MULTIPLE INTERACTIONS OF IGG WITH ITS CORE OLIGOSACCHARIDE CAN MODULATE RECOGNITION BY COMPLEMENT AND HUMAN FC-GAMMA RECEPTOR-I AND INFLUENCE THE SYNTHESIS OF ITS OLIGOSACCHARIDE CHAINS

Glycosylation at Asn(297) within the C(H)2 domains of IgG is important for recognition by the effector ligands Fc gamma R and C. Protein eng ineering has been used to replace amino acid residues within the exten sive oligosaccharide interaction site that contact the core hexasaccha ride (GlcNAc(2)Man(3)GlcNAc). Replacement of residues Phe(241), Val(26 4), or Asp(265), in particular, results in reduced recognition of huma n chimeric anti-nitroiodophenacetyl IgG3 produced in Chinese hamster o vary cells, by guinea pig C and human C1q, Replacement of residues Val (264) or Asp(265), in particular, results in reduced superoxide produc tion triggered through human Fc gamma RI expressed on U937 cells, Thes e results suggest that noncovalent interactions of multiple amino acid residues of IgG with oligosaccharide residues that include the primar y and secondary GlcNAc are necessary for optimal recognition of IgG by human Fc gamma RI and C1q, Replacement of residues 241, 243, 264, 265 , or 301 with alanine in each case resulted in increased galactosylati on and sialylation relative to the wild-type oligosaccharide chains, I n particular, for the mutant FA243 there was much increased sialylatio n of its oligosaccharide chains (73%) relative to the wild-type (4%), Thus, even single residue replacements within the oligosaccharide inte raction site of the C region can influence galactosylation and sialyla tion of its oligosaccharide chains, These data suggest a protein engin eering route to the production of more homogeneously glycosylated Ige molecules with or without compromised biologic activities.