Nextran Inc., an affiliate of Baxter Healthcare Corporation, San … · 2001. 3. 20. · Core...

Lennarz W.J. and Hart G.W., eds. 1994. Guide to techniques in glycobiology. Methods Enzymol., vol. 230. AcademicPress, San Diego, California.

Iozzo R., ed. 2000. Proteoglycans: Structure, biology and molecular interactions. Marcel Dekker, Inc., New York.

Jeffrey Esko, Professor of Cellular and Molecular MedicineAssociate Director, Glycobiology Research and Training CenterUniversity of California, San Diego, La Jolla, California

Adriana Manzi, Director, Analytical Research and Development/Quality ControlNextran Inc., an affiliate of Baxter Healthcare Corporation, San Diego, California

N-CAM neural cell adhesion moleculeNeu5Ac N-acetyl neuraminic acidNGF nerve growth factor

From Page xi

FromContributors,Page xiii

FromAbbreviations,Page xvii

FIGURE 1.4. Recommended symbols and conventions for drawing glycan structures. The exam-ple

From Chapter 1,Page 6


FIGURE 1.5. Basic core structures of the common classes of animal glycans. (For the monosac-charide symbol code used for the depictions, see Figure 1.4.)

polylactosamine and is therefore attached to a N- or O-glycan core rather than a typical proteogly-can core region. One type of glycosaminoglycan, hyaluronan (GlcNAcβ1-4GlcA)n, appears to existprimarily as a free sugar chain, unattached to any aglycone. The glycosaminoans



FIGURE 1.6. Biosynthesis, utilization, and turnover of a common monosaccharide. This schemat-ic shows the biosynthesis, fate, and turnover of one common monosaccharide constituent of ani-mal glycans, galactose. Although small amounts of galactose can be taken up from the outside ofthe cell, most is either synthesized de novo from glucose or recycled from degradation ofglycoconjugates in the lysosome. The schematic presents a simplified view of the generation ofthe UDP sugar nucleotide, its equilibrium state with UDP-glucose, and its uptake and utilizationin the Golgi apparatus for synthesis of new glycans. (Solid lines) Biochemical pathways; (dashedlines) pathways for the trafficking of membranes and glycans.

runs for most of the length of one surface of the protein. This cleft is astonishingly large, consid-ering that lysozyme has only 129 amino acids, and is capable of accommodating a hexasaccharideand cleaving it into a disaccharide product and a tetrasaccharide product.

From Chapter 4,2

N-acetylglucosamine (22,23)

Synthesis of UDP-GlcNAc begins with the formation of GlcN-6-P from Fru-6-P by transamidationusing glutamine as the –NH2 donor. Glucosamine-6-P is then N-acetylat

but appears optimal on N-glycans bearing the GlcNAcT-V branch. GlcNAcT-V requires the prioractivity of GlcNAcT-II. GlcNAcT-III and GlcNAcT-V are sometimes exclusive, as the action ofGlcNAcT-III inhibits the action of GlcNAcT-V. GlcNAcT-VI action is uncommon, but it may requireprior branching by GlcNAcT-II and GlcNAcT-V.

N-glycosylation sites on the same protein may contain different glycan structures, a finding referredto as microheterogeneity. It appears that factors other than protein sequence may influence N-glycandiversification. Such factors may include sugar nucleotide metabolism, transport rates in the ER andGolgi, and the localization of glycosyltransferases in the






FIGURE 8.1. Human small intestinal mucin. Large numbers of O-glycan chains (wavy lines) arefound throughout the polypeptide (encoded MUC2 gene), also termed the apomucin. A few N-glycosylation sites are indicated (tridents). Disulfide bridges may occur in an intra- or intermole-cular fashion. (Reprinted with permission, from [56] Toribara et al. 1991.)

FIGURE 8.2. Multiple polypeptideGalNAcTs initiate O-glycosylation in allmulticellular organisms studied. At leasteight highly homologous genes exist in thegenomes of Caenorhabditis elegans, Musmusculus, and Homo sapiens. (Adapted,with permission, from [55] Marth 1996 [© Oxford University Press].)

