Effect of glycosylation on antibody function: implications for genetic engineering

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22 Beveridge, T.j. (1981) Int. Rw. C@. 72, 229-317

23 Sira, M. and Sleytr, U. 8. (1987)~. Xfcmbr. Sci. 33, 27-49 24 Sleytr, U. B. and Sira. M. (1989) L&I Patent h’o. 4 886 604

25 Kiipcii, S., Sira, M. and Sleytr, U. B. (1991) 1. Mewbr. %I. 61, 165-175

26 Kiipcii, S., Sira, M. and Sleytr, U. B. (1993) Da&wow 90,

65-76 27 Weigert, S. and Sira, M. (1995)). :%4+&r. Sri. 106, 147-159

28 Sira, M., Kiipcii, S., Weiner, C., Weigert, S. and Sleytr, U. B. (1993) m Immobilised Macromolectrles: Application Pofentiak (S&r, U. U.,

Messner, P., Pum, D. and Sara, M., eds), pp. 71-86. Springer 29 Sira, M., Kiipcii, S. and Sleyn, U. B. (1996) in Crystalline Bactt~tial

Cell Sutface Proteins (Sleytr, U. B., Messner, P., Pum, D. and S?~ra, M., eds), pp. 133-159, Academic Press

30 Pum, D., Sira, M. and Sleytr, U. B. (1993) in Imnrobilrscr~ Mwt)-

mok~uks: Apphion Potentials (Sleytr, U. B., Messenr, P., Pum, 1). and Sira, M., eds), pp. 141-160, Springer

31 Pm% D. and Sleytr, U. B. (1996) in Crystalline Bactenol G/l Slr+o Layer Proteirts (Sleytr, U. B., Messner, P., Pum, 1). and Sira, M., rds), pp. 175-209, Academic Press

32 Breitwieser, A. et al. BioTechnqrtcr (in press)

33 Weiner, C.. Sira, M.. Dasgupta, G. and Sleytr, U. B. (lYY1) Boot& nol. Bioq. 44, 5-5

34 Kay, W. W. and Trust, T. J. (1991) E.xperiEtifia 47, 412-414 35 Bla\er, M. J, and Gotnchlich, E. C. (1990) /. Biol. Chem. 265,

11529-14535

36 Ford, L. A. and Thune, R. L. (1992) Biomed. Lett. 47,355-362 37 Evenberg, D., De GraafX P., Lugtenberg, B. and van

Muiswinkel, W. B. (1988)J. Fish Dixvse 11, 337-350

38 Trust, T. J. (1986) Annu. Rev. Miicrobiol. 40, 479-502

39 Messnrr, P., Unger, F. M. and Sleytr, U. B. (1996) in Crpaiiinr Barkml Cell Su$w F’mtems (Sleytr, U. B., Messner, P., Pum, D. and

%-a, M., eds), pp. 161-173, Academic Press 40 Sleytr, U. B., Mundt, W., Mezsner, P., Smith, R. M. and

Unger, F. M. (1991) US‘4 P&nt I%. 5 043 158

41 Jahn-Schmid, B. ef al. (1996) In~rnlrnotechnolo~y 2, 10>113 42 Jahn-Schmid. B. rf al. (1996)J. Biotechnol. 44, 225-231 43 Pum, D. and Sleytr, U. B. (1994) 7%~ So/id Films 244, 882-886

44 Pum, D., Weinhandl, M., HGdl, C. and Sleyrr, U. B. (1993)

J. Bactetiol. 175, 2762-2766 45 Kiipch, S., S&a, M. and Slrytr, U. B. (1995) Bio&m. Biophys. rlcta

1235, 263-269

46 Pum, D. and Sleytr, U. B. (1995) Supramol. Sci. 2, 193-197 47 Pum. D., Stangl, G., Sponer, C., Falhnann, W. and Sleytr, U. 8.

