

The up-regulation of MMPs results in the degradation

of extracellular matrix proteins such as laminin and type

IV collagen, which may facilitate the development of lung

emphysema inmice and humans. In Fut8 knock-outmice,

the phosphorylation of SMAD proteins was markedly

down-regulated, suggesting an impairment in the TGF-�

cascade. The core fucosylation of the TGF-� receptor was

completely abolished in Fut8 knock-out mice, indicating

that the binding affinity for the ligand, TGF-�, was

impaired, and therefore, the negative regulation of TGF-�

signalingwas also suppressed (34). The Fut8 nullmutation

inmice also leads to the impairment of LRP-1 (low density

lipoprotein receptor-related protein-1) (35) and an

impaired pre-B-cell re-population due to dysfunction of

VECAM-2 (vascular endothelial cell adhesion mole-

cule-1) and integrin �4�1 (36).

Some natural killer cells contain receptors for the Fc

domain of IgG and bind to the Fc portion of the IgG anti-

body on the surface of a target cell, such as tumor cells, and

release cytolytic components that kill the target cell. This

killing mechanism is referred to as antibody-dependent

cell-mediated cytotoxicity.



It is well known that mouse monoclonal antibodies

and humanized antibodies, including clinically effective

agents such as trastuzumab (Herceptin�) and rituximab

(Rituxan�), require both activation via Fc�RIII and

inhibition via Fc�RIIB antibody receptors. The expres-

sion of antibodies with altered glycoforms, especially

the addition of bisecting GlcNAc, leads to an increase

in antibody-dependent cell-mediated cytotoxicity

through a higher affinity for FC�RIII by up to 10–20-

fold. Similar to the line of evidence described above, two

groups reported that binding of fucose-deficient IgG1

to human Fc�RIIIA was improved by up to 50–100-fold

(37, 38). Core fucosylation also plays important roles in

the adhesion of tumor cells via integrins (39). These

findings strongly suggest that the �1,6-fucosylation of

N-glycans modifies the function of the glycoproteins.

The Glycomics Approach Is One of the More Promising

Approaches for the Discovery of Cancer Biomarkers—In

2003, the International Union of Biochemistry andMolec-

ular Biology (IUBMB) Congress in Toronto was cancelled

due to the SARS problem, and a joint meeting of the

Human Proteome Organization (HUPO) with IUBMB

was held in Montreal. I attended the council meeting of

HUPO and was asked by several participants to launch a

glycomics initiative because there was no official initiative

on glycomics under HUPO. We finally launched the

Human Disease Glycomics/Proteome Initiative (HGPI),

with myself as the chair, along with many glycobiologists

and biochemists who agreed to collaborate.Many experts,

especially experts in mass spectrometry, joined this initia-

tive and participated in the steering committee meeting.

Among them was the Nobel Laureate Koichi Tanaka and

many others. Since the launch of HGPI, two pilot studies

of N-glycan (40) and O-glycan analyses have been per-

formed with 26 participating laboratories. In 2006, with

Jim Paulson, Sudhir Srivastava, Pamela Marino, and Ram

Sasisekharan, I organized a joint meeting at the National

Institutes of Health entitled “Frontiers in Glycomics:

Bioinformatics and Biomarkers in Disease,” and on this

occasion, we proposed a white paper emphasizing the

importance of glycomics (41). In terms of identifying can-

cer biomarkers using glycomics, Dr. Sen-ichiro Hakomori

has made many pioneering discoveries. The fucosylation

of �-fetoprotein is markedly increased in patients with a

primary hepatoma, and affinoelectrophoresis using lectin

and antibody can be used to distinguish between patients

with hepatocellular carcinoma and those with chronic

hepatitis and liver cirrhosis as reported by Taketa et al.

(42). In 2006, the Food andDrugAdministration approved

the use of fucosylated �-fetoprotein for the differential

diagnosis of patients with liver cirrhosis and primary hep-

atoma. Glycan patterns of the �-fetoprotein L3 fraction

and its enzymatic basis were reported by our group (43).

Using a similar approach, fucosylated haptoglobin was

identified as a possiblemarker for pancreatic cancer.Most

fucosylated proteins are found in the bile duct under nor-

mal conditions, but in the case of cancer, polarity changes

occur, which are possibly due to the expression of carrier

FIGURE 6. Schematic drawing of the glycan cycle (fucose cycle). Glycosylation is affected by various factors as described in text, and the pro-formis converted to the mature form of glycoproteins by glycosyltransferases and then localized on the cell surface, such as growth factor receptors bybinding to lectins. The similar cycles such as the GlcNAc cycle, sialic acid cycle, etc., will be present. TGF�-R; TGF-� receptor; GMD, GDP-mannose4,6-dehydratase; FX, GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase; ER, endoplasmic reticulum.



protein(s) or the disorganization of tumor cells (44). The

levels of GDP-fucose, as well as synthetic enzymes such as

GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase

and GDP-mannose 4,6-dehydratase, play an important

role in core fucosylation.

