Oligosaccharide-protein interactions in IgG can modulate recognition by Fc gamma receptors.
X-ray crystal structures of IgG-Fc provide evidence of extensive noncovalent interactions between the protein and carbohydrate moieties, and glycosylation, at Asn-297 within the Fc, has been shown to be important for effector functions mediated through Fc gamma receptors expressed on leukocytes. We have applied protein engineering in an attempt to define protein/carbohydrate interactions essential to wild-type biological activity. We demonstrate that replacement of Lys-246, Asp-249, and 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 make contacts with the primary GlcNac sugar residue and is covalently attached to Asn-297, resulted in loss of recognition of both Fc gamma RI and Fc gamma RII. Similarly, replacement of Asp-265 in mouse IgG2b resulted in loss of recognition by mouse Fc gamma RII. These results suggest that noncovalent contacts of Asp-265 with the primary GlcNac residue are important for maintenance of recognition of IgG by Fc gamma receptors whereas contacts with GlcNac and Gal on the alpha [1-->6] arm do not have a measurable effect. This conclusion was supported by experiments 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.
Lund
J
J
Department of Immunology, Medical School, Edgbaston, Birmingham, United Kingdom.
Takahashi
N
N
Pound
J D
JD
Goodall
M
M
Nakagawa
H
H
Jefferis
R
R
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005690:Galactose / Q000032:analysis; D006241:Haptens / Q000276:immunology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011961:Receptors, Fc / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012397:Rosette Formation; D013329:Structure-Activity Relationship
D006241:Haptens; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011506:Proteins; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D005690:Galactose