Human IgG subclass proteins exhibit more than 95% primary amino acid sequence homology in their Fc regions, but each has a unique profile for recognition by the 3 human Fc gamma receptors. The Fc gamma Rs are themselves highly homologous members of the immunoglobulin supergene family. Consistent with these data we have proposed that Fc gamma RI, Fc gamma RII and Fc gamma RIII recognise overlapping non-identical interaction sites in the lower hinge region of the C(H)2 domain of the IgG molecule. Evidence in support was provided by protein engineering effecting single amino acid replacements in the proposed site. Alternatively, we have demonstrated that the primary amino acid sequence alone is not sufficient for IgG molecules to fold with the generation of Fc gamma 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 does not affect this primary activity; although in vivo it could influence other essential biological activities. These findings have opened up a new approach to engineering antibody function - by protein engineering of amino acid residues that form contacts with the oligosaccharide moiety. In the present report we demonstrate that replacement of contact 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.