O-GLYCAN DIVERSIFICATION IN VERTEBRATES: CORE SUBTYPE FORMATION (29–35)

The glycosyltransferase responsible is known as the Core 1 β1-3 galactosyltransferase (Core 1 GalT)(Figure 8.3). None of the cDNAs encoding the Core 1 GalTs have been identified, although it appearsbased on enzyme activities that multiple Core 1 GalT enzymes may exist in vertebrates. Like thepolypeptide GalNAcT family, the Core 1 GalTs appear to


Core 2-type O-glycans can be generated by addition of GlcNAc to the GalNAc in a β1-6 linkage.The production of Core 2 O-glycans requires the Core 1 structure as a substrate, so the Core 2 struc-ture also contains the Core 1 structure. For this reason, use of the term Core 1 O-glycans refers to anO-glycan in which Core 2 GlcNAcT has not acted. Recent




FIGURE 8.4. Core 3 and Core 4 O-glycan subtype formation is controlled by the activity of theCore 3 GlcNAcT and Core 4 GlcNAcT enzymes. Further O-glycan biosynthesis can yield bi-antennary forms sometimes similar to those of the Core 2 subtypes. Additional linkages may sub-sequently occur (vertical arrow).

FIGURE 8.6. Early and alternate pathways in O-glycan biosynthesis leading to the formation oftumor-associated (T) antigens. Specific sialyltransferases can act to produce the sialyl Tn anddisialyl T antigens. These appear to be biosynthetic “dead ends” (boxed) that cannot be furthermodified. High levels of various T antigens are frequently associated with cancer cells (seeChapter 35).

O-glycans perform a role in the formation of the ABO blood group antigens, a system that hasclinical relevance in blood transfusions, although glycolipids are also known to carry these antigens(see Chapter 16). The blood group antigens are enriched on the erythrocyte surface and their diversi-ty is a product of mutations in a glycosyltransferase encoded by the H locus. Mutations in another gly-cosyltransferase generate either null alleles (O) or actual changes in substrate specificity (A and B) (seeChapter 16). Antibodies to specific blood group oligosaccharides have previously defined blood typesby agglutination activities, but the endogenous roles of these O-glycan structures are not presentlyknown.

O-GLYCAN FUNCTIONS IN ENDOGENOUS LECTIN-LIGAND INTERACTIONS (44–57)

O-glycans have been reported to function in sperm binding to the egg. The mammalian egg coat (thezona pellucida) contains a large number of O-glycans, as well as some N-glycans. Removal of egg N-glycans by glycosidase treatment does not destroy sperm binding, but loss of O-glycans followingmild alkali treatment ablates sperm binding. Of those O-glycans found on the mammalian egg, theZP3 glycoprotein of the mouse zona pellucida has been reported to have the primary role of thesperm receptor. Analyses of the O-glycan structures on ZP3 have revealed terminal α1-3Gal residueson O-glycans. Although this structure was previously believed to be responsible for sperm-egg bind

From Chapter 8,Pages 108-109



TABLE 9.1. Names and abbreviations for major core structures of vertebrate glycosphingolipids

Name (Series) Abbreviation Core Structure

Lacto (LcOSe4) Galβ3GlcNAcβ3Galβ4Glcβ1CeramideLactoneo (LcnOSe4) Galβ4GlcNAcβ3Galβ4Glcβ1CeramideGlobo (GbOSe4) GalNAcβ3Galα4Galβ4Glcβ1CeramideIsoglobo (GbiOSe4) GalNAcβ3Galα3Galβ4Glcβ1CeramideGanglio (GgOSe4) Galβ3GalNAcβ4Galβ4Glcβ1CeramideMuco (MucOSe4) Galβ3Galβ3Galβ4Glcβ1CeramideGala (GalOSe2) Galα4Galβ1CeramideSulfatide 3-O-Sulfo-Galβ1Ceramide

FIGURE 9.4 Pathways for the biosynthesis of the ganglio series glycosphingolipids. Note that theaction of the GalNAc transferase commits the molecules to further extension along one of fourdifferent pathways; the consequences of genetic disruption of this enzyme are discussed in thetext. Further additions of sialic acids occur in some cell types and species, giving polysialogan-gliosides (not shown).

