Dataset Information

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

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

SUBMITTER: Wojcik I

PROVIDER: S-EPMC7547861 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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

Wojcik Iwona I Sénard Thomas T de Graaf Erik L EL Janssen George M C GMC de Ru Arnoud H AH Mohammed Yassene Y van Veelen Peter A PA Vidarsson Gestur G Wuhrer Manfred M Falck David D

Analytical chemistry 20200922 19

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 ...[more]

PMID: 32886488

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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 Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors.

Publications

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

Similar Datasets

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