GLYCOSYLATION OF THE VARIABLE REGION OF IMMUNOGLOBULIN-G - SITE-SPECIFIC MATURATION OF THE SUGAR CHAINS

The structure of the N-linked sugar chains attached to three IgG antib odies, identical in amino acid sequence except for the changes require d to introduce the carbohydrate addition sites, has been determined. A ll three antibodies are specific for dextran but differ in their abili ty to bind antigen. The heavy chains with a murine variable region (V region) attached to the human gamma 4 constant region were expressed i n a murine hybridoma synthesizing the specific light chain. In additio n to the glycosylation site in the Fc portion, each antibody has a dif ferent glycosylation site in the second complementarity determining re gion (CDR2) of the heavy chain (Asn54, Asn58, or Asn60). The sugar cha ins were released from purified Fab and Fc fragments by hydrazinolysis and converted to radioactive oligosaccharides by reduction with sodiu m borotritide. The structures of these radioactive oligosaccharides we re determined by a combination of sequential exoglycosidase digestion and Bio-Gel P-4 and lectin column chromatography. For all three antibo dies, the carbohydrate attached to the Fc portion was a mixture of com plex-type biantennary sugar chains. The variable region carbohydrate s tructures attached at Asn54 and Asn58 were also complex-type but more highly sialylated than were the Fc-associated sugars. Moreover, unlike the Fc-associated sugars, a significant population of Fab-associated sugars contained a Gal alpha 1-->3 residue as a non-reducing terminus. In contrast, the carbohydrate attached at Asn60 was a high mannose st ructure. These results demonstrate that slight changes in the position of carbohydrate attachment within CDR2 of the variable region of the heavy chain can substantially alter carbohydrate processing and that c omplex-type carbohydrates contained within the same polypeptide chain can have different structures.