Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding

The variable-domain-attached oligosaccharide side chains of a human IgG pro duced by a human-human-mouse heterohybridoma were analysed. In addition to the conserved N-glycosylation site at Asn-297, an N-glycosylation consensus sequence (Asn-Asn-Ser) is located at position 75 in the variable region of its heavy chain. The antibody was cleaved into its antigen-binding (Fab) a nd crystallizing fragments. The oligosaccharides of the Fab fragment were r eleased by digestion with various endo- and exoglycosidases and analysed by anion-exchange chromatography and fluorophore-assisted carbohydrate electr ophoresis. The predominant components were disialyl-bi-antennary and tetra- sialyl tetra-antennary complex carbohydrates. Of note is the presence in th is human IgG of oligosaccharides containing N-glycolylneuraminic acid and N -acetylneuraminic acid in the ratio of 94:6. Furthermore, we determined N-a cetylgalactosamine in the Fab fragment of this antibody, suggesting the pre sence of O-linked carbohydrates. A three-dimensional structure of the glyco sylated variable (Fv) fragment was suggested using computer-assisted modell ing. In addition, the influence of the Fv-associated oligosaccharides of th e CBGA1 antibody on antigen binding was tested in several ELISA systems. De glycosylation resulted in a decreased antigen-binding activity.