Position Effects of Variable Region Carbohydrate on the Affinity and In Vivo Behavior of an Anti-(1→6) Dextran Antibody This information is current as M. Josefina Coloma, Ryan K. Trinh, Alexander R. Martinez of September 27, 2021. and Sherie L. Morrison J Immunol 1999; 162:2162-2170; ; http://www.jimmunol.org/content/162/4/2162 Downloaded from References This article cites 45 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/162/4/2162.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Position Effects of Variable Region Carbohydrate on the Affinity and In Vivo Behavior of an Anti-(136) Dextran Antibody1 M. Josefina Coloma, Ryan K. Trinh, Alexander R. Martinez, and Sherie L. Morrison2 IgG is a glycoprotein with an N-linked carbohydrate structure attached to the CH2 domain of each of its heavy chains. In addition, the variable regions of IgG often contain potential N-linked carbohydrate addition sequences that frequently result in the at- tachment of V region carbohydrate. Nonetheless, the precise role of this V region glycan remains unclear. Studies from our laboratory have shown that a naturally occurring somatic mutant of an anti-dextran Ab that results in a carbohydrate addition 58 site at Asn of the VH has carbohydrate in the complementarity-determining region 2 (CDR2) of the VH, and the presence of carbohydrate leads to an increase in affinity. However, carbohydrate attached to nearby positions within CDR2 had variable Downloaded from affects on affinity. In the present work we have extended these studies by adding carbohydrate addition sites close to or within all the CDRs of the same anti-dextran Ab. We find that carbohydrate is attached to all the novel addition sites, but the extent of glycosylation varies with the position of the site. In addition, we find that the position of the variable region carbohydrate influences some functional properties of the Ab, including those usually associated with the V region such as affinity for Ag as well as other characteristics typically attributed to the Fc such as half-life and organ targeting. These studies suggest that modification of variable region glycosylation provides an alternate strategy for manipulating the functional attributes of the Ab molecule and http://www.jimmunol.org/ may shed light on how changes in carbohydrate structure affect protein conformation. The Journal of Immunology, 1999, 162: 2162–2170. ntibodies are glycoproteins that have at least one showed that carbohydrate addition sites present within the CDR2 N-linked carbohydrate added to the constant regions of of the VH gene 19.22.1 were actually used, and N-linked carbo- A the heavy chains. Many Abs also have V region-associ- hydrate attached (7, 15). This was true both for a naturally occur- ated carbohydrate. In fact, it has been calculated that human serum ring somatic mutation at position 60 (Asn3Thr) resulting in gly- 58 IgG has, on the average, 2.8 N-glycoside-type sugar chains/protein cosylation of Asn in the VH CDR2 (hybridoma 14.6b.1) as well molecule (1). Two of these carbohydrate moieties represent the as for glycosylation sites introduced at residues 54 and 60 within by guest on September 27, 2021 conserved N-linked carbohydrate in the Fc region, while the re- the CDR2 using site-directed mutagenesis. Interestingly, glycosyl- 58 mainder reflect V region glycosylation. Analysis of human my- ation of Asn of the VH increased the affinity of the Ab for Ag 60 eloma proteins and Abs of other species has indeed verified the approximately 10-fold, while carbohydrate at Asn of the VH only 54 frequent presence of V region carbohydrate (2–7). About 18% of increased the affinity 3-fold, and carbohydrate at VH Asn actu- 3 the VH sequences in Kabat’s database (8) contain a potential N- ally blocked the binding of Ag (15). In the anti-dextran Ab one of linked glycosylation site, Asn-X-Ser/Thr (9). The presence of this the surprising observations was that the carbohydrate added at po- site, however, does not guarantee that it is used for carbohydrate sition 60 remains in the high mannose form, while position 58, attachment (10, 11). Although asymmetric utilization of the V re- only two amino acids apart in sequence, contains carbohydrate that gion glycosylation sites has been reported (6, 12, 13), both V re- is processed to complex glycans. While differential accessibility gions can be glycosylated (7). has been proposed as the cause