ner that would be indistinguishable from that of molecules that were endogenously synthesized. Theextent to which such intercellular transfer can take place in vivo is not known. When fluorescentlylabeled ceramide is added to culture medium, it is taken up into the


It has also been suggested that gangliosides are involved in thermal adaptation of neuronal mem-branes. The data suggest a general rule that “the lower the environmental temperature the more polaris the composition of brain gangliosides.” Studies with model bilayer membranes also indicate thatgangliosides can modulate basic membrane properties in a thermosensitive manner. Hakomori andcolleagues have also provided evidence that glycosphingolipids can mediate low-affinity but high-specificity carbohydrate-carbohydrate


the polymer has formed (cf. Chapter 6). The GalNAc 6-sulfotransferase, GalNAc 4-sulfotransferase,and iduronic acid 2-O sulfotransferase have been purified and cloned. Progress in this area is expect-ed to accelerate as more genome sequences become available from various organisms.

CS assembly can occur on virtually all proteoglycans, depending on the cell in which the core pro-tein is expressed. The major proteoglycans that typically contain CS or DS chains

FIGURE 15.5. Biosynthesis and turnover of the N-acyl group of sialic acids. The general pathwaysfor biosynthesis, activation, and transfer of N-acetylneuraminic acid are shown. The step at whichthe N-acetyl group can be hydroxylated is indicated.




FIGURE 16.4. Modification of exposed GlcNAc moieties by galactosylation. Modification by β1-4-linked galactose residues (top) is generally found in all mammalian tissues. This reaction is cata

FIGURE 16.6. Examples ofpolylactosamine chains on vari-ous glycans.


FIGURE 16.11. Type-4 A, B, and O(H) blood group structures.


LEWIS BLOOD GROUP STRUCTURES (72–94)

The Lewis blood group antigens correspond to a structurally similar set of α1-3(4)-fucosylated gly-can structures (Figure 16.14). The term Lewis refers to the family name of indi


the Le(a–b–) phenotype. In contrast, in individuals who are Lewis-negative nonsecretors, type-1 pre-cursors are not substituted by either the Lewis or the Secretor fucosyltransferases, accounting forabsence of Lea, Leb, or H structures, and their Le(a–b–) phenotype.

Expression of Lea and Leb molecules, and the Lewis α1-3/1-4 fucosyltransferase, is




FIGURE 16.14. Representative type-1 and type-2 Lewis structures. (Top) Type-2-based structures;(bottom) type-1-based structures. Type-1 and type-2 structures differ in the linkage of the outer-most Gal (β1-3 and β1-4, respectively) and in the linkage of the fucose moiety to the internalGlcNAc (α1-4 and α1-3, respectively). R represents N-linked, O-linked, or glycolipid-based sub-structures.

FIGURE 16.15. Lewis blood group phenotypes. The structures of the Lewis blood-group-activeglycolipids are determined by the presence or absence of the Se locus α1-2 fucosyltransferaseand the Lewis locus α1-4 fucosyltransferase. R represents underlying glycoconjugates.

Lex (SSEA-1) and Ley molecules and forms of the Lea and Lex determinants that are sialylatedand or sulfated (see Figure 16.14). These structures are formed through the actions of one or moreα1-3 fucosyltransferases in addition to the Lewis α1-3(4) fucosyltransferase.



FIGURE 16.16. Antigens of the P blood group system.

the P precursor (Pk) is not present. However, p phenotype red cells express low levels of a P-like anti-gen reactivity distinct from that of P antigen. This reactivity likely corresponds to the recognition ofa different complex glycolipid whose structure is currently unknown.

These considerations assume that the P, P1, and Pk transferases represent the products of three




FIGURE 16.18. P1 antigen biosynthesis via paragloboside.

The P blood group antigens have also been assigned a role as a receptor for the human parvovirusB19. This virus causes erythema infectiosum and leads to congenital anemia and hydrops fetalis fol-lowing infection in utero. It is also associated with transient aplastic crisis in patients with hemolyt-ic anemia and with cases of pure red cell aplasia and chronic anemia in immunocompromised indi-viduals. Parvovirus B19 replication is restricted to erythroid progenitor cells; an adhesive interactionbetween the virion and P-antigen-active glycolipids is involved in viral infection of erythroid pro-genitors. Individuals with the p blood group phenotype are apparently resistant to parvovirus B19infection.

