OLIGOSACCHARIDE PROTEIN INTERACTIONS IN IGG CAN MODULATE RECOGNITION BY FC-GAMMA RECEPTORS

X-ray crystal structures of IgG-Fc provide evidence of extensive nonco valent interactions between the protein and carbohydrate moieties, and glycosylation, at Asn-297 within the Fc, has been shown to be importa nt for effector functions mediated through Fc gamma receptors expresse d on leukocytes. We have applied protein engineering in an attempt to define protein/carbohydrate interactions essential to wild-type biolog ical activity. We demonstrate that replacement of Lys-246, Asp-249, an d Glu-258, which make contacts with GlcNac and Gal on the outer alpha[ 1-->6] arm, do not affect recognition of human chimeric IgG3 by human Fc gamma RI and Fc gamma RII. However, replacement of Asp-265, which m ake contacts with the primary GlcNac sugar residue and is covalently a ttached to Asn-297, resulted in loss of recognition of both Fc gamma R I and Fc gamma RII. Similarly, replacement of Asp-265 in mouse IgG2b r esulted in loss of recognition by mouse Fc gamma RII. These results su ggest that noncovalent contacts of Asp-265 with the primary GlcNac res idue are important for maintenance of recognition of IgG by Fc gamma r eceptors whereas contacts with GlcNac and Gal on the alpha[1-->6] arm do not have a measurable effect. This conclusion was supported by expe riments in which galactose-deficient and fully galactosylated forms of a human IgG4-Fc fragment were shown to be equivalent in their ability to inhibit superoxide generation by IgG4 stimulated U937 cells.