Colloid.c Su$ B: Biointe$xcs (in press) 48 Sira, M. and Sleytr, U. B. Pq. Biophys. .W. Btol. (in press)

Effect of glycosylation on antibody function: implications for

genetic engineering Ann Wright and Sherie L. Morrison

Antibodies are able to both bind antigens and trigger the responses that eliminate

them from circulation. All antibodies are glycosylated at conserved positions in their

constant regions, and the presence of carbohydrate can be critical for antigen

clearance functions such as complement activation. The structure of the attached

carbohydrate can also affect antibody activity. Antibody glycosylation can be

influenced by the cell in which it is produced, the conformation of the antibody and

cell culture conditions. These variables should be considered in the design and

production of antibodies with selected specificity and function.

Antibodies, or immunoglobulins (Igs), are serum composed of two identical light chains and two proteins that play a central role in the humoral immune identical heavy chains (Fig. 1). The light chains response, binding antigens and inactivating them fold into two domains and the heavy chains into four or triggering the inflammatory response which results or five’. The N-terminal domains of each chain in their elimination. The antibody core structure is constitute the variable (V) regions which recognize

antigens, while the other domains make up the

A. CVtij$t and S. L. ,I!omkon (Fax: + I 3 IO 206 223 1) an’ al th constant (C) regions. Antibodies form a Y-shaped

Department ofMicrobiology aMd ,WI/~~II~A~ Genetics and rhr .%folert~lar structure consisting of two identical Fab ‘arms’, each Biology Institrctc, tlniversity o~Cni$7fnrriia, tar An&~, 405 Hi/pd composed of a light chain and the variable and first At~enue, LAS Aqeles, CA YOOY.5, ciS.4. constant region of a heavy chain, and an Fc stem with

TIBTECH JANUARY 1997 (VOL 15) CopyrIght 0 1997. Elsevier Science Ltd. All rights reserved. 0167 - 7799/97/$17.00 PII: SO167-7799(96)10062-7

the remaining heavy chain constant domains (see Fig. 1). The Fab regions contain the antigen-binding sites, while the elimination of antigen is mediated by the Fc through the activation of host immune mechanisms such as complement and antibody- dependent cell-mediated cytotoxicity (ADCC). In humans, there are five classes of immunoglobulins: IgG, IgA, IgM, IgE and IgD, with four further subclasses of IgG and two of IgA. These nine iso- types differ with respect to biological activity, structure and relative abundance in serum and secretions.

All antibodies contain carbohydrate at conserved positions in the constant regions of the heavy chains, with each isotype possessing a distinct array of N-linked carbohydrate structures, which variably affects protein assembly, secretion or functional activ- ityl. In addition, up to 30% of rabbit and human IgGs possess Fab-associated carbohydrate2~“. The structure of the attached N-linked carbohydrate varies consid- erably, depending upon the degree of processing+z5, and can include high-mannose, multiply-branched as well as biantennary complex oligosaccharides (Fig. 2). IgAl and IgD molecules also possess several O-linked sugars6.7. IgG, the most abundant antibody in serum, has a single N-linked biantennary structure at Asn297, which is buried between the C,2 domains, forming extensive contacts with amino acid residues within C,2 (Ref. 8; Fig. 3). The oligosaccharide is approximately as large as the C,2 domain$.

Antibodies have many structural and functional features that make them valuable reagents for research or therapy. They are capable of highly specific interac- tions with a wide variety of ligands, both in their Fab and Fc regions. Functional domains providing specific antigen-binding (Fabs or Fvs) or effector functions (Fcs) can be exchanged between molecules, expressed as separate, biologically active, fragments, or expressed as components of novel fusion proteins’O,‘l. Such manipulations have been instrumental in elucidating the biological functions of the different Igs and have contributed to improvements in the design of genetically engineered antibodies.

Innovative gene expression systems have been developed to allow the production of intact antibodies or fragments in a variety of heterologous hosts such as lymphoid and non-lymphoid mammalian cells, bacteria, yeast, plants and insect cellsta. The challenge is to define an expression system that produces antibodies with the desired functional properties. Because antibodies are glycoproteins, an additional concern is the contribution of the attached carbohydrate to their functional properties. Antibodies produced in prokaryotic cells lack carbohydrate’“. Although all higher organisms are capable of glycosylation, both the structure of the attached oligosaccharide and the efficiency of glycosylation site utilization may vary.

This review will focus on IgG and will address the effects of the absence or alteration of carbohydrate structures on the functional properties of antibodies.

Antigen- combining region

;i:

Figure 1 Schematic of a human IgGl antibody molecule1 composed of two identical light chains (each composed of two domains, labeled V, and C,) and two identical heavy chains (each with four domains, labeled Vu, C,,l, C,2 and C&3). IgG molecules can be cleaved enzymatically Into several functional subunits: Fvs, composed of V, and V,; Fab fragments, which consist of the lrght charn and the V, and C,l of the heavy chain; and the Fc, consisting of heavy chain constant domains. A flexible hinge separates Fabs from the Fc. Amino acid residues in the V, and V, domains form the antigen combining sites. Effector functions, such as complement actrvation and binding to cytotoxic cells, are mediated by the Fc region. The carbohydrates between the C,2 domains are represented by forked structures. Inset: The composite, fully-processed carbohydrate structure attached to Asn297 in C,2 (Ref. 55). Sugar residues include: Nacetylglucosamine (GlcNac; H), mannose (Man; cl, fucose (Fuc; A), galactose (Gal; 0) and sialic acid (SA; l ).The sugar residues are attached in invariant order to the core structure GlcNac,Man,GlcNac, but the actual numbers of the outer residues will vary.

The role of carbohydrate in IgG function As mediators of the humoral immune response,

antibodies trigger biological responses after binding to specific antigens by interacting through their Fc regions with both cellular and soluble effector systems. The binding site on IgG for Clq, the first component of the complement cascade, has been localized to the C,2 domains (Fig. 3); antigen-mediated aggregation of IgGl, IgG2 or IgG3 initiates complement activation’.‘. Binding of IgG to the high- affinity Fc receptors on monocytes can stimulate those cells to eliminate the antigen to which the Ig is bound. Other cells such as natural killer (NK) cells and neu- trophils express low-affinity Fc receptors that produce a cytotoxic response when engaged by aggregated antibodies”.

Removal of carbohydrate and its e&t on function Two general approaches have been used to deter-

mine the effect of total removal of oiigosaccharide: tunicamycin treatment of the antibody-producing cells to inhibit the attachment of the oligosaccharide pre- cursor to asparagine’“.“, or removal of Asn297 by mutation to Gln (Ref. 18). Although both manipulations profoundly depressed certain antibody functions, subtle differences were observed. In both cases interaction with antigen and Sfuphylorocclrs Protein A was unaffected. However, both C 1 q binding and monocyte- associated Fc receptor binding were undetectable in

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Endoplasmic reticulum

High-mannose

+ + cis Golgi

I---- i

--

J i medial Golgi

1

4 Vans Golgi

Figure 2 An abbreviated schematic representation of the processing pathway of ohgosacchande to complex biantennary form51. The newly synthesized species Glu,Man,GlcNac, (1) is transferred from dolichyldiphosphate to the Asn-X-Ser/Thr sequence in the peptide as it emerges from the ribosome. The arrows Indicate sequential enzymatic reactions through which sugar residues are trimmed as the glycoprotein passes through the endoplasmlc reticulum. After removal of the three terminal glucoses, the glycoprotein then moves to the cis Golgl, where it undergoes a series of steps through which mannose residues are trimmed by a-mannosidases. Processing can stop at this point, yielding glycoprotelns with high-mannose sugars attached. Alternatively, processing can proceed to yield Man,GlcNac, (2). This intermediate is the preferred substrate for Nacetylglucosaminyltransferase I, whose action, in the medial Golgi, is the committed step in complex oligosaccharide synthesis. The CHO glycosylation mutant Lecl is deficient In this enzyme so the sugars produced by these cells bear this structure. In the medial (3) and trans (4) Golgi the oligosaccharide undergoes further processing steps in which mannose residues are trimmed and the sugar residues are sequentially added. The newly synthesized glycoprotein then exits the Golgi and is transported to the cell membrane or IS secreted. Symbols: glucose (A); mannose (:,I; Nacetylglucosamine (WI; fucose (A); galactose (0) and slalic acid (0).

