RECOGNITION SITES ON HUMAN-IGG FOR FC-GAMMA RECEPTORS - THE ROLE OF GLYCOSYLATION

Human IgG subclass proteins exhibit more than 95% primary amino acid s equence homology in their Fc regions, but each has a unique profile fo r recognition by the 3 human Fc gamma receptors. The Fc gamma Rs are t hemselves highly homologous members of the immunoglobulin supergene fa mily. Consistent with these data we have proposed that Fc gamma RI, Fc gamma RII and Fc gamma RIII recognise overlapping non-identical inter action sites in the lower hinge region of the C(H)2 domain of the IgG molecule. Evidence in support was provided by protein engineering effe cting single amino acid replacements in the proposed site. Alternative ly, we have demonstrated that the primary amino acid sequence alone is not sufficient for IgG molecules to fold with the generation of Fc ga mma R interaction sites and that glycosylation of Asn 297 of the C(H)2 domain is essential. We have further defined a 'core' oligosaccharide structure that provides for the generation of Fc gamma R interaction sites which suggests that the addition of outer-arm sugar residues doe s not affect this primary activity; although in vivo it could influenc e other essential biological activities. These findings have opened up a new approach to engineering antibody function - by protein engineer ing of amino acid residues that form contacts with the oligosaccharide moiety. In the present report we demonstrate that replacement of cont act residues for galactose on the alpha((1-6)) arm does not affect Fc gamma RI and Fc gamma RII recognition while replacement of Asp 265, a contact for a 'core' N-acetylglucosamine residue, results in a loss of Fc gamma RI and Fc gamma RII recognition.