Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of health donors

Abstract

Fc gamma receptors (FcγR) 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. In this study, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation from primary human material. In contrast to recently published alternatives, we assessed all glycosylation sites 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 FcγRIII glycosylation differences between individuals, cell types, subcellular locations and pathophysiological conditions.

Fc γ receptor compositional heterogeneity: Considerations for immunotherapy development

“…N162 -The primary CD16b N162-glycan was a core-fucosylated complex-type structure with two branches and a single a2-6 N-acetylneuraminic acid, consistent with CD16a, though no evidence for less processed hybrid or oligomannose forms were found and one report analyzed material from 50 donors (27,49,50). The comparison to 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).…”

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

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

“…glycan processing for the soluble form of the NA2 allele, comparable to N38 and N74 but with less fucosylation and sialylation (49) in contrast to Washburn et al and Wojchik et al that noted limited glycan occupancy at this site (27,50,51). It is unclear if N64 glycosylation impacts CD16b function.…”

“…N169 -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).…”