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

Abstract

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 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.

Introduction

Fc γ (gamma)-receptors (FcγRs) on leukocytes bind immunoglobulin G (IgG) immune complexes to mediate effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP).¹ FcγRs additionally act as intermediaries, during phagocytosis and endocytosis of opsonized antigens, to facilitate antigen degradation and/or integration into major histocompatibility complex (MHC) class I and class II membrane protein complexes.² The cellular effects of IgGs are initiated by the interaction between the crystallizable fragment (Fc) region of an IgG and the extracellular domain of an Fc gamma receptor (FcγR) on the plasma membrane of leukocytes.^3,4 In humans, multiple Fcγ receptors with differing affinities for IgGs are expressed in cell-specific patterns, including FcγRI (CD64), FcγRIIA-C (CD32a-c), and FcγRIIIA-B (CD16a-b).^5,6 Pro-inflammatory (activating) FcγRs include FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, and FcγRIIIB. Most activating FcγRs rely on interactions with adaptor molecules containing immunoreceptor tyrosine-based activation motifs (ITAMs) to propagate intracellular signaling, while the cytoplasmic regions of FcγRIIA and FcγRIIC contain ITAMs that directly initiate downstream signaling.¹ Activating FcγRs promote the release of pro-inflammatory cytokines, ADCC, and ADCP.^2,7 FcγRIIB is the only inhibitory FcγR and contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that opposes activating signals originating from the ITAM pathway via inhibition of phospholipase-Cγ (PLCγ), AKT, and Ras.^2,7

The extracellular domains of FcγRs are co- and post-translationally modified by asparagine N-linked glycosylation. Over the past two decades, an understanding of how N-linked glycans regulate FcγR-IgG interactions has emerged.⁵ Among FcγRs, N-linked glycans have the most pronounced role in regulating FcγRIIIA-B function.^3,4 Specific carbohydrate-carbohydrate interactions between the crystallizable fragment (Fc) region of IgG and the extracellular domain of FcγRIIIA-B modulate receptor affinity for IgGs.^3,4,8 Human FcγRIIIA has five N-glycosylation sites within its extracellular domain (Figure 1, Supplemental Figure S-1), with N-linked glycans at site N162 playing an outsized role in the regulation of its affinity for IgG.⁹ Evidence suggests that FcγRIIIA site N162 glycans directly interact with N297 glycans on the Fc region of IgG to promote ligand binding.¹⁰⁻¹² FcγRIIIA forms with high mannose N-linked glycans at site N162 have the highest IgG binding affinity compared to other receptor glycoforms.^9,11,13−15 Opposing this, FcγRIIIA site N45 glycans sterically block FcγR-IgG interactions.¹⁰ Similarly, within the Fc region of IgG, the presence of site N297 core fucosylated N-linked glycans decreases IgG binding to human FcγRIIIA by up to 20-fold,³ while the presence of terminal galactose residues doubles IgG recognition by FcγRIIIA.³ The effect of IgG-Fc glycosylation on FcγRIIIA binding affinity has also been defined in relation to its impact on ADCC and antigen presentation.²⁻⁴ While much has been learned regarding how N-glycans regulate FcγR-IgG interactions, the role of FcγR N-glycans in human disease has not been defined.

Figure 1.

Murine and human Fcγ receptor N-glycosylation sites. Diagrams representing the sequences and N-glycosylation sites of the murine (m) and human (h) Fcγ receptors (FcγRs) analyzed in this study are shown, with the N-glycosylation site locations labeled with black circles and numbered based on the amino acid sequence inclusive of the signal peptide, as these positions are listed in the protein sequence database Uniprot.¹⁶ The position of the N-glycosylation site in the mature protein without the signal peptide is given in parentheses. Signal peptides are displayed in gray. Immunoglobulin-like domains (D1, D2, ...) are labeled. The position of the common hFcγRIIIA variant V/F176(158) is noted in gray.

