Characterization of endogenous human FcRIII by mass spectrometry reveals site, allele and sequence specific glycosylation

Abstract

The importance of IgG glycosylation, Fc-gamma receptor (FcR) single nucleotide polymorphisms and FcR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcRs in modulating the FcR-IgG interaction based on the association between the glycosylation of recombinant FcRs and the kinetics and affinity of the FcR-IgG interaction. Although glycosylation of recombinant FcRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcRs. In order to improve the structural understanding of FcRs expressed on human cells we characterized the site specific glycosylation of native human FcRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcRIII from a single donor, identified FcRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcRIIIb. Identification of FcRIIIb specific glycosylation allows for assignment of FcRIIIb alleles and relative copy number of the two alleles where DNA/ RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.