the mutant aglycosylated antibodies, while these functions were reduced but not eliminated in the tunicamycin-treated antibodies. Tunicamycin-treated IgG3 showed loss of rosette formation through Fc receptors expressed on cultured tumor cells)’ and NK cells’“. For Gln297 variants, the irl kJo half-life of IgG 1 was not affected by lack of carbohydrate, while that of IgG3 was shortened. Compared with wild type, aglycosylated IgGl and IgG3 showed increased sensitivity to proteases and altered reactivity to a panel of monoclonal antibodies, suggesting that alterations in protein conformation had occurred.

More recently, when mouse IgG2a antibodies either treated with tunicamycin or deglycosylated by virtue of an Asn297+Ala mutation were compared directly for irl vivo clearance and binding of the Fc receptor from neonatal rat (FcRn; Ref. 20), the genetically aglycosylated antibody was indistinguishable horn wild type, whereas the tunicamycin-treated antibody was less

stable, cleared more rapidly and was transported by FcRn less efficiently. It was concluded that tunicamycin treatment caused alterations in the protein structure in addition to removing the carbohydrate, suggesting that antibiotic treatment may introduce unintended variables.

Effect of altered carbohydrate structure on antibody function

Typically there is heterogeneous processing of the core oligosaccharide structures attached at a particular glycosylation site so that even monoclonal antibodies exist as multiple glycoformsz1-z3. While such heterogeneity is normal, certain structures appear to be associated with aberrant antibody function. The serum IgG of some patients with certain auto-immune diseases such as rheumatoid arthritis, Crohn’s disease and osteoarthritis contains a higher than normal proportion of sugar structures

lBTECHJANUARY1997(VOL151

terminating in ,V-acetylglucosamine (Fig. 2). It has been suggested that such antibodies might be more susceptible to aggregation and formation of insoluble immune complexesa4. To determine whether defined alterations in carbohydrate structure actually affect antibody properties, terminal residues have been selectively removed by treatment with specific glycosidases, or antibodies have been expressed in cells that attach sugars that are incompletely processed.

Exoglycosidase treatment of IgG antibodies To obtain a population of serum IgG that was totally

lacking galactose, Tsuchiya et ~1.‘~ treated polyclonal IgG with neuraminidase and P-galactosidase. Com- pared with untreated samples, these antibodies revealed a significant reduction in Clq binding and Fc receptor binding, but were unaffected in their capacity for Staphylococcus Protein A and rheumatoid factor binding. Desialation of the CDw52-specific human monoclonal antibody Campath-1H produced no effect on any function tested’“. Removal of galactose was found to reduce complement-mediated lysis of antigen- bearing target cells but did not affect ADCC. Although these experimental systems are not com- pletely equivalent they suggest that degalactosylation of IgGl will have a deleterious effect on complement activation but that the reduction in Fc-receptor- mediated activity varies depending on the subclass and assay.

Using human IgG that was enzymatically degalac- tosylated, Malhotra et al.” reported that the terminal 1%acetylglucosamine residues thus exposed were accessible and could interact with mannose-binding protein (MBP), a serum protein with a structure similar to Clq. The deposition of C4b, an intermediate in the complement cascade, was observed when immobilized Fc fragments produced from these antibodies were incubated with MBP and serum. It was suggested that this unconventional capability to activate complement could contribute to the chronic inflammation associated with rheumatoid arthritis.

Treatment of cells with the carbohydrate-processing inhibitor swainsonine (SW) results in the synthesis of ‘hybrid’ carbohydrate structures that possess one fully processed arm and one containing mannose exclusively. When hybridoma cells producing a sheep red blood cell (SRBC)- p .fi s eci c monoclonal antibody of the murine IgG2a subclass were treated with SW (Ref. 28), the capacity of these antibodies to be secreted, bind antigen, mediate ADCC ofSRBCs and perform complement-mediated hemolysis of target cells was unaffected. However, the new carbohydrate structure was accessible to lectin binding, whereas the biantennary structure is normally buried between the C,2 domains”. Therefore, in this system, possession of even one fully processed arm of the sugar structure sufficed for retention of CH2-associated function, although the aberrant sugar may introduce variations in conformation.

