Springer Semin Immunopathol (1993) 15:259-273 Spdnoer Seminars in Immunopathology �9 Springer-Verlag 1993

Antibody variable region glycosylation: biochemical and clinical effects

Ann Wright and Sherie L. Morrison

Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, CA 90024, USA

Introduction

All antibodies are glycoproteins containing at least one N-linked carbohydrate in their constant region [51]. Constant region carbohydrate contributes to the bio- logic properties of the antibody molecule and has been shown to play an impor- tant role in the maintenance of its effector functions [55]. Less well appreciated is the fact that many antibodies also contain carbohydrate in their variable re- gions. The occurrence of this variable region carbohydrate and the impact of its presence on the biochemical properties of the antibody molecule and on the ability of the antibody to interact with its cognate antigen will be the focus of this review.

N-linked glycosylation is a widespread post-translational modification, occur- ring among mammalian, yeast, insect and plant cells [29]. While they share com- mon processes of oligosaccharide biosynthesis and processing in the endoplasmic reticulum (ER), the processing steps in the Golgi apparatus vary among cell types. Sialic acid is commonly found in oligosaccharides produced in mammalian cells but not in plants; moreover, plant-derived oligosaccharides frequently contain xylose, which is not produced by mammalian cells [17]. In contrast, yeast cells produce elongated oligosaccharide structures through stepwise addition of man- nose, with final structures containing as many as 100 mannose monomers [30]. Insect cells such as Spodopterafrugiperda Sf 9 cells apparently possess the enzymes to trim the lipid-linked precursor to Man 3 GlcNAc2, but it is not clear whether insect cells are capable of further processing to complex-type oligosaccharides [39].

Among mammalian cells, oligosaccharide processing is species dependent and, within a given species, cell-type dependent. Cell-type dependent differences are apparently due to differences in the presence of specific glycosyltransferase activities. Numerous examples of differences in sialylation, fucosylation and galactosylation among human and rodent cell types have been reported [15, 24,

Correspondence to: A. Wright

260 A. Wright and S.L. Morrison

Fuca 1 Neu5Aco~2---~ 6Galfl l---~ 4GIcNAcJM ---~2Manc~l,~

6 6 GIcNAcfl 1--~ 4ManJM --~ 4GIcNAc~I~ 4GIcNAc

f 3 Neu5Aca2--~ 6Galfl l - -~ 4GleNAc~ I --~2Man~l

Fig. 1. A composite drawing of the biantcnnary o[igosacchafidr associated with immunog]obu- ]in. Heterogeneity of the structure is derived from the presence or absence of the italicized residues

53]. Variances in oligosaccharide processing can also be attributed to differences among cell types in the concentrations of competing glycosyltransferases. For example, mammalian cells that have been transformed by oncogenes or isolated from carcinomas demonstrate an increased glycosyltransferase activity which results in increased branching of N-linked oligosaccharides [11, 28]. Since recom- binant mammalian proteins are routinely produced in all these cell types, and are glycosylated according to the mechanisms of the host cell [24], it is of interest to determine the impact of species-specific oligosaccharide structures on protein function.

Accessibility of an Asn-X-Ser/Thr tripeptide sequence is critical but not suffi- cient for N-linked glycosylation. Glycosylation is inhibited if proline is located at position X of the tripeptide, and glycosylation sites at the carboxy terminus of a glycoprotein are less likely to be used [42]. Unused glycosylation sites which fit neither of these criteria have also been reported [16]. Folding of the peptide as it is translated within the ER or the tertiary structure of the protein itself, may inhibit access of the oligosaccharyltransferase to the carbohydrate attachment sequence.