Historically, in vivo mouse models have been utilized to study the role(s) of FcγRs in inflammation and inflammatory diseases and may therefore offer valuable insight into the glycobiology of FcγRs in inflammatory disease, with limitations.^1,3 Four FcγRs have been described in mice: FcγRI, FcγRIIB, FcγRIII, and FcγRIV.^1,17,18 As in humans, mouse FcγRI is the only high affinity receptor for IgG, with the remaining receptors demonstrating lower, subclass specific affinity for IgG ligands.^1,18 Murine FcγRIV is the orthologue of human FcγRIIIA and recognizes IgG2a and IgG2b, though it differs from human FcγRIIIA in that it also binds IgE immune complexes. Several clear differences exist between human and mouse FcγRs. For example, human FcγRs show a greater evolutionary complexity compared to their murine counterparts as evidenced by the presence of three homologous human FcγRI genes FcγRIA, FcγRIB, and FcγRIC.¹ 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.^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.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. Variations of this strategy have been applied to characterize the N-glycosylation of FcγRs derived from human immune cells and serum.²⁶⁻³⁰ These approaches have characterized one or a limited number of FcγRs in a single analysis, and each has used slightly different analytical workflows, limiting researchers’ ability to directly compare results between FcγRs and between studies. Furthermore, while human FcγR N-glycosylation has been characterized in recombinant FcγRs and native FcγRs from human immune cells, the glycosylation of murine FcγRs is not well described.

To advance the study of FcγR N-glycosylation function and its role in autoimmune diseases, we sought to develop a rapid and high-throughput nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) method for the site-specific analysis of FcγR N-glycosylation in humans and mice. Given the frequent use of murine models of disease in biomedical research, we applied the method to compare the glycosylation of murine and human FcγR orthologues. Here, we document the site-specific glycosylation of multiple murine and human activating FcγRs, including human FcγRI, FcγRIIIA V158, and FcγRIIIA F158, and murine Fcγ receptors FcγRI, FcγRIII, and FcγRIV. Based on these analyses, we compare the glycosylation patterns of (i) a conserved site on murine FcγRIV and human FcγRIIIA, and (ii) human FcγRIIIA V/F158 polymorphic variants. Our findings demonstrate the high potential of these methods to advance functional and disease-specific studies of FcγR N-glycosylation.

Experimental Section

Reagents

Recombinant Fcγ receptors, including human FcγRI, FcγRIIIA V158, and FcγRIIIA F158 expressed in Chinese hamster ovary (CHO) cells, and murine Fcγ receptors FcγRI, FcγRIII, and FcγRIV expressed in mouse myeloma cell line NS0, were purchased from R&D Systems (Minneapolis, MN, USA). Dithiothreitol (DTT), Pierce C18 100 μL tips, sequencing grade chymotrypsin, and Glu-C protease were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Ammonium bicarbonate and iodoacetamide were purchased from Millipore Sigma (Burlington, MA, USA).

Proteolysis

In silico proteolysis was performed on the canonical protein sequences of each Fcγ receptor from UniProt (uniprot.org) using the MS-Digest tool from Protein Prospector (prospector.ucsf.edu) to guide the selection of proteases, with the goal of maximizing the number of observed N-glycosylation sites. Chymotrypsin and GluC proteases were selected based on these analyses. The most notable advantage of combining the proteolytic specificity of chymotrypsin and GluC is that it enables the separation of FcγRIIIA (CD16a) N-glycosylation sequons at N162 and N169. Lyophilized recombinant FcγRs were reconstituted in phosphate buffered saline (PBS, pH 7.4) to a concentration of 1 μg/μL. For each FcγR, 5 μg of receptor was diluted into 50 mM ammonium bicarbonate to achieve a final volume of 100 μL and then reduced in the presence of 5 mM DTT at 60 °C for 1 h. Next, samples were carbamidomethylated in the presence of 15 mM iodoacetamide for 45 min at room temperature in the dark. To quench the reaction, 30 mM DTT was subsequently added. Chymotrypsin (1:40 w/w) and Glu-C (1:30 w/w) were added simultaneously, and samples were incubated overnight at 37 °C. Following overnight digestion, Glu-C (1:30 w/w) was added again and samples were incubated at 37 °C for 3 h to maximize proteolytic cleavage efficiency, based on a published protocol.²⁵ In particular, the addition of GluC again following the initial digestion step was performed to maximize proteolysis of the C-terminal to FcγRIIIA residue E166, to separate N-glycosylation sequons located at N162 and N169. Samples were then dried under vacuum, resuspended in water with 1% acetonitrile and 0.1% formic acid, and desalted using Pierce C18 100 μL tips. Peptides were eluted in 50% acetonitrile/50% water with 0.1% formic acid and dried under vacuum.