FIGURE 16.20. Forssman antigen biosynthesis. Globoside serves as the substrate for the Forssmanα1-3 N-acetylgalactosaminyltransferase (α1-3 GalNAcT) that forms globopentosylceramide, alsotermed the Forssman glycolipid.

THE FORSSMAN ANTIGEN (117–123)

The Forssman antigen, also known as globopentosylceramide, is a glycolipid structure formed by theaddition of GalNAc in α1-3 linkage to the terminal GalNAc residue of globoside (Figure 16.20). There



FIGURE 16.22. Production of the Sda or CT antigen and the glycolipid GM2.

Structures bearing α2-3 sialic acid linkages have also been implicated in contributingto bacterial pathogenesis. For example, α2-3 sialic acid structures support adhesion of H.pylori, the spirochete implicated in the pathogenesis of gastritis, gastric ulcers, and lym-phoma of the gastrointestinal tract mucosa. However, it remains to be determined if thesein vitro observations have a physiological correlate. In contrast, there is strong evidence fora physiological role for the ganglioside GM1 (GM1a; Galβ1-3GalNAcβ1-4[Siaα2-3]Galβ1-4Glcβ1-Cer) as a receptor for cholera toxin, produced by Vibrio cholerae, and heat-labileenterotoxin (LT-1), produced by enterotoxigenic E. coli (see Chapter 28). Cholera toxin isresponsible for the severe enteropathogenicity that accompanies infection with V. cholerae.

α2-6-SIALYLATED STRUCTURES (139,140,149,155–157)

Sialic acid in α2-6 linkage is expressed by various vertebrate cells and tissue types anddisplayed on a wide variety of glycoproteins and glycolipids. Synthesis of α2-6-linked sial-ic moieties is directed by members of a family of at least five different α2-6 sialyltrans-ferases (see Figure 16.24). The genes of these have been defined by molecular cloning



FIGURE 16.24. Synthesis of α2-6-sialylated termini on N-glycans, O-glycans, and glycolipids(and see Chapters 8 and 9) by the ST6GalNAc series of sialyltransferases. Enzymes in parenthe-ses contribute at relatively low levels in vitro to the reactions indicated.

approaches (ST6Gal-I, ST6GalNAc-I, ST6GalNAc-II, ST6GalNAc-III, and ST6GalNAc-IV). A gene corresponding to a sixth activity, ST6GlcNAc-I, has not yet been geneticallyisolated. Surveys of tissues and cells in a variety of mammals, and some lower vertebrates,indicate that α2-6-sialylated glycans may be found in various (but not all) cell types, asthey appear less ubiquitous than the α2-3-linked sialic-acid-bearing glycans.

hepatocytes and lymphocytes and is responsible for α2-6 sialylation of serum glycoproteinsand glycoproteins of the antigen receptor complex. In contrast, the α2-6-sialylated productsof ST6GalNAc-I and ST6GalNAc-II and the enzymes themselves are restricted to structuresdisplayed on O-glycans. The ST6GalNAc-III enzyme appears to use glycosphingolipid pre-cursors as the preferred acceptor, whereas the ST6GalNAc-IV enzyme is responsible for sub-stitution of core N-acetylgalactosamine moiety on O-glycans.

Few definitive functions have been assigned to α2-6-linked sialic acid modifications. Asnoted above (and see Chapter 28), α2-6-linked sialic acid serves as a receptor for human


From Chapter 16,Page 240Closed DiamondDeleted


FIGURE 16.26. Structure and synthesis of polysialic acid on glycolipids. Enzymes in parenthesescontribute at low levels in vitro to the reactions indicated.

FIGURE 16.27. Sulfation of α2-3-sialylated, α1-3 fucosylated glycans represented by the sialylLex tetrasaccharide have been observed at the 6-hydroxyl positions of the galactose residue andthe GlcNAc residue. Both may contribute to L-selectin ligand activity. The biosynthesis of thesestructures, the enzymes that participate in this process, and their functional attributes are dis-cussed further in Chapter 26.