The degree of oligosaccharide processing observed in glycoproteins appears to be site specific [7, 21], and can differ between N-glycosylation sites on the same protein. Among immunoglobulins, both IgM and IgD possess high mannose and complex carbohydrates on their heavy chains. IgG molecules, which have a single N-linked glycosylation site at Asn 297, possess a characteristic complex, bianten- nary oligosaccharide which is largely conserved among species [41, 52]. A com- posite diagram of the biantennary structure is shown in Fig. 1. Oligosaccharide processing typically leads to site microheterogeneity because of the presence of multiple competing glycosyltransferases in the Golgi [29]. In the IgG-associated oligosaccharide, heterogeneity appears to occur in the latter steps of processing, with variability in the degree of sialylation, fucosylation, and the addition of a bisecting N-acetylglucosamine [41]. Species differences observed to date in IgG- associated oligosaccharide structure appear to be largely confined to linkage specificities of sialyltransferases and the presence of the bisecting N-acetylglu- cosamine [13, 44, 54]. The presence of carbohydrate affects a number of biological properties of IgG, and abnormal IgG oligosaccharides are reported to have clinical implications [43].

Occurrence of V region glycosylation

Approximately 25% of the Fab fragments [1] and 15% of the light chains [49] isolated from human myeloma proteins contain an N-linked oligosaccharide.

Variable region glycosylation 261

Since neither the kappa nor lambda constant region contains a carbohydrate addition sequence, the light chain carbohydrate is presumed to be present on the variable region. In an early analysis of five myeloma proteins, carbohydrate addition sites were located in three [48]. The kappa chain HBJ4 contained a carbohydrate addition site at Asn 28, while the lambda chain Ful possessed a site at Asn 26. Both light chains possess a carbohydrate attachment site in CDR1; in contrast, the kappa chain Sup contained two potential glycosylation sites at Asn 65 and Asn 70, at least one of which was used. In another study, six human IgG myeloma proteins were examined, four of which were glycosylated in the variable region of the light chain, one in the variable region of heavy chain and one in both [49]. Carbohydrate was probably attached to Asn 25 of the lambda light chains Bal and Pet and to residue 91 in the lambda chain Hug. Arguments from homol- ogy suggested that carbohydrate was at Asn 70 in the kappa proteins Bou and Ste. For the heavy chains, carbohydrate was probably attached to Asn 30 in Bou and at approximately the same position in Wil.

More recently, a series of monoclonal immunoglobulin light chains that make up the fibrils observed in primary amyloidosis has been sequenced and shown to contain carbohydrate addition sites in CDR3 [14, 20, 57]. One amyloid-fibril protein, ES492, contained two glycosylation sites, at Asn 30 and Asn 95, at least one of which is used. Human light chain variable region glycosylation has been demonstrated in myeloma, Bence-Jones and amyloid fibril proteins. Glycosyla- tion appears to be common but not universal in amyloid fibril proteins and speculation that glycosylation may play a role in "amyloidogenic" behavior has not yet been proven [14].

When myeloma (intact) and Bence-Jones proteins were isolated from the same patient, the light chains associated with the myeloma IgG were more likely to be glycosylated than the Bence-Jones proteins; it was postulated that the heavy chains preferentially combined with light chains with the carbohydrate moiety on the light chain [49]. Where glycosylation has been demonstrated, to the extent that proteins have been analyzed, the carbohydrate addition sites appear most fre- quently at position Asn 95 on the light chain. In most cases the role of the carbohydrate has yet to be demonstrated, but its attachment position may con- tribute to the properties of the immunoglobulin.

For both human and murine heavy chains, Vn subgroups have been identified in which a substantial number of the members contain a potential N-linked glycosylation site at the same position. Several members of the human V n III group have a potential glycosylation sequence at Asn 72, in FR 3 [23]. Among murine V n genes, the V n 441 gene family, which encodes the V n region of antibod- ies against levan, galactan, and the anti-dextran W3129, contains a carbohydrate addition sequence in CDR2 at Asn 58. This sequence also occurs in the Vn gene of the anti-dextran 14.6b.1, a member of the J558 family, and was shown to be glycosylated [59]. Lectin-binding studies indicated that the carbohydrate was exposed on the immunoglobulin surface.