Mass Spectrometry

An Orbitrap Eclipse Tribrid mass spectrometer coupled with an EASY nLC 1200 system (Thermo Fisher Scientific, Waltham, MA) was used for the analysis of FcγR proteolytic products by online nanoliquid chromatography-mass spectrometry (nLC-MS). For chromatographic separation, an Acclaim PepMap 100–75 μm × 2 cm trapping column and a PepMap RSLC C18 analytical column −2 μm, 100 Å, 75 μm × 15 cm were employed with the following gradient: initial conditions 2% B, 2–6% B from 0 to 5 min, 6–35% B from 5 to 75 min, 35–60% B from 75 to 80 min, 60–95% B for 30 s, and 95% B for 9.5 min (solvents A and B consisted of 1% acetonitrile/99% water +0.1% formic acid and 80% acetonitrile/20% water +0.1% formic acid, respectively). For all nLC-MS experiments, four technical replicates (n = 4) were performed unless otherwise stated. Initially, 500–750 ng of each FcγR sample (peptides and glycopeptides) were analyzed via nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis for glycopeptide discovery, including glycopeptide assignment and retention time determination. To facilitate determination of glycopeptide relative abundance, a second set (n = 4) of MS analyses were performed in which 250 ng of sample/injection were used, and MS1 profile spectra were acquired (MS only) with identical chromatographic conditions as above.

All MS spectra were acquired in positive mode with source voltage: 1900 V; transfer tube temperature: 275 °C; RF lens: 30%. The mass spectrum analyzer was the Orbitrap with the following settings: resolution of 120,000; m/z range: 375–2000; cycle time: 3 s; maximum injection time: auto; normalized AGC target (%) of 200; AGC target: custom; 2 μscans. For MS2 scans, an HCD spectrum was acquired with the following parameters: 35% collision energy, peptides with charge states 2–7 were selected; min intensity: 2 × 10⁴; dynamic exclusion of 10 s. An isolation window of 1.2 nm and a maximum injection time of 35 ms were set.

The detection of ≥2 glycan oxonium ion peaks (m/z 138.055, 144.065, 168.066, 186.076, 204.087, 274.092, 292.103, 366.139, 512.198; with 25 ppm mass tolerance) in the HCD spectrum triggered an EThcD MS2 scan and an sceHCD scan. EThcD was performed with the following parameters: ETD with 20% supplemental activation, 30,000 resolution, 1 μscan, custom AGC target, 200 normalized AGC target (%), auto maximum injection time. Stepped collision energy HCD (sceHCD) was performed with 15, 30, and 40% collision energies, 30,000 resolution, 1 μscan, custom AGC target, 200 normalized AGC target (%), and 100 ms maximum injection time. MS1 and MS2 spectra were recorded as profile spectra. 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.

Data Analysis

Data analyses was performed using Proteome Discoverer 2.4 (Thermo Fisher Scientific) with an integrated Byonic v4.0.12 node (Protein Metrics Inc., Cupertino, CA, USA), using human and mouse protein sequence databases, inclusive of hFcγRIIIA isoforms V158 and F158, from Uniprot (Release 2023_03).¹⁶ Parameters considered for the search include: an N-glycan database consisting of 309 mammalian glycans, 1 N-glycan per peptide (variable), and carbamidomethylation of cysteine (fixed). For the PD/Byonic searches, the settings were as follows: precursor tolerance: 10 ppm, fragment tolerance: 20 ppm, maximum number of missed cleavages: 2, cleaved residues: DEFLWY, common modifications max: 2, rare modification max: 1, false discovery rate (FDR): 1%. Following the Byonic analyses, glycopeptide assignments were filtered (Byonic score ≥200) and manually curated.

Extracted ion chromatograms (EICs) of glycopeptide precursor ion m/z values were generated from MS (only) RAW data using FreeStyle 1.8 SP2 (Thermo Fisher Scientific), with a 5 ppm tolerance. The area under the curve (AUC) was calculated for each precursor m/z (inclusive of multiple charge states and glycopeptide) using the integration function in FreeStyle. EICs were manually reviewed. The areas of glycopeptides with multiple detected charge states were determined by summing the areas of each charge state, while precursors representing <10% of the most abundant precursor for each glycoform were excluded. Further, when multiple glycopeptides share a common N-glycosylation site but differ in the length of the peptide (arising from missed cleavage(s)), the areas of glycoforms differing in peptide length were summed. Retention time and intensity correlations were measured between runs to ensure consistency between replicates. Finally, AUCs from each glycopeptide were summed, and the summed area was used as the denominator to determine the relative abundance of each glycoform sharing a common N-glycosylation site. Coefficients of variation (CVs) and statistical significance were calculated using Microsoft Excel. Student’s paired t test was used to compare glycopeptide relative abundances in Excel. Protein structures were visualized with Mol*Viewer.³¹