Figure 3 Crystal structure of the Fc portron of a human IgGl molecule52 depicted using the

program Maclmdad. The carbohydrate chains attached to Asn297 in each chain are

shown in space-filling format, extending between the C,2 domains. The residue

Pro331, which is critical for high-affinrty Fc receptor interaction and for complement

activation”3, is located on a loop near the lower hinge region and is indicated at the

top of the drawing. Also labeled are the charged residues Glu318, Lys320 and

Lys322, which constitute a motrf that is thought to play a role in Fc binding to Clq,

the initial step in the classical complement activation pathway54. The lower hinge

region is not resolved in this structure. For simplicity the amino acids of only one

chain are labeled.

Rothman et u1? tested the capacity for ADCC of monoclonal murine IgG antibodies that were purified from hybridomas grown in the presence of glycosidase inhibitors that acted at different steps in the oligosaccharide-processing pathway. These inhibitors included SW (see above) and castanospermine (Cs), which inhibits the removal of glucose residues from the oligosaccharide newly attached to the peptide (Fig. 2; compare Structure 1 to Structure 4). Compared with wild-type antibodies, those treated with Cs showed enhanced ADCC mediated by NK cells but not by other types of effector cells such as monocytes. By contrast, SW-treated antibodies failed to induce enhanced NK-cell-mediated ADCC. Through lectin- binding analysis the oligosaccharides on SW-treated and wild-type IgGs were shown to contain fucose, which was lacking on the Cs-treated antibodies. It was suggested that recognition by IgG Fc ofthe type of Fc receptor present on NK cells, leading to enhanced ADCC, was glycosylation dependent, requiring the absence of fucose. However, both Sw-treated and wild-type oligosaccharides contain at least one complex ‘arm’, which would produce an overall conformation, as well as several sugar residues, that differs from the oligosaccharide produced by Cs treatment.

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Studies with antibodies produced in glycosylation mutants

Chinese hamster ovary (CHO) cells are commonly used to produce many recombinant proteins, includ- ing immunoglobulins. These cells attach sugar structures that resemble those found on human cells3l.32. Immunoglobulins have been produced in CHO cells that have defined defects at distinct steps in the oligosaccharide biosynthesis pathway33, resulting in the accumulation of intermediate structures with reduced overall carbohydrate heterogeneity. The CHO glycosylation mutants are use&l for analyzing specific alterations in carbohydrate structure in normal proteins, without resorting to the use of glycosidase treatment that might affect the protein backbone.

In initial studies, IgGl antibodies were produced in Lecl cells, which lack N-acetylglucosaminyltransferase I activity, leading to the accumulation of high- mannose intermediates not normally found on IgG (Fig. 2; Ref. 34). When compared with IgG produced in wild-type myeloma or CHO cells, the Lecl- produced antibodies retained antigen specificity but were incapable of complement-mediated hemolysis, were greatly deficient in Clq binding and complement consumption, and showed reduced but significant affinity for the high-affinity Fc receptors. The in viva half-life of the Lecl-produced antibody was shorter than that of the normally glycosylated antibodies, although clearance could be temporarily blocked by the co-injection of yeast-derived mannan. More recently, antibodies have been expressed in CHO glycosylation mutants deficient in the attachment of either sialic acid or galactose, resulting in glycoforms that are normally seen on serum IgG.

Glycosylation profoundly affects the effector functions of IgG antibodies and the glycosylation status of IgG can be altered either enzymatically or by manipu- lating the glycosylation machinery of cells. Such studies will help determine whether particular carbohydrate structures definitively affect antibody function and may lead to the optimization of expression systems or growth conditions for the production of such glycoproteins.

