Site-specific N-glycosylation analysis of soluble Fcγ receptor IIIb in human serum

Abstract

Fc-receptors for immunoglobulin G (FcγRs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of FcγRs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous FcγRIIIa and FcγRIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant FcγRIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of FcγRIIIb purified from human serum based on mass spectrometric analysis. Our data indicate a distinct and common tendency of the glycoforms exhibited at each N-glycosylation site between the native and the previously reported recombinant FcγRIII glycoproteins. Among the six N-glycosylation sites of serum soluble FcγRIIIb, Asn45 was shown to be exclusively occupied by high-mannose-type oligosaccharides, whereas the remaining sites were solely modified by the complex-type oligosaccharides with sialic acid and fucose residues. The results of our endogenous FcγRIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.

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

“…The occupancy of site N64 appears to be the most prominent difference between the membrane-bound and the soluble version of the receptor IIIb ( Supplementary Table 6). In contrast to the neutrophil-derived version, soluble FcγRIIIb has been described to display highly branched glycan structures on site N64 32 . The limited m/z range up to 1500 may not allow us to detect those complex structures.…”

“…(H5N4F1S1). The MS/MS spectrum was generated for three stepping energies (32,37,42 V) at retention time 11.0 min. Interestingly, ammonia adducts were exclusively observed for glycopeptides carrying an oligomannose glycan, while iron adducts were detected for both oligomannose and complex structures.…”

“…While ground-breaking, the two previous studies on site-specific glycosylation of FcγRIIIb also had some limitations 31,32 . Both studies relied on two independent proteolytic cleavages to assess different sites, thus necessitating multiple liquid chromatography -mass spectrometry (LC-MS) runs to cover the whole receptor.…”

“…Both studies relied on two independent proteolytic cleavages to assess different sites, thus necessitating multiple liquid chromatography -mass spectrometry (LC-MS) runs to cover the whole receptor. Yagi et al focused on the soluble version of FcγRIIIb which is released from neutrophils during activation and whose function is largely unknown 32 . They used pooled blood from multiple donors, loosing inter-donor variability.…”

“…Nonetheless, we still know too little about the functional and clinical impact of FcγR glycosylation to prefer a method focussing only on certain glycosylation sites. Other existing strategies covering all sites of FcγRIIIb or FcγRIIIa are quite complicated and difficult to apply to clinical investigations where eventually large numbers of samples need to be detected in a robust way 29,32 .…”