Results

Murine and Human FcγRI Glycoproteomic Analyses Reveal Site-Specific Differences in N-Glycosylation

FcγRI is a high-affinity receptor for the Fc region of IgGs and functions as a bridge between innate and adaptive immune responses. To provide site-specific evidence of murine FcγRI N-glycosylation and compare the findings to the better-characterized human FcγRI, recombinant murine (mFcγRI) and human (hFcγRI) Fc gamma receptor I, expressed in NS0 and CHO cells, respectively, were subject to proteolysis followed by nLC-MS/MS using higher-energy collisional dissociation (HCD) and triggered electron transfer dissociation with supplemental activation (EThcD). The predicted protein structure of murine FcγRI (mFcγRI, Figure 2A),^32,33 and the orthologous human FcγRI (hFcγRI) protein structure determined via X-ray crystallography,³⁴ are shown (Figure 2B), with asparagine residues in blue denoting the sites of N-glycopeptides detected in this study. The relative abundances of mFcγRI N-glycopeptides detected at sites N24, N45, and N144 are shown (Figure 2C–E, Supplemental Tables S1 and S2). At site N24, within the Ig-like C2-type 1 (D1) domain, four N-glycan compositions were detected, with the glycan compositions HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 59%) and HexNAc₂Hex₅ (N₂H₅, 30%), accounting for most of the relative abundance (Figure 2C).

Figure 2.

Site-specific N-glycosylation of recombinant mFcγRI and hFcγRI. (A, B) A protein structure model of mFcγRI (AlphaFold)^32,33 based on UniProt sequence P26151, and the crystallographic protein structure of hFcγRI (PDB ID 3RJD),³⁴ respectively, shown as space fill models. Only the extracellular domains that bear the N-glycosylation sites are shown. Asparagine (N) residues bearing N-glycans detected in this study are labeled (blue). (C–E) mFcγRI site N24, N45, and N144 glycopeptide relative abundances are shown. (F) The relative abundance of high mannose (High Man.), hybrid, and complex type N-linked glycans at mFcγRI sites N24, N45, and N144, are shown. The abundance of each glycan type was calculated by summing the relative abundances of glycopeptides with compositions consistent with high mannose, hybrid, or complex N-linked glycans based on nLC-MS analyses. (G, H) hFcγRI site N35 and N216 glycopeptide relative abundances are shown. (I) The relative abundance of high mannose (High Man.), hybrid, and complex type N-linked glycans at hFcγRI sites N35 and N216, are shown. Glycoform relative abundances are shown in bar plots for each N-glycosylation site. The height of the bar reflects the average of the relative abundance (technical replicates, n = 4) ± standard deviation (SD). N = N-acetylhexosamine, H = hexose, F = fucose, and S = N-acetylneuraminic acid. Glycopeptides were assigned based on precursor m/z, peptide backbone fragment ions, carbohydrate fragment ions, and retention time.

hFcγRI does not have an equivalent N-glycosylation sequon in its D1 domain. A peptide spanning the predicted sequon at site N4 in the D1 of mFcγRI was not detected in these analyses. Of all mFcγRI sites, N45 is of particular interest, given that N-glycosylation sites N45 in mFcγRI and N35 in hFcγRI occupy similar positions in the mouse and human receptors (Figure 2A,B), and they may therefore have similar structural and functional significance. Both residues fall within the D1 domains, occupy the same faces of the extracellular domain, and are positioned equivalently in their respective receptors.

At mFcγRI site N45, 21 distinct glycan compositions were detected via MS, with the most abundant being HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 28%), HexNAc₆Hex₇Fuc₁ (N₆H₇F₁, 12%), and HexNAc₂Hex₅ (N₂H₅, 11%) (Figure 2D). At mFcγRI site N45 only two glycans with compositions consistent with high mannose type N-linked glycans, namely, HexNAc₂Hex₅ (N₂H₅) and HexNAc₂Hex₅ (N₂H₆), were detected and account for a minor share of the relative abundance, while glycan compositions consistent with highly processed complex N-linked glycans account for most of the relative abundance. Eight distinct glycan compositions were detected at mFcγRI site N144 within the Ig-like C2-type 2 (D2) domain. The most abundant compositions were HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 38%) and HexNAc₅Hex₈Fuc₁ (N₅H₈F₁, 25%) (Figure 2E). Glycan compositions consistent with the presence of high mannose and hybrid structures accounted for 3.5% and 8.4% of the relative abundance at this site, respectively.

