Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure.
The presence of N-linked carbohydrate at Asn58 in the VH of the antigen binding site of an antibody specific for alpha(1----6)dextran (TKC3.2.2) increases its affinity for dextran 10- to 50-fold. Site-directed mutagenesis has now been used to create novel carbohydrate addition sequences in the CDR2 of a non-glycosylated anti-dextran at Asn54 (TST2) and Asn60 (TSU7). These antibodies are glycosylated and the carbohydrates are accessible for lectin binding. The amino acid change in TSU7 (Lys62----Thr62) decreases the affinity for antigen; however, glycosylation of TSU7 increased its affinity for antigen 3-fold, less than the greater than 10-fold increase in affinity seen for glycosylated TKC3.2.2. The difference in impact of glycosylation could result either from the position of the carbohydrate or from its structure; unlike the other antibodies, TSU7 attaches a high mannose, rather than complex, carbohydrate in CDR2. In contrast, glycosylation of TST2 at amino acid 54 inhibits dextran binding. Thus slight changes in the position of the N-linked carbohydrate in the CDR2 of this antibody result in substantially different effects on antigen binding. Unlike what was observed for the anti-dextrans, a carbohydrate addition site placed in a similar position in an anti-dansyl is not utilized.
Wright
A
A
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Tao
M H
MH
Kabat
E A
EA
Morrison
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002460:Cell Line; D003208:Concanavalin A / Q000378:metabolism; D003911:Dextrans / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D003911:Dextrans; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D037102:Lectins; D003208:Concanavalin A