The presence of a large, bulky carbohydrate on the surface of a glycoprotein contributes to, and likely alters, the conformation of the protein at that site. For example, the biantennary oligosaccharide associated with immunoglobulin is similar in size to an immunoglobulin domain [39]. A computer model depicts in Fig. 2 the relative size of a carbohydrate compared to the anti-dextran Fv region in which it is attached at Asn 58 of the V n gene. The carbohydrate shown is the

262 A. Wright and S.L. Morrison

I I I VL VH

Fig. 2. Space-filling model of the Fv region of an anti-dextran with attached carbohydrate, visualized with the computer graphics program Maclmdad. The coordinates for the Fv are from the model of Padlan and Kabat [38], and the model is oriented so that the proposed antigen contact surface is at the top of the figure. V L is to the left (darker), and V n to the right. The carbohydrate is derived from the model of the human IgG1 Fc [12], and the resolved structure contains N-acetylglucosamine, fucose, and mannose residues. The carbohydrate is oriented so that the first N-acetylgtucosamine is "attached" to Asn 58 in the V n domain. For clarity, it is extended up and away from the protein surface

t r u n c a t e d o l igosacchar ide resolved in the crys ta l s t ruc ture o f h u m a n IgG1 F c [12]; a l t h o u g h it lacks the t e rmina l sugar res idues it is s imi lar to the s t ruc ture tha t w o u l d n o r m a l l y be seen in the var iab le region. H o w the o l igosacchar ide is ac tu- al ly o r i en t ed on the p ro t e in surface is a m a t t e r o f conjec ture , as there is p r o b a b l y a d y n a m i c in te rac t ion be tween the two s t ructures .

Comparison of the structure of constant and variable region carbohydrate

The c a r b o h y d r a t e in the var iab le region appea r s to differ in s t ructure f rom tha t p resen t in the cons t an t region. W h e n the s t ruc ture o f the sugar chains p resen t on r a b b i t I g G was de te rmined , b o t h F a b and F c f ragments con ta ined b i a n t e n n a r y o l igosaccha r ides [54]. Whi le the F a b - a s s o c i a t e d c a r b o h y d r a t e con ta ined neut ra l , m o n o - a n d d i s i a ly la ted o l igosacchar ides , the F c con ta ined only neu t ra l and

Variable region glycosylation 263

monosialylated sugars. The Fab-associated carbohydrates also contained more galactose and bisecting GlcNAc than those of the Fc. The position of the oligosaccharide in the rabbit Fab is not known, but the restriction of disialylated sugars to the Fab region of immunoglobulin has also been observed in human Ig [41, 46].

The absence of disialylation in the Fc has been attributed to the structural role assumed by the Fc-associated carbohydrate in IgG. In rabbit Fc, the opposing oligosaccharides in the CH2 domains have different conformations. One of the ~1 --,3 arms lacks galactose, exposing the GlcNAc residue to interact directly with the Man fll ~4GlcNAc fll ~4Gal portion of the opposing oligosaccharide. The ~1 ~3 arm of that oligosaccharide extends between the domains and can termi- nate in GlcNAc or Gal. The 0tl ---,6 arm terminates in galactose or sialic acid [52]. Such a structure appears to fulfill best the steric requirements of the interactions between protein and carbohydrates of the Cn2 domains.

Disialylated oligosaccharide structures have also been described for light chain-associated oligosaccharides from other species. A human lambda im- munoglobulin light chain was isolated which is glycosylated at Asn 25 [8]. The oligosaccharide was predominantly disialylated, although a minor monosialylat- ed component was also observed. None of the oligosaccharides contained a bisecting GlcNac residue. Interestingly, this protein contained a disialylated O-linked tetrasaccharide at Ser21; both oligosaccharides occurred in the CDR1 region just preceding an 81-amino acid deletion. The presence of an O-linked oligosaccharide in the light chain is quite uncommon; the unusual structure of the light chain may confer access to the transferase that initiates O-linked glycosyla- tion.

More recently the carbohydrate found at Asn 107 of the light chain of the human IgG1 kappa myeloma protein Hom was analyzed [45, 46]. One-third of the Hom light chains were not glycosylated; this was attributed to competition be- tween protein folding and the transfer of the oligosaccharide precursor to the nascent polypeptide [45]. Comparison of the light chain- and Fc-associated car- bohydrates showed the Fc-associated sugars to be biantennary but not bisected, and neutral or monosialylated while two oligosaccharides isolated from the Fab were bisected, fucosylated, and either mono- or disialylated, with sialic acid residues linked c~2 ~ 6, The investigators suggest that the differential glycosylation at the Asn 107 and Asn 297 sites may be due to the stabilization of the Asn 297 oligosaccharide in such a conformation that it is not a substrate for the trans- ferase that attaches the bisecting GlcNAc. Meanwhile the light chain-associated carbohydrate remains readily accessible to other transferases which attach galac- tose and sialic acid [46]. Variable glycosylation of a VH gene was observed in a murine monoclonal antibody specific for a rat endothelial membrane glyco- protein MRC-OX45 [3].