For each site, mFcγRI N-glycan compositions were categorized as either high mannose (least processed), hybrid, or complex (Figure 2F). In Figure 2F, it is possible to appreciate the distinct patterns of glycan expression across the three mFcγRI N-glycosylation sites. Site N24 displays a mixture of compositions consistent with high mannose (36%) and complex (64%) glycan types. Site N45 displays a higher abundance of complex type glycans (75%) and low levels of high mannose (12%) and hybrid type glycans (13%). Finally, site N144 displays predominantly complex glycans (88%) with only 3% of detected glycan compositions consistent with high mannose and 8% of compositions assigned as hybrid type glycans. Glycopeptides spanning N225, a predicted N-glycosylation site, were not detected, while a nonglycosylated peptide was detected. To compare the N-glycosylation of mFcγRI with hFcγRI, we next analyzed hFcγRI proteolytic products via nLC-MS/MS. The relative abundance of N-glycopeptides detected at hFcγRI sites N35 and N216 are shown (Figure 2G–I). At hFcγRI site N35, compositions consistent with high mannose type N-glycans account for all detected glycoforms, including five distinct compositions. The most abundant glycan compositions were HexNAc₂Hex₅ (N₂H₅, 42%), followed by HexNAc₂Hex₆ (N₂H₆, 26%) and HexNAc₂Hex₈ (N₂H₈, 15%) as shown in Figure 2G. In contrast, site N216 located within Ig-like C2-type 3 (D3) domain, displayed glycan compositions consistent with complex type N-glycans. Twelve distinct glycan compositions consistent with complex type glycans were detected, including HexNAc₆Hex₇Fuc₁NeuAc₃ (N₆H₇F₁S₃, 17%) and HexNAc₅Hex₆Fuc₁NeuAc₃ (N₅H₆F₁S₃, 17%) as shown in Figure 2H. Conspicuously, in the hFcγRI, the two modified asparagine residues show opposite glycan type profiles with site N35 displaying 100% high mannose type glycans and site N216 displaying 100% complex type glycans (Figure 2I). At D1, an unmodified peptide spanning the predicted N-glycosylation site N54 was detected, while the remaining predicted hFcγRI sites N128, N139 and N171 were not detected. These results show evidence that mFcγRI and hFcγRI display different sets of N-glycan compositions. In the D1 domain of mFcγRI, two glycosylated asparagine residues (N24 and N45) display a mixture of high mannose, hybrid, and complex glycans. In contrast, site N35 within hFcγRI D1 displays exclusively high mannose type glycans. hFcγRI site N216 displays a high percent of complex sialylated glycans (98%), in contrast to mFcγRI glycosylation sites and hFcγRI site N35.

Site-Specific N-Glycosylation of Murine FcγRIII

Murine Fc gamma receptor III (mFcγRIII) is a low-affinity Fc receptor that activates neutrophils redundantly with mFcγRIV when bound to IgG immune complexes.³⁵ mFcγRIII does not have a human orthologue. To provide site-specific evidence of mFcγRIII N-glycosylation, recombinant mFcγRIII expressed in NS0 cells, was subject to proteolysis and nLC-MS/MS. The predicted protein structure of mFcγRIII (Figure 3A)^32,33 is shown, with asparagine residues labeled in blue reflecting the sites of N-glycopeptides documented in this study. mFcγRIII N-glycopeptides spanning all four predicted N-glycosylation domains were detected. Based on our analyses, mFcγRIII N-glycan compositions from sites N34, N61, N135, and N142 were categorized as either high mannose, hybrid, or complex and their relative abundances are shown (Figure 3B). At all four mFcγRIII N-glycosylation sites, glycan compositions consistent with complex glycans were the predominant species. Site N142 displays low amounts of high mannose (10%) and hybrid (3%) glycans with complex glycans accounting for the remaining relative abundance. The relative abundances of mFcγRIII site N34, N61, N135, and N142 N-glycopeptides are shown (Figure 3C–F, Supplemental Table S2). At site N34, within the Ig-like C2-type 1 (D1) domain, 30 distinct N-glycan compositions were detected, with HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 27%) and HexNAc₅Hex₈Fuc₁NeuAc₁ (N₅H₈F₁S₁, 20%) compositions accounting for the two most abundant (Figure 3C). HexNAc₆Hex₇Fuc₁ (N₆H₇F₁, 9%) and HexNAc₅Hex₇Fuc₁NeuGc₂ (N₅H₇F₁T₂, 8%) were also detected.

