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

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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 N-glycan 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 disease-associated outcomes. The multi-Fcγ receptor glycoproteomic workflow reported here will empower studies focused on the role of FcγR N-glycosylation in autoimmune diseases.", "claims": [{"id": "claim-method", "statement": "Student’s paired t test was used to compare glycopeptide relative abundances in Excel.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:38363599", "locator": "sections[6] \"Data Analysis\" paragraph 3", "quote": "Student’s paired t test was used to compare glycopeptide relative abundances in Excel."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry data have been deposited to the ProteomeXchange Consortium (Deutsch et al. 2017) via the PRIDE (Perez-Riverol et al. 2019) partner repository with the data set identifier PXD043966.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:38363599", "locator": "sections[5] \"Mass Spectrometry\" paragraph 4", "quote": "The mass spectrometry data have been deposited to the ProteomeXchange Consortium (Deutsch et al. 2017) via the PRIDE (Perez-Riverol et al. 2019) partner repository with the data set identifier PXD043966."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:38363599"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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