The structures of Fab-associated carbohydrates have been most extensively surveyed on human IgG and most of the permutations on complex biantennary carbohydrates have been observed [39]; nevertheless, all had the same core struc- ture. In contrast, a high-mannose carbohydrate was identified in the VH region of a mouse-human chimeric anti-dextran that was glycosylated at Asn 60 [61]. Other anti-dextrans with carbohydrate attachment sequences at different positions at- tached complex (i.e., endoglycosidase H-resistant) oligosaccharides, although their exact structure has not been determined. Whether different constraints

264 A. Wright and S.L. Morrison

affect V n and VL-associated glycosylation as well as the effect of the position of the attachment site on carbohydrate structure are open questions.

Asymmetric glycosylation of Fab regions

Observations that some IgG myeloma protein contained odd numbers of sugar moieties led to the speculation that IgG may be asymmetrically glycosylated [54]. It was found that both sheep and rabbit non-precipitating anti-DNP (dinitrophe- nol) antibodies were unable to precipitate or fix complement with DNP-BSA but were able to do so with DNP-GABA-BSA, while precipitating antibodies would fix complement with both antigens [32]. Treatment of the nonprecipitating anti- bodies with endoglycosidase H restored their ability to fix complement with DNP-BSA. Further, F(ab)' 2 from the non-precipitating antibodies were retained by ConA Sepharose, while fragments from precipitating antibody were not; when the F(ab')2 from the non-precipitating antibodies was reduced to F(ab), half were retained by Con A. The glycosylated variable region functioned, at least in part, to block access to the combining site because spacing the DNP groups further apart, as in DNP-GABA-BSA, restored reactivity with both sites. The presence of carbohydrate appeared to render the antibody molecule functionally univalent.

Subsequent studies showed that the carbohydrate is localized on the Fd' fragments of the F(ab')2 [33]. Approximately 10% of the antibodies induced by the T-dependent response to DNP had this structure, and the proportion of asymmetrically glycosylated antibodies could be increased through administra- tion to the animal of particulate, rather than soluble, antigen. The finding that 10-15% of antibodies obtained from nonimmune human serum showed asym- metric glycosylation [5] suggested that the asymmetric glycosylation is neither species nor antigen specific. Recently a murine monoclonal anti-CEA antibody was also described as asymmetrically glycosylated in the Fab region [18]. The carbohydrate was localized to the heavy chain; in this instance, the presence of carbohydrate did not inhibit antigen binding.

The occurrence of asymmetric Fab glycosylation appears to be widespread among species and may have a kinetic explanation. As the peptide is translated the competitive reactions of protein folding and translocation of the oligosaccha- ride precursor to the peptide may result in the glycosylation of only a minority of heavy chains in this region. These would then pair with the more abundant heavy chains which are not glycosylated in Fab. Interestingly, the anti-dextrans appear to be symmetrically glycosylated [59, 61]. The precise location of the glycosylation sites has not been identified in any of the asymmetric antibodies; their position in the peptide may provide insights into the criteria for access to the glycosylation enzymes which determine complete or partial usage of glycosylation sites.

While asymmetric glycosylation in the Fab region of antibodies appears to be commonplace, the biological significance of these antibodies is not known. The murine anti-CEA monoclonal IgG1 Fab retained the ability to bind antigen regardless of the state of glycosylation. In contrast, the antibodies identified by Margni and coworkers [5, 37]. Were functionally univalent, with diminished antigen binding and complement activation. It was proposed that these antibod- ies serve a blocking or regulatory role in the immune response. However, rat monoclonal antibodies raised against human tumor cell-surface antigens and

Variable region glycosylation 265

rendered monovalent through somatic cell hybridization to produce antibodies with one specific and one irrelevant light chain mediated enhanced complement cytotoxicity, perhaps as monovalent antibodies they could aggregate on the cell surface [9]. Therefore, the biologic activity of monovalent antibodies may depend upon the nature of the antigen.

Glycosylation and antibody specificity

Potential glycosylation sites are present in the CDR2 of the VH region of many antibodies. Many representations of murine V n heavy chains of subgroup III [23], which includes many anti-carbohydrate antibodies, contain glycosylation sites at the same position at Asn 58 in CDR2. The germ-line gene Vn441, a member of the X24 family from subgroup III, contains the potential glycosylation site at Asn 58 and encodes antibodies against several carbohydrates, including 3-fucosyUac- tosamine, levan, galactan, dextran and galactosylgloboside [25-27]. J539, an anti-galactan antibody from this family is glycosylated [4]. To date the anti-dex- trans, members of the J558 family, have been most extensively studied [34, 35, 69, 51] and suggest that glycosylation at this site may contribute to the anti-carbohy- drate properties of this V H gene.

VHX24, also a member of the X24 gene family, contains a second potential glycosylation site at Asn 19 in addition to that at Asn 58. This gene is used infrequently compared to VH441; of 28 monoclonal antibodies from the X24 gene family sequenced to date, only the anti-galactan XRPC24 is encoded by VHX24. It is possible that glycosylation at Asn 19 alters the antigen combining site in an unfavorable manner [26]. For most of the antibodies, however, the presence of VH-associated carbohydrate has yet to be demonstrated, so the impact of carbo- hydrate on antigen binding remains a matter of speculation.

Four murine monoclonal IgM anti-galactosylgloboside antibodies obtained in a single fusion were virtually identical [47] and had VH441-encoded heavy chains in which the glycosylation site was eliminated by a single base change resulting in a Thr-Ala substitution at position 60. It was suggested that glycosy- lation at Asn 58 is incompatible with binding of this antigen.

The myeloma tumor cells producing the ABPC48 (A48) antibody specific for bacterial levan are capable of responding to immunoregulatory stimuli directed to the idiotypic determinants present on their surface immunoglobulin. Mono- clonal proteins bearing the A48 cross-reactive idiotype can be divided into non- protective, moderately protective, and highly protective categories based upon the degree of protection against subsequent growth of the myeloma tumor [58]. In the system studied all of the proteins belong to the X24 gene family with the majority encoded by Vn441. One highly protective protein is encoded by VHX24. In one of the non-protective antibodies, somatic mutation has introduced two additional potential glycosylation sites at Asn 54 and Asn 86. The possible role of carbohydrate in the protective response has not been investigated.

That VH-associated carbohydrate does affect antigen binding was positively demonstrated by Wallick et al. [59], who demonstrated that the anti-dextran 14.6b.1, which is glycosylated at Asn 58, has a higher affinity for antigen than anti-dextrans lacking this site (see below). It was suggested that carbohydrate linked to the antibody at this site alters the conformation of the binding site.

266 A. Wright and S.L. Morrison

Alternatively there could be direct interaction between the two hydrophilic carbo- hydrate moieties. Examination of a computer model of the Fv of the anti-dextran 19.1.2, which is also a member of the J558 family [38] and is identical to 14.6b.1 except for a Thr-Asn substitution at position 60, indicates that Asn 58 is posi- tioned at the top of the molecule, with the combining site depicted as a shallow groove in a basically flat surface. Thus, Asn 58 is apparently accessible for glycosylation, and the attached carbohydrate can interact with antigen.

Recently human VH4 germ-line genes were analyzed from amplified DNA of two unrelated individuals [60]. Whereas the human immunoglobulin VH4 gene family had been thought to exhibit little polymorphism, this analysis identified at least nine new germ-line genes. All were reported to have potential glycosylation sites at Asn 60; however, since the residue at position 61 is invariably proline, it is unlikely that any of these sites are in fact glycosylated. Some of the VH4 genes encode a second glycosylation site at Asn 52. VH4 genes encode a number of autoantibodies, and immunoglobulin gene polymorphisms may contribute to variations in the immune response, including the predisposition to autoimmune diseases. Glycosylation is only one manifestation of immunoglobulin structure, but it has indeed been implicated in the pathogenesis of certain diseases (see below).

Association of variable region glycosylation with disease states

Under certain conditions structural alterations of IgG may lead to certain conse- quences, such as reduced solubility or formation of immune complexes. One way cryoimmunoglobulins, or cold-insoluble immunoglobulins, acquire this property is through glycosylation. The monoclonal IgG cryoimmunoglobulin Ger contains a carbohydrate addition site at Asn 32 in CDR1 of the V. gene. Neuraminidase treatment of Ger abolishes cryoprecipitation, thus establishing that the presence of VH-associated carbohydrate and specifically sialic acid confers the cryoim- munoglobulin character on this protein [36]. Abnormal levels of sialic acid, rather than the presence of an unusual glycosylation site, was responsible for cryoprecip- itation. Since Fab-associated carbohydrates have a higher sialic acid content than those of Fc, antibodies glycosylated in Fab may have reduced solubility.

F(ab')2 fragments of IgG anti-globulins isolated from a patient with rheuma- toid arthritis fragments retained the ability to form complexes with normal IgG [22]. The anti-globulin activity was localized to light chains and was abolished by neuraminidase treatment of the F(ab')2. Thus, sialic acid was required for IgG complex formation. It has subsequently been documented that IgG from patients with rheumatoid arthritis have a higher than normal incidence of agalactosylated Fc-associated carbohydrate [39, 43]. The presence of sialylated light chains on anti-globulins from arthritis patients has not been widely reported, so it is not known if these two structures can exist on the same molecule (or if the patient can present with both kinds of carbohydrate structures, whether or not on the same molecule). Nevertheless, it is clear that either condition will markedly affect the conformation of the immunoglobulin as well as its biochemical properties.

The presence of light chain-associated carbohydrate has also been reported in diseases featuring tissue deposition of monoclonal light chains, such as primary amyloidosis and light chain deposition disease (LDCC). Glycosylated light chains

Variable region glycosylation 267

have been found in amyloid tissue deposits and glycosylation is common, but not universal, among amyloid proteins, particularly in the CDR3 of the light chain [14, 20, 57]. In one case of light chain deposition disease, a closely related syn- drome, the V~ gene was from the V~IV subgroup, but was unusual in that it had a glycosylation site at Asn 70 [10]. While glycosylation is not essential for either disease, it is apparent that glycosylation can induce structural abnormalities in the light chain that lead to tissue deposition.

It has been suggested that Vn-associated carbohydrate may play a role in the etiology of rheumatoid arthritis. The V H genes associated with rheumatoid factor (RF) fall into two groups: one is restricted primarily to Vnl heavy chains and MIIb light chains, while the second group utilizes Vn3 heavy chains and an assortment of light chains [19, 56]. In the first category of RF variable region glycosylation has not been demonstrated. However, many VH3 genes in the sec- ond category contain potential N-linked glycosylation sites at Asn 72, in FR3, although it has not yet been determined whether any of these sites are in fact glycosylated [23]. In patients with rheumatoid arthritis galactosylation of the IgG Fc-associated carbohydrate is reduced, producing a "pocket" between the Cn2 domains which is normally filled by the 0~(1--*6) arm. It has been suggested that these "pockets" may be filled by the sugars projecting from the immunoglobulin Fabs, thereby forming immune complexes. Variable region glycosylation may, thereby, contribute to disease by altering certain biochemical properties of the antibody [19].

The anti-dextrans, a model system for variable region glycosylation

Antibodies specific for dextran have provided the best system to date for the analysis of the impact of glycosylation on the interaction between antibody and antigen and the contribution of variable glycosylation to the biochemical proper- ties of antibodies.

The issue was first addressed with the isolation of a variant of the BALB/c myeloma protein J558 (IgA, 2) with specificity for ~t(1 ~3)dextran. The variant, L187, showed reduced reactivity with polymeric dextran. The Ig produced by L187 was indistinguishable in molecular weight from that of J558; however, a difference in charge between the two proteins was observed and could be localized to the Fab portion of the molecule. Peptide map analysis failed to show any differences; instead the carbohydrate content of the two chains differed with the glycopeptides derived from L187 containing more sialic acid [34]. The altered glycosylation appeared to be the consequence of changes in glycosyl transferase expression because viral proteins grown in the mutant and wild-type cells showed alterations in their carbohydrate content which mirrored that of the Ig [35].