“Glycan Cycles” as Functional Units of Glycan Functions

for Understanding the Systemic Approach: To Make Mov-

ies Rather Than to Take a Snapshot—Here, I propose the

concept of the “glycan cycle” as a functional unit of glycans

that will permit the integration of glycan functions in anal-

ogy with the �-glutamyl cycle for understanding glutathi-

one metabolism (Fig. 1). Jim Dennis reported thatMgat5,

a gene that encodes GnT-V, plays a key role in growth and

arrest via the glycan structure of growth factor receptors

and GLUT1 by elucidating the binding of galectin-3 (45).

However, glycosylation is affected not only by a single gly-

cosyltransferase such as GnT-V but also by various com-

ponents such as the levels of mono-oligosaccharides,

nucleotide sugars, nucleotide transporters, the localiza-

tion of glycosyltransferases, nucleotide sugar levels in

the Golgi, transcription factors, the pro-form and

mature form of glycoproteins, and the structure of cell-

surface glycoproteins (46). All of these components

should be considered in detecting changes in more

dynamic ways; namely, it is necessary not only to take a

snapshot but also to make movies of the dynamic

changes in glycan metabolism.

For example, glucose enters the cell via a glucose trans-

porter and is converted to UDP-GlcNAc via the hexosa-

mine pathway.UDP-GlcNAcplays a key role in the cytosol

and is then incorporated into the Golgi via the UDP-

GlcNAc transporter and serves as a donor substrate for

various GlcNAc-transferases from GnT-I to GnT-VI.

UDP-GlcNAc also serves as a donor forO-GlcNAc-trans-

ferase in the cytosol. These enzymes function to modify

target proteins such as TGF-�, the EGFR, and the glucose

transporter. These receptors are localized on the cell sur-

face and bind to lectins such as galectin-3.When the affin-

ity of lectin binding is decreased due to changes in recep-

tor glycosylation, the receptors are endocytosed, and the

free monosaccharide is probably recycled. This concep-

tual “functional unit of the glycan cycle,” such as just

described for GlcNAc, is intended to help develop an

understanding of the integrative and dynamic analysis of

glycan functions. The same is true for the “fucose cycle,”

in which GDP-fucose, GDP-mannose 4,6-dehydratase,

GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase,

the GDP-fucose transporter, and fucosyltransferases are

key components for core fucosylation as depicted in Fig. 6.

Similarly, the “galactose cycle” and “sialic acid cycle,” etc.,

would be present.

Among the components of the glycan cycle, nucleotide

sugars play key roles in the function of glycans. Very

recently, our group developed a method for the simulta-

neous assay of nucleotide sugars using ion-pair reversed-

phase high pressure liquid chromatography and observed

dramatic changes in nucleotide sugar levels in cells cul-

tured under dense or sparse conditions (K. Nakajima, S.

Kitazume, R. Fujinawa, E. Miyoshi, and N. Taniguchi,

unpublished data). This indicates that nucleotide sugars

may control glycan structures and function via the glycan

cycle (Fig. 6). To clarify glycan functions in a more

dynamic way, after I retired from the Osaka University

Medical School in 2006 after reaching retirement age, I

became an EndowedChair Professor of theDepartment of

Disease Glycomics in the same university, with support

from Seikagaku Corp., and we subsequently launched a

Systems Glycobiology Research Group at RIKEN (Wako,

Japan).We expect that our understanding of glycan cycles

will be enhanced considerably in the near future.

Acknowledgments—I thank my many colleagues who collaborated with

me over three decades, many whose names also appear in the list of

literature citations. During my stay at Osaka University, many eminent

scientists came to visit as visiting professors and contributed to our

research and education in ourM.D./Ph.D. programaswell. Among them,

I thank Professors Dirk van den Eijnden, John Gutteridge, Kunihiko

Suzuki, Harold F. Deutsch, Kosaku Uyeda, Takashi Yonetani, Peng

GeorgeWong, Mary Anderson, OwenW. Griffith, andWilliam Lennarz,

who spent from three weeks to one year as visiting professors and sup-

ported our research and the M.D./Ph.D. program in our faculty. I thank

Drs. Vincent C.Hascall, Etorre Appella,Milton Feather, JianguoGu, and

Eiji Miyoshi and Lisa Jenkins for reading this manuscript prior to sub-

mission and for valuable suggestions. I thank Dr. Kazuki Nakajima and

FumiOta for help in preparing thismanuscript. I acknowledge continued

grant support from the Japan Society for the Promotion of Science and the

Ministry of Education, Sports, Culture, Science, and Technology, Japan

(from 1973 to the present).


Author’s Choice—Final version full access.

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VOLUME 284 (2009) PAGES 34469 –34478

DOI 10.1074/jbc.A109.023150

From the �-glutamyl cycle to the glycan cycle: a road

with many turns and pleasant surprises.

Naoyuki Taniguchi

On page 34472, right column, second paragraph, line 3, the sentence

should read as follows: “We first prepared a large amount of non-bi-

sected biantennary sugar chains from human serum transferrin.”

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 5, p. 3524, January 29, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 5 • JANUARY 29, 2010

ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.org

We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the originalarticle. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carrynotice of these corrections as prominently as they carried the original abstracts.

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