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.

Figure 1

Sequence alignments of the extracellular regions of human FcγRIIIa and sFcγRIIIb NA1 and NA2 forms. N-glycosylation sites are shown in red. The residues substituted between NA1 and NA2 are shown in blue.

Figure 2

MS profiling of site-specific glycoforms of the serum sFcγRIIIb, covering six N-glycosylation sites. (A) Asn38, (B) Asn45, (C) Asn64, (D) Asn74, (E) Asn162, and (F) Asn169. The spectra are based on the averaged mass scans of the glycopeptides containing individual N-glycosylation sites, which eluted in time ranges of (A) 27.8–29.1 min, (B) 32.7–33.4 min, (C) 31.0–33.8 min, (D) 21.8–24.8 min, (E) 32.0–39.0 min, and (F) 44.0–50.0 min. The identities of Man, Gal, Fuc, and GlcNAc were inferred from the literature and are represented by symbols according to the Symbol Nomenclature for Glycans (SNFG) system, the details of which can be found at NCBI (http://www.ncbi.nlm.nih.gov/books/NBK310273/): GlcNAc, ; Man, ; Gal, ; Neu5Ac, ; Fuc, .

Figure 3

MS/MS spectra of the major glycoforms of glycopeptides covering six N-glycosylation sites. (A) Asn38, (B) Asn45, (C) Asn64, (D) Asn74, (E) Asn162, and (F) Asn169. The identities of Man, Gal, Fuc, and GlcNAc were inferred from the literature and are represented by symbols according to the Symbol Nomenclature for Glycans (SNFG) system, the details of which can be found at NCBI (http://www.ncbi.nlm.nih.gov/books/NBK310273/): GlcNAc, ; Man, ; Gal, ; Neu5Ac, ; Fuc, .