Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors.

ABSTRACT: Fc gamma receptors (Fc?Rs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and Fc?R impacts their interaction dramatically. Regarding Fc?R glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map Fc?RIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for Fc?RIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying Fc?RIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous Fc?RIIIa on natural killer cells. This method will allow assessment of differences in Fc?RIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

SUBMITTER: Wojcik I 

PROVIDER: S-EPMC7547861 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature