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

Abstract

Fc gamma receptors (FcγRs) 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 FcγR impacts their interaction dramatically. Regarding FcγR 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 FcγRIIIb 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 FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

Fc γ receptor compositional heterogeneity: Considerations for immunotherapy development

“…The primary CD16b N162-glycan was a core-fucosylated complex-type structure with two branches and a single α2-6 N -acetylneuraminic acid, consistent with CD16a, although no evidence for less-processed hybrid or oligomannose forms was found and one report analyzed material from 50 donors ( 27 , 49 , 50 ). The comparison with CD16a is notable because the IgG1 Fc binding affinity for the CD16b NA2 allotype, despite a high sequence identify to CD16a, is not affected by changing its N-glycan composition, unlike CD16a ( 30 ).…”

“…and Wojcik et al. who noted limited glycan occupancy at this site ( 27 , 50 , 51 ). It is unclear if N64 glycosylation impacts CD16b function.…”

“…This N-glycan shows substantial processing with a high degree of complex-type forms but less branching than glycans at N38 and N74 ( 49 , 50 ).…”

“…N38 and N74 showed high levels of processing with branch sialylation and LacNAc repeats comparable with CD16a ( 49 , 50 ).…”

“…The CD16b NA2 N45 glycosylation site contains predominantly oligomannose-type N-glycans identified in contrast to the hybrid forms found on CD16a, although two reports differ in the proportion. One study using CD16b isolated from serum reported only oligomannose forms ( 49 ), whereas two others reported 80 to 90% oligomannose forms from neutrophils ( 27 , 50 ). One report also identified lower percentages of oligomannose forms in soluble CD16 (40–60%) likely because of the presence of both CD16a and CD16b in the solution ( 27 ).…”