FIGURE 17.1. A generic glycosylation reaction. A glycosyltransferase utilizes a glycosyl donorand an acceptor substrate. Glycosyl donors can include nucleotide sugars and dolichol-phos


GENERAL PROPERTIES OF GLYCOSYLTRANSFERASE REACTIONS (1,2,4,5,7,12)

Most glycosyltransferase-dependent transglycosylations involve a divalent cation as a cofac-tor (typically Mg++ or Mn++), and the enzymes tend to be most active in the pH range of5.0 to 7.0, which reflects pH values found in various parts of the ER-Golgi-plasmalemmapathway. Glycosyltransferases typically exhibit Michaelis-Menten constants (Kms) fornucleotide sugar substrates in the low micromolar range, when assayed in vitro. These val



FIGURE 17.2. Strict acceptor substrate requirement of the human B blood group α1-3 galacto

FIGURE 19.1. Lipid-linked oligosaccharide biosynthesis and mutants in yeast. Lipid-linkedoligosaccharide synthesis and transfer to protein in the ER is genetically defined by a wealth offunctional mutations that effect lipid-linked oligosaccharide structure and transfer to protein.Some of the steps in the synthesis of the earliest precursors such as Man-6-P and Man-1-P havealso been detected but are not shown here. Defects in dolichol biosynthesis and recycling areexpected to also produce mutations that would reduce glycosylation efficiency. (Dark gray)Mannose residues that originate from GDP-Man; (light gray) mannose residues fromdolichylphosphomannose. (Modified, with permission, from [3] Burda and Aebi 1999 [© ElsevierScience].)


8. Kuwabara P.E. 1997. Worming your way through the genome. Trends Genet. 13: 455–460.9. Chen S., Zhou S., Sarkar A., Spence A., and Schachter H. 1999. Expression of three Caenorhabditis elegans N-

acetylglucosaminyltransferase I genes during development. J. Biol. Chem. 274: 288–297.10. Haslam S.M., Coles G.C., Munn E.A., Smith T.S., Smith H.F., Morris H.R., and Dell A. 1996. Haemonchus con-

tortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylat


12. Lindahl U., Kusche-Gullberg M., and Kjellén L. 1998. Regulated diversity of heparan sulfate. J. Biol. Chem. 273:24979–24982.

13. Beyer T.A., Rearick J.I., Paulson J.C., Prieels J.P., Sadler J.E., and Hill R.L. 1979. Biosynthesis of mammalian gly-coproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences. J. Biol. Chem. 254:12531–12534.

14. Watkins W.M. 1980. Biochemistry and genetics of the ABO, Lewis, and P blood group systems. Adv.


FIGURE 20.8. Structures of a high-mannose N-glycan andthree complex N-glycans found in plant glycoproteins.


FromChapter24,Page364

TABLE 24.1. Established and putative members of the I-type lectin family

Minimal Alternate Tissue/Cell type Domain carbohydrate

Lectin name distribution structure structure(s) recognized

SiglecsSiglec 1 sialoadhesin macrophages in spleen, (V)1-(C2)16 Siaα2-3Galβ1-3(4)GlcNAc-

lymph nodes, and Siaα2-3Galβ1-3GalNAc-bone marrow

Siglec 2 CD22 B cells (V)1-(C2)6 Siaα2-6Galβ1-4GlcNAc-Siglec 3 CD33 myeloid cell lineage (V)1-(C2)1 Siaα2-3Galβ1-3(4)GlcNAc-

Siaα2-3Galβ1-3GalNAc-Siglec 4a MAG PNS (V)1-(C2)4 Siaα2-3Galβ1-3GalNAcSiglec 4b SMP Schwann cells in quail (V)1-(C2)4 Siaα2-3Gal-Siglec 5 granulocytes and (V)1-(C2)3 Siaα2-3Gal- and Siaα2-6-Gal-

monocytes

Non-SiglecsPECAM platelets heparinP0 PNS (V)1 SO3GlUAβ1-3Galβ1-R (HNK1 epitope)a

N-CAM PNS and CNS (C2)5 high-mannose oligosaccharidea

ICAM-1 blood cells, (C2)5 hyaluronan (GlcNAcβ1-3GlUAβ1-4)nendothelium, etc. leukosialin (sialylated mucin)a

CD48 activated B cells (V)1-(C2)1 heparin and heparan sulfate

a In these cases, the evidence for specific binding involving carbohydrates is indirect.

lectins, whereas the subset recognizing sialylated structures is termed the Siglecs (sialicacid/Immunoglobulin/lectin) and has been numbered sequentially by common agreement. Thischapter reviews structural and functional data about this group of lectins, concentrating on theSiglecs, about which the most is known.

COMMON FEATURES OF SIGLECS

Domain Structure and Genetics(13-21)

The primary amino acid sequence of the I-type lectins indicates that they are members of the IgSF,containing an amino-terminal V-set domain followed by a variable number of C2-set domains (Vl-C2n) (see Table 24.1). The C2-set domains are a variation of the more common C1-set domains andare believed to represent a primordial motif predating the



FIGURE 24.1. Sequence alignments of the first two domains of the murine forms of CD22,sialoadhesin, MAG, and CD33 are shown, with regions of identity included in boxes and regionsof similarity are shaded. Above the sequences are the regions of predicted and confirmed (forsialoadhesin) β strands. Asterisks indicate the residues known to be involved in binding of sialyl-lactose in sialoadhesin and are identified in Figure 24.2. The leader peptides have been deletedfrom each sequence.

In contrast to HS, one might expect that a better consensus would exist for HA-binding proteinsdue to the uniformity of the repeats in HA ([GlcAβ1-3GlcNAcβ1-4]n). Indeed, a consensus sequencefor HA binding was deduced by comparing a number of HA-binding proteins: BX7B, where B is argi


Heparin is solely the product of mast cells, and although it has proven to be of great therapeuticuse, the natural oligosaccharide that participates in the control of antithrombin/thrombin has notbeen identified. Endogenous heparan sulfate present on endothelial cells can contain small amountsof antithrombin binding sequence, unlike heparan sulfates from most other tissues and cells thatcontain none. The location of these binding


sequences, however, appears to be on the ablumenal side of the cells, which would not be exposed toblood except after injury. The nature of the endogenous activator of antithrombin remainsunknown.


The structure of FGF-2 cocrystallized with a heparin hexasaccharide has been obtained (Figure29.5). The heparin fragment ([GlcNS6Sα1-4IdoA2Sα1-4]3) was helical and bound to a region of thebFGF surface containing residues Asn-28, Arg-121, Lys-126, and Gln-135 and an additional bindingsite formed by Lys-27, Asn-102, and Lys-136. The linear seg


The PMM2 locus encodes an enzyme that catalyzes an essential step in the biosynthesis of GDP-Man and Dol-P-Man (Figure 32.2). These two compounds are essential substrates for the mannosyl-transferases required for the synthesis of the lipid-linked precursor for N-glycosylation (Figure 32.2).In CDGS type Ia, a deficiency of phosphomannomutase activity reduces the levels of GDP-Man andDol-P-Man. The reduced levels of these compounds reduce synthesis of Glc3Man9GlcNAc2-PP-Dol,the physiological substrate for oligosaccharyltransferase. Reductions in GDP-Man and Dol-P-Man


It is important to point out that Dol-P-Man is also required for the synthesis of the four mannoseresidues of glycophosphotidylinositol anchors (see Chapter 10) and is the major source of GDP-Fucrequired for terminal fucosylation (see Leukocyte Adhesion Deficiency



FIGURE 32.3 N-glycans that accumulate in CDGS type II and in HEMPAS. (Normal) N-glycansthat are present on red cell bands 3 and 4.5; “n” may be from 1 to several, and represents thenumber of lactosamine units within the polylactosamine chain. CDGSII glycans are not modified

Structural analyses of the band-3 and band-4.5 glycans indicate that these vary to somesignificant extent among different individuals with HEMPAS. In some such patients, theabnormal glycans feature a truncation of the antenna attached to the α1-6-linked man-nose residue of the trimannosyl core structure in the N-glycan (Figure 32.3). This struc-ture is the immediate synthetic precursor to GlcNAcT-II, and its excessive accumulationhas implied a defect in the expression or activity of this enzyme in some HEMPAS patients(Figure 32.3). Although it has been reported that the cells in some HEMPAS patientsexhibit 70–90% reduction in GlcNAcT-II activity, the pathophysiological relevance of


