The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites

Lampros, Elizabeth A; Kremer, Paul G.; León, Jesús S Aguilar Díaz de; Roberts, Elijah T; Benavente, Maria C. Rodriguez; Amster, I. Jonathan; Barb, Adam W.

doi:10.1016/j.crimmu.2022.05.005

Cited by 2 publications

(3 citation statements)

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“…1,2 For example, the FcγRIIIA Val176(158)Phe substitution is associated with increased susceptibility to systemic lupus erythematosus (SLE) 19 and rheumatoid arthritis, 20 yet the role of FcγR glycosylation in this context is unclear. 21,22 A lack of high-throughput methods for site-specific analysis of immunomodulatory FcγR N-glycosylation in humans and mice, is a bottleneck for the study of FcγR N-glycosylation in human autoimmune diseases and precludes the analysis of clinical samples and of FcγRs derived from mouse models of autoimmune disease. Recent studies have begun to bridge this gap via the analysis of released N-linked glycans derived from recombinant FcγRs 23,24 and FcγRs purified from human NK cells.…”

Section: ■ Introductionmentioning

confidence: 99%

“…14 However, the release of glycans prior to analysis results in the loss of valuable contextual information about the site of glycosylation. Tandem mass spectrometry has been utilized to measure FcγR N-glycosylation site occupancy, via the analysis of peptide:N-glycosidase F (PNGase F)-treated peptides, 22 and characterize FcγR N-linked glycosylation in a site-specific manner, via the analysis of chymotrypsin + GluC glycopeptides. 25 The latter approach is advantageous in that it maintains a relationship between the glycan modifications and the site(s) of glycosylation.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Human FcγRIIA, FcγRIIC, and FcγRIIIB also lack murine orthologs. Human FcγRs also have greater allelic variation compared to their mouse counterparts, and FcγR polymorphisms are associated with autoimmune disorders including Guillain-Barré syndrome and Wegener’s granulomatosis. , For example, the FcγRIIIA Val176(158)Phe substitution is associated with increased susceptibility to systemic lupus erythematosus (SLE) and rheumatoid arthritis, yet the role of FcγR glycosylation in this context is unclear. , …”

Section: Introductionmentioning

confidence: 99%

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Site-Specific Glycosylation Analysis of Murine and Human Fcγ Receptors Reveals High Heterogeneity at Conserved N-Glycosylation Site

Pavan,

Abdoollah,

Marrero Roche

et al. 2024

J. Proteome Res.

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Fc γ-receptors (FcγRs) on leukocytes bind immunoglobulin G (IgG) immune complexes to mediate effector functions. Dysregulation of FcγR-mediated processes contributes to multiple inflammatory diseases, including rheumatoid arthritis, lupus, and immune thrombocytopenia. Critically, immunoregulatory N-glycan modifications on both FcγRs and IgGs alter FcγR-IgG binding affinity. Rapid methods for the characterization of N-glycans across multiple Fcγ receptors are needed to propel investigations into disease-specific contributions of FcγR N-glycans. Here, we utilize nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) to characterize FcγR glycosylation and report quantitative and site-specific Nglycan characterization of recombinant human FcγRI, FcγRIIIA V158, and FcγRIIIA F158 from CHO cells and murine FcγRI, FcγRIII, and FcγRIV from NS0 cells. Data are available via ProteomeXchange with identifier PXD043966. Broad glycoform distribution (≥30) was observed at mouse FcγRIV site N159 and human FcγRIIIA site N162, an evolutionarily conserved site. Further, mouse FcγRIII N-glycopeptides spanning all four predicted N-glycosylation sequons were detected. Glycoform relative abundances for hFcγRIIIA V/F158 polymorphic variants are reported, demonstrating the clinical potential of this workflow to measure differences in glycosylation between common human FcγRIIIA allelic variants with diseaseassociated outcomes. The multi-Fcγ receptor glycoproteomic workflow reported here will empower studies focused on the role of FcγR N-glycosylation in autoimmune diseases.

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