The additional sialic acid present in L187 interfered with its ability to interact with polymeric dextran, but did not appear to alter its combining site size and reactivity pattern with oligosaccharides. Based on a report of variable region carbohydrate in J558 initially it was concluded that it was an alteration in variable region carbohydrate that altered the ability of L187 to interact with antigen. However, because of the lack of a canonical carbohydrate addition sequence in the variable region of the heavy chain of J558, it seems more probable that there was in fact an alteration in the structure of the carbohydrate present in Cnl which influenced the antigen-antibody reaction.

268 A. Wright and S.L. Morrison

Table 1. Sequences of CDR2 regions of anti-dextrans (A) and apparent binding constants (aKa) for dextran of glycosylated and deglycosylated transfectomas (B)

A. Transfectoma CDR2 sequence a

50 51 52 52A 53 54 55 56 57 58 59 60 61 62 63 64 65 THV8.3 E I L P G S G S T N Y N E K F K G

TKC3.2.2 N Y T

TST2 N G S

TSU7 N E S

B. Antibodyb Carbohydrate Tunicamycin aKa (___ SD) ~ attachment site treatment

THV8.3 None -- 1.5 x 10 s (0.58)

TKC3.2.2 Asn 58 -- 1.1 x 106 (0.15) + 1.17 • l0 s (0.44)

TST2 Asn 54 - < 1 • 10 '~ + 1.15 • 103 (0.53)

TSU7 Asn 60 - 1.24 x 105 (0.64) 4.0 x 104 (0.12)

a Sequences of CDR2 regions of the anti-dextrans described in the text. The CDR2 sequences of the glycosylated anti-dextrans are compared to that of THV8.3, which is not glycosylated. The tripeptide carbohydrate acceptor sequence is indicated; all other amino acids identical to those found in THV8.3 are indicated by dashes. The residue that varies from THV8.3, thereby creating a glycosylation site, is underlined b Deglycosylated antibodies were prepared by incubating transfectomas in medium containing 8 Ixg/ml tunicamycin for 24 h. Supernatants were harvested and treated with Con-A-Sepharose to remove trace carbohydrate. Microtiter plates were coated with constant amounts of dextran B512. Test supernatants were incubated with increasing amounts of free dextran, and antibody binding was detected by ELISA " The aK a was calculated as the reciprocal amount of free ligand required to cause 50% inhibition of antibody binding to fixed antigen [61]

Analys is of the sequence of a series of mur ine monoc lona l ant ibodies specific for a(1 ~ 6 ) dext ran showed that the presence of a glycosylat ion sequence in C D R 2 of V n correlated with an increased affinity for dextran. Gene t ransfect ion and expression demons t ra t ed that the ca rbohydra te addi t ion sequence at Asn 58 was indeed used and that it was the presence of ca rbohydra te at that residue which lead to the greater than tenfold increase in affinity for ant igen. The variable region ca rbohydra te was exposed on the outside of the an t ibody and was acces- sible to b ind ing by lectin. Therefore, these studies demons t ra ted that no t only the a m i n o acid sequence of the variable region but also its ca rbohydra te moieties can de te rmine the magn i tude of the an t igen-an t ibody in terac t ion [59]. Sequence anal- ysis suggests that the ca rbohydra te addi t ion site was generated by somatic muta - t ion of the V n [2] and would, thus, con t r ibu te to the increase in affinity observed dur ing the m a t u r a t i o n of the i m m u n e response.

