The N-linked oligosaccharide at Fc RIIIa Asn-45: an inhibitory element for high Fc RIIIa binding affinity to IgG glycoforms lacking core fucosylation

Abstract

Human leukocyte receptor IIIa (FcγRIIIa) plays an important role in mediating therapeutic antibodies’ antibody-dependent cellular cytotoxicity (ADCC), which is closely related to the clinical efficacy of anticancer processes in humans in vivo. The removal of the core fucose from oligosaccharides attached to the Fc region of antibodies improves FcγRIIIa binding, allowing the antibodies to enhance dramatically the antibody effector functions of ADCC. In this study, the contribution of FcγRIIIa oligosaccharides to the strength of the FcγRIIIa/antibody complex was analyzed using a serial set of soluble human recombinant FcγRIIIa lacking the oligosaccharides. A nonfucosylated antibody IgG1 appeared to have a significantly higher affinity to the wild-type FcγRIIIa fully glycosylated at its five N-linked oligosaccharide sites than did the fucosylated IgG1, and this increased binding was almost abolished once all of the FcγRIIIa glycosylation was removed. Our gain-of-function analysis in the FcγRIIIa oligosaccharide at Asn-162 (N-162) confirmed that N-162 is the element required for the high binding affinity to nonfucosylated antibodies, as previously revealed by loss-of-function analyses. Interestingly, beyond our expectation, the FcγRIIIa modified by N-162 alone showed a significantly higher binding affinity to nonfucosylated IgG1 than did the wild-type FcγRIIIa. Attachment of the other four oligosaccharides, especially the FcγRIIIa oligosaccharide at Asn-45 (N-45), hindered the high binding affinity of FcγRIIIa to nonfucosylated IgG1. Our data clearly demonstrated that N-45 is an inhibitory element for the high FcγRIIIa binding affinity mediated by N-162 to nonfucosylated antibodies. This information can be exploited for the structural-based functional study of FcγRIIIa.