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 4 publications

(3 citation statements)

References 47 publications

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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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Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface

Kremer

Barb

2022

Journal of Biological Chemistry

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One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure

Kremer,

Lampros,

Blocker

et al. 2024

eLife

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Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

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

Cited by 4 publications

References 47 publications

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

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

The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure

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