Factors affecting antibody glycosylation Expression systems and glycosylation

Comparative studies of IgGs obtained fi-om different species have reported species-specific differences in IgG-associated oligosaccharide structures. For example, whereas a significant proportion of human IgG antibodies attach an N-acetylglucosamine (Glc- NAc) residue that bisects the biantennary sugar35, antibodies produced in murine cells lack that structure”,“. Furthermore, CHO cells attach sialic acid through (a2+3) linkages exclusively, whereas human and murine cells can attach a mixture of (~~2-3) and (ol2-+6)-linked sialic acid residues. While human Igs preferentially attach galactose residues to the Man(al-+6) branch of the sugar, galactosylation of the Man(cyl-+3) branch predominates in bovine Igs (Ref. 36).

lBTECHJANUARY1997NOL15)

In a few instances, antibodies produced in murine cells contain terminal (al+3)galactose23*37, which has been identified in both the Fc and constant regions of antibodies. This carbohydrate residue is widely dis- tributed among non-primate mammals and in man is recognized by up to 1% of circulating Ig (Ref. 38). Thus, antibodies bearing that residue would probably be subject to rapid immune clearance, greatly com- promising their effectiveness as in viva therapeutic agents39.

While subtle differences in IgG glycosylation are observed among mammalian cells, they all attach a biantennary structure with a common core at Asn297. However, production of immunoglobulins in non- mammalian expression systems can introduce consid- erable variation in carbohydrate structure. Insect cells produce truncated sugars and also are reported to attach xylose4”. Yeast attach high-mannose sugars of up to 200 residues”‘. Antibodies thus glycosylated retained ADCC but lost the ability to activate complement-“.

Plants have been proposed as an economical method for the large-scale production of recombinant antibodies”3. Plant cells elaborate complex carbohydrates through a biosynthetic pathway similar to that of mammalian cells, but can attach a xylose residue - which is not found in mammals - linked to the p- linked mannose of the glycan core. Sialic acid is absent in plants44. To date, the question of whether these novel structures affect antibody function has not been adequately addressed. Antibodies produced in plants reportedly bind antigen effectively; however, antigen binding is generally not influenced by Fc-associated glycosylation. It is not clear whether Fc-associated functions have actually been tested in the plant- produced antibodies.

E$ect of protein conformation on glycosylation The degree of processing of a carbohydrate at a par-

ticular attachment site on a protein depends on the accessibility of that site to the relevant glycosyltransferases. Microheterogeneity of Fc-associated carbohydrate structures exists with polyclonal human serum IgG containing approximately 30 different biantennary structure@. Studies of glycosylation of different IgG subclasses have reported that the distribution of galactose on IgG2 appears to differ from the other subclasses”sr46. It was suggested that differences in protein conformation might affect the accessibility of the arms of the biantennary structure to galactosyltransferases; however, only a small number of proteins has been analyzed and clonal variation cannot be discounted. As a rule, novel sugar structures have not been observed, although in one comparative study of IgG myeloma proteins4h small amounts of CH2-associated high-mannose sugars were found to be attached to IgG2 and IgG3 paraproteins.

The adjacent immunoglobulin structure itself can affect glycosylation. Comparison of glycosylated Bence-Jones (free light-chain) proteins with Fab hag- ments produced from IgGs containing a glycosylation

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site in the light chain showed the Bence-Jones proteins to be much more uniformly glycosylated, suggesting that the glycosylation site in the free light chain is more accessible to glycosyltransferases”5. Indeed, Fab-associated glycosylation sites tend to be more fully processed than Fc-associated sites within the same molecule35.“5, suggesting a difference in accessibility. Thus the direct expression of antibody fi-agments may lead to differences in glycosylation that might have functional or physiological significance.

The carbohydrate associated with C,2 in IgG forms extensive contacts with amino acids within that region. In a recent study these amino acid residues were altered by site-directed mutagenesis. It was found that mutation of a contact residue for a core N-acetylglucosamine, but not for galactose, resulted in a loss of recognition of that Fc region by Fc receptorsa. More- over, mutations of several contact residues resulted in increased sialation and galactosylation of the oligosaccharide chain?.

