Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure.

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Abstract
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.