Dataset Information

Site-specific N-glycosylation analysis of soluble Fc? receptor IIIb in human serum.

ABSTRACT: Fc-receptors for immunoglobulin G (Fc?Rs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of Fc?Rs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous Fc?RIIIa and Fc?RIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant Fc?RIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of Fc?RIIIb 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 Fc?RIII glycoproteins. Among the six N-glycosylation sites of serum soluble Fc?RIIIb, 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 Fc?RIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.

SUBMITTER: Yagi H

PROVIDER: S-EPMC5807427 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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

Yagi Hirokazu H Takakura Daisuke D Roumenina Lubka T LT Fridman Wolf Herman WH Sautès-Fridman Catherine C Kawasaki Nana N Kato Koichi K

Scientific reports 20180209 1

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 ...[more]

PMID: 29426894

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Project description:Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

Project description:Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-Fc?RIIA and Fc?RIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding Fc?RIIA and Fc?RIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding Fc?RIIA p.H166R and Fc?RIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. Fc?RIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between Fc?RIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p=0.0061; recessive: p=0.097; dominant: p=0.0076) of inheritance. The Fc?RIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p=0.049). The Fc?RIIIB-NA2*03 allotype (CTGCGA), a variant of the classical Fc?RIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p=0.0092). The present study is the first to report an association between a variant of Fc?RIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical Fc?RIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.

Dataset Information

Site-specific N-glycosylation analysis of soluble Fc? receptor IIIb in human serum.

Publications

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

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