Culture conditions and their e$ct on glycosylation A pressing concern of commercial antibody pro-

duction is the delivery of large quantities with high and consistent quality while minimizing cost. Cell culture methods for IgG production have been developed and compared. Both human18 and chimeric mouse- human IgG23 were more heavily galactosylated when grown in still culture than in hollow fiber reactors. The highly-galactosylated human IgGs showed enhanced ADCC involving both low- and high-afkity Fc receptors. However, it is not clear whether in vim clearance or any other biological functions were affected. Cell culture conditions such as pH, concentration ofNH,+ and hormonal supplements have been shown to affect N-linked glycosylation in several systemsqY.““.

Conclusion: manipulation of glycosylation Glycosylation is an inherently complex process

whose purpose appears to be to introduce diversity into cells and proteins. Although it is clear that cor- rect glycosylation is critical for effector activity of antibodies, the experimental systems used are not entirely comparable so the results may be incomplete and are not always entirely consistent. Nevertheless, some findings suggest that some alterations in glycosylation may allow the ‘uncoupling’ of certain functions (e.g. antibodies that can bind Fc receptors but cannot activate complement). These observations may eventually add a new dimension to the deliberate design of antibodies with a selected specificity and function. More- over, the ability to produce quantities of antibodies with specific alterations in carbohydrate structure may help resolve the role of carbohydrate in antibody function.

Acknowledgements Research in this laboratory was supported by grants

CA-16858, AI-39187 and AI-29470 f?om the National Institutes of Health and ACS-IM-77313 from the Cancer Society.

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J. Immnnol. 129. 2016-2020

‘Interfacial activation’ of lipases: facts and artifacts

Robert Verger

An important aspect of lipolytic enzymes is the unique physicochemical character

of the reactions they catalyse at lipid-water interfaces, involving inter-facial

adsorption and subsequent catalysis sensu stricto. Lipases are now used as

catalysts in aqueous as well as low-water media and accept various molecules as

substrates. They were previously defined in kinetic terms, based on ‘interfacial

activation’. This phenomenon was not found among esterases. Recently determined

3D structures of some, but not all, lipases show a ‘lid’ controlling access to the

active site. Thus, the presence of a lid, and ‘inter-facial activation’, are unsuitable

criteria for classifying specific esterases. Consequently, lipases can be

pragmatically redefined as carboxyl-esterases acting on long-chain acylglycerols:

they are simply fat-splitting ‘ferments’.

Three of the four main classes of biological substances, carbohydrates, proteins and nucleic acids, have been clearly defined on the basis of their structural features. whereas the property that is common to all lipids, the fourth main class, is a physicochemical one. Lipids are in fact a group of structurally heterogeneous molecules that are all insoluble in water but soluble in apolar and slightly polar solvents such as ether, chloroform and benzene. Lipids have been classified by Small’ accord- ing to their behaviour in the presence of water.

Conventional enzyme kinetics is of little relevance under heterogeneous conditions. To determine kinetic parameters, in vitro experiments have traditionally been carried out using synthetic short- and medium-chain esters as putative lipase substrates, such as methyl

butyrate, triacetin (TCZ), tripropionin (TC3) and tri- butyrin (TC4). In aqueous media, these esters can exist either as an isotropic solution in the form of monomers, micelles and adsorbed monomolecular films, or as a turbid emulsion, which appears at higher substrate concentrations. These various physicochemical forms of ester molecules are schematically depicted in Fig 1 and they usually coexist as a complex equi- librium between potential lipase ‘monomeric substrate’ (S,) or ‘super-substrates’ (Sa, S, and S,).

Definition of lipases and ‘interfacial activation’ What exactly is a lipase? Is it enough to define it as

a carboxyl-esterase that specifically hydrolyses acylglycerols? In 1958, Sarda and Desnuellea defined lipases in kinetic terms, based on the phenomenon of ‘inter-facial activation’. This property was not to be found, for example, among enzymes that have been classified as esterases, i.e. those acting only on

TlBTECHJANUARY1997lVOL 15) CopyrIght 1997. Elsewer Science Ltd. All rights reserved. 0167 7799/97/$17.00. PII: SO167-7799(96)10064-O

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