These studies have now been extended. Site-directed mutagenesis was used to create novel ca rbohydra te addi t ion sequences at Asn 54 and Asn 60 of C D R 2 of

Variable region glycosylation 269

Fig. 3. SDS-PAGE analysis of Fab, aglycosylated Fab, and Fc from anti-dextran transfectomas TKC3.2.2, TST2, and TSU7. Immunoglobulins were labeled by growing ceils in [3SS]methionine in the presence or absence of tunicamycin and digested with papain, immunoprecipitated and analyzed by SDS-PAGE. The positions of light chains, Fds and Fcs are indicated

the anti-e(1 ~ 6 ) dextran antibodies (Table 1 A) [61]. These engineered glycosyla- tion sites were utilized and the added carbohydrate was accessible to binding by lectin. Depending on the position of the added carbohydrate, different results were observed. Addition of carbohydrate to Asn 54 resulted in an antibody which no longer was able to bind dextran. This lack of ability to bind antigen was the consequence of the presence of carbohydrate and not the amino acid change necessary to produce the glycosylation site. Addition of carbohydrate to Asn 60 increased the affinity of the interaction with dextran three fold, less than the ten fold increase seen with carbohydrate addition at Asn 58 (Table 1 B).

The position of carbohydrate addition also appears to influence the structure of the added carbohydrate, as is apparent from the different mobilities through SDS-PAGE of the Fd fragments of the anti-dextrans (Fig. 3). The carbohydrate at Asn 60 remains in the high mannose form even though the Fc carbohydrate on the same antibody is processed to the complex form as the antibody traffics through the Golgi [61]. SDS-PAGE analysis also suggests that a novel complex carbohydrate structure is added at Asn 54, although this remains to be directly determined, These variable region carbohydrates, therefore, provide evidence in support of the concept of site-directed processing of N-linked oligosaccharides [6] and are an ideal model system to investigate the contribution of three-dimension- al structure to this process.

In contrast to the invariant Fc-associated glycosylation sites, those associated with the variable region occur at different positions, and may be associated with a variety of carbohydrate structures. Since variable region glycosylation has been associated with changes in antibody solubility, it is possible that certain charac-

270 A. Wright and S.L. Morrison

teristics of the oligosaccharide structure may contribute to changes in the bio- chemical properties. For example, the presence of sialic acid has been associated with self aggregation [21, 36] and cryoprecipitation. It would therefore be useful to produce antibodies with defined carbohydrate structures to assess their effect on antibody properties.

To address this question, recombinant IgG antibodies have been produced in this laboratory in glycosylation mutants of Chinese hamster ovary (CHO) cells. Wild-type CHO cells have been shown to attach a complex carbohydrate struc- tures to glycoproteins similar to those produced by human or murine myeloma cells [17, 53]. CHO cells mutant at all stages of carbohydrate biosynthesis, pro- cessing, and terminal glycosylation have been identified [50]; using these cells for expression it is possible to evaluate the effects of defined carbohydrate structures on antibody properties. Studies in this laboratory have emphasized IgG-Fc-asso- ciated activity to date, and have indicated that attachment of a truncated carbo- hydrate to Cn2 in IgG1 abolishes complement activation. (Wright and Morrison, unpublished). This system can also be applied to the study of V region glycosyla- tion. Because the presence of carbohydrate effects a quantifiable effect on antigen binding, the anti-dextrans are an appropriate system with which to initiate studies of the impact of carbohydrate structure on interaction with antigen.

Conclusions

There is a substantial body of evidence that implicates antibody glycosylation in disease. Several reports have documented that the presence of carbohydrate in the variable region contributes to alterations in protein solubility, leading to forma- tion of immune complexes or tissue deposition. It has been suggested that disrup- tions in the regulation of glycosylation can lead to the expression of altered carbohydrate structure (such as agalactosyl sugars) with the resulting glyco- proteins exhibiting properties (such as a tendency towards aggregation) that contribute to disease [40].

The conservation of a potential glycosylation site in many anti-carbohydrate antibodies in the V H gene at Asn 58 suggests that, for this group of antibodies, the presence of carbohydrate may contribute to antigen binding. For anti-dextrans, it has been demonstrated that the presence of carbohydrate at this site enhances antigen affinity, but that even slight differences in the position of the carbohy- drate attachment site in CDR2 result in quite different outcomes. This system will prove useful to study the contribution of V region-associated carbohydrate to the antigen-combining site.

Acknowledgements. This work was supported in part by grants CA 16858 and AI 129470 from the National Institutes of Health to S.L.M.A.W. was supported in part by the Tumor Immunol- ogy Training Grant CA-09120.

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