52. Marquardt T., Brune T., Lühn K., Zimmer K.-P., Kömer C., Fabritz L., van der Werft N., Vormoor J., Freeze H.H.,Louwen F., Biermann B., Harms E., von Figura K., Vestwever D., and Koch H.G. 1999. Leukocyte adhesion defi-ciency II syndrome, a generalized defect in fucose metabolism. J. Pediatr. 13: 681–688.

53. Marquardt T., Lühn K., Srikrishna G., Freeze H.H., Harms E., and Vestweber D. 1999. Correction of leukocyteadhesion deficiency type II (LAD II) with oral fucose. Blood 94: 3976–3985.

54. Crookston J.H., Crookston M.C., Burnie K.L., Francombe W.H., Dacie J.V., Davis J.A., and Lewis S.J. 1969.

58. Fukuda M.N., Bothner B., Scartezzini P., and Dell A. 1986. Isolation and characterization of poly-N-acetyllac-tosaminylceramides accumulated in the erythrocytes of congenital dyserythropoietic anemia type II patients.Chem. Phys. Lipids 42: 185–197.



From Chapter 33,Page 507New Art

From Chapter 33,Page 510New Art

FIGURE 33.8. Site-directed recombination and conditional gene mutagenesis in vivo. Mice bear

FIGURE 33.9. Branch specificity is observed in N- and O-glycan structure-function relationships.

AJIT

Once the number and relative ratios of glycan forms present in a glycoprotein are known, it is pos-sible to calculate from the analytical chromatography result whether a sufficient quantity of individualglycans can be prepared to completely identify their structures by physicochemical approaches (i.e.,NMR and MS). Quantity of purified material permitting, the best choice is to perform 1H NMR analy-sis first. Because this method is nondestructive, the same sample can later be used for other, destructiveapproaches (e.g., MS and methylation analysis). The limitations on the use of NMR spectroscopy arethe cost of equipment and the level of expertise required for interpreting NMR spectra. However, accessto high-field (i.e., 500 MHz and above) NMR spectrometers fitted with very sensitive probes (e.g.,Nano-NMR probes) allows one to obtain 1H NMR profiles of individual HPLC fractions using minutequantities of sample (2–5 nmoles per oligosaccharide). As an example, the 1H NMR spectrum of a mix-ture of two trisialyl triantennary oligosaccharides obtained from bovine fetuin is shown in Figure 38.5.



FIGURE 38.6. Methylation analysis: A chemi-cal approach to elucidating the glycosidiclinkage positions in glycans.

FIGURE 38.8. MALDI-TOF mass spectra of N-glycan mixtures obtained from 5 µg of bovinefetuin after treatment at pH 7.5 with (A) PNGase F and neuraminidase; (B) PNGase F,neuraminidase, and β1-4 galactosidase; (C) PNGase F, neuraminidase, β1-4 galactosidase, and β-N-acetylglucosaminidase (adapted, with permission, from Mechref and Novotny, Anal. Chem.70: 455–463 [1998].) The m/z values measured for the various peaks are compatible with themonosodium adducts of oligosaccharides with the given schematic structures (for explanation ofsymbolic notation, see Chapter 1).


FromChapter41, Page632

TABLE 41.3. Examples of enzyme-based inhibitors as potential therapeutics

Clinical situation Drug/Mechanism Company

Influenza A infection GS4104 (neuraminidase inhibitor) Gilead/Hoffman LaRocheInfluenza A infection Zanamivir (Relenza) (neuraminidase inhibitor) Glaxo Wellcome/BiotaTumors and hepatitis swainsonine (α-mannosidase II inhibitor) GlycoDesignGlycolipid accumulation

in lysosomal storage diseases glycosyltransferase inhibitors Oxford GlycoSciencesDiabetes Acarbose (intestinal α-glucosidase inhibitor) BayerFlea infestation in pets Lufenuron (Program®) (chitin synthesis inhibitor) Ciba-Geigy

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