of differential processing, it seems The role of the V region carbohydrate remains unclear, but sev- likely that the protein itself plays an important role in determining eral studies suggest that its presence may influence affinity and/or the specificity of processing. specificity (4, 9, 14). Previous studies of anti-(136) dextran Abs The structure of the V region carbohydrate is frequently differ- ent from that of the constant region carbohydrate, and the structure Department of Microbiology, Immunology, and Molecular Genetics, The Molecular of the V region carbohydrate can be associated with differences in Biology Institute, University of California, Los Angeles, CA 90095 binding specificity and affinity (16, 17). For the anti-dextrans, car- 54 58 Received for publication June 11, 1998. Accepted for publication November 6, 1998. bohydrate attached at Asn and Asn was a biantennary complex a 3 The costs of publication of this article were defrayed in part by the payment of page structure containing Gal 1 3Gal as a nonreducing terminus, charges. This article must therefore be hereby marked advertisement in accordance while the carbohydrate attached to the Fc of the same Ab lacked with 18 U.S.C. Section 1734 solely to indicate this fact. Gala133Gal at its terminus (18). Recent evidence suggests that 1 This work was supported by National Institutes of Health Grants CA16858, carbohydrate can influence characteristics such as organ localiza- AI39187, and AI29470. tion, clearance rates, and receptor binding (19, 20). Variation 2 Address correspondence and reprint requests to Dr. Sherie L. Morrison, Department of Microbiology, Immunology, and Molecular Genetics, University of California, in glycoform structure has been associated with some disease 1602 Molecular Sciences Building, 609 Circle Drive East, Los Angeles, CA 90095- states (21, 22). 1489. E-mail address: [email protected] In the present work we have extended our studies of V region 3 Abbreviations used in this paper: VH and VL, heavy and light chain variable regions; carbohydrate by adding carbohydrate addition sites close to or CDR, complementarity-determining region; IMDM, Iscove’s modified Dulbecco’s b b within CDR1 and CDR3 of the VH as well as the CDR1, CDR2, medium; Endo-H, endoglycosidase H; aka, apparent affinity constant; t1/2, phase half-life. and CDR3 of the VL of the same anti-dextran Ab. The sites chosen Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 2163 were predicted to be on the surface of the native protein based on Ab analysis the molecular model of the binding site of the 19.22.1 and were The Abs were analyzed by metabolic labeling and immunoprecipitation. introduced as single amino acid changes to minimize the effect of Between 3 and 5 3 106 cells were washed, resuspended in 1 ml of labeling amino acid substitution on the binding site (23). We joined the medium (high glucose DMEM deficient in methionine: Life Technologies, k Grand Island, NY) containing 25 mCi of [35S]methionine (Amersham, Ar- mutant VL and VH to human IgG1 and constant regions and expressed different combinations of the mutations as chimeric Abs lington Heights, IL), and allowed to incorporate label for 3–4 h or over- night with the addition of 1% FCS or a-calf serum at 37°C under tissue in the cell line Sp2/0. culture conditions. The Abs were immunoprecipitated with 2.5 mlofa We found that the position of the V region carbohydrate can rabbit anti-human IgG Fc and anti-human Fab polyclonal antiserum and a influence many of the functional properties of the Ab. These in- 10% suspension of IgGSorb (Enzyme Center, Woburn, MA). The precip- clude not only properties usually associated with the V region, itated labeled Ab was then analyzed by SDS-PAGE on 5% sodium phos- phate-buffered polyacrylamide gels. To examine heavy and light chains such as affinity for Ag, but other characteristics as well, such as separately, the labeled sample was reduced by treatment with 0.15 M 2-ME half-life and organ targeting, and may shed light on how changes at 37°C for 30 min and analyzed on 12.5% Tris-glycine buffered poly- in carbohydrate structure affect protein conformation and create acrylamide gels. potentially pathogenic molecules. These studies suggest that mod- Tunicamycin treatment of transfectomas ification of V region glycosylation provides an alternate strategy for manipulating the functional attributes of the Ab molecule.