Figure 3.

Site-specific N-glycosylation of recombinant murine Fcγ receptor III (mFcγRIII). (A) A space fill model of the predicted mFcγRIII protein structure is shown. Asparagine (N) residues bearing N-glycans profiled in this study are shown (blue). (B) The relative abundance of high mannose (High Man.), hybrid, and complex type N-linked glycans at mFcγRIII sites N34, N61, N135, and N142, are shown. The abundance of each glycan type was calculated by summing the relative abundances of glycopeptides with compositions consistent with high mannose, hybrid, or complex N-linked glycans based on nLC-MS analyses. (C–F) The relative abundances of mFcγRIII glycopeptide glycoforms are shown at sites N34, N61, N135, and N142. Glycopeptides were generated by treatment of the receptor with chymotrypsin and Glu-C. Replicate (n = 4) nLC-MS/MS analyses were performed to facilitate glycopeptide assignment. Paired replicate (n = 4) nLC-MS (MS only) analyses were subsequently performed, and extracted ion chromatograms (EICs) for each glycopeptide m/z were generated to determine the area under the curve (AUC) of each glycoform. In panels B–F, relative abundance values are derived from nLC-MS technical replicates (n = 4). Error bars representing + / – standard deviation (S.D.) are shown. N = N-acetylhexosamine, H = hexose, F = fucose, S = N-acetylneuraminic acid. T = N-glycolylneuraminic acid.

The amino acid sequence motif Asn-Gly (residues 42–43), close to site N34, is prone to asparagine deamidation to form aspartic acid, followed by partial isomerization to isoaspartic acid.³⁶ This affects the chromatographic behavior of glycopeptides. In the present study, site N34 glycopeptides were detected predominantly in the deamidated form and exhibited a chromatographic pattern consistent with aspartic/isoaspartic acid isomerization (see Supplementary Figure S-2).

Sites N61 within mFcγRIII Ig-like domain D1 and N135 within domain D2 both display the high abundance glycoform HexNAc₅Hex₉Fuc₁ (N₅H₉F₁), with values of 66% (N61) and 95% (N135) relative abundance, respectively. The remaining relative abundance at site N61 is accounted for by HexNAc₆Hex₇Fuc₁ (N₆H₇F₁, 34%). Finally, at site N142 within domain D2, 32 distinct N-glycan compositions were detected, with HexNAc₅Hex₈Fuc₁NeuAc₁ (N₅H₈F₁S₁, 12.7%) and HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 10%) being the most abundant. HexNAc₂Hex₅ (N₂H₅), consistent with a less processed high mannose N-glycan, was detected at a relative abundance of 9%.

Murine FcγRIV and Human FcγRIIIA Exhibit Distinct Sets of N-Glycan Compositions while Human Variants Display Similar N-Glycan Compositions

Human FcγRIIIA (hFcγRIIIA) is a low-affinity Fc receptor that promotes antibody-dependent cellular cytotoxicity (ADCC) following binding to IgG within an antigen-IgG complex.²⁶ For murine FcγRIV (mFcγRIV), a similar function promoting ADCC has also been described.³⁷ Recombinant mFcγRIV and hFcγRIIIA expressed in NS0 and CHO cell lines, respectively, were subject to proteolysis followed by nLC-MS/MS. The predicted protein structure of mFcγRIV (Figure 4A) and hFcγRIIIA (Figure 4B),^32,33 are shown with asparagine residues in blue indicating the sites of detected N-glycopeptides. The relative abundances of mFcγRIV N-glycopeptides detected at sites N144 and N159 are shown (Figure 4C,D, and Supplemental Table S2). At site N144, within the Ig-like C2-type 2 (D2) domain, four glycan compositions were detected, with HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 52%) and HexNAc₄Hex₇Fuc₁ (N₄H₇F₁, 30%) accounting for most of the relative abundant (Figure 4C). At site N159, within the same domain, 33 different glycan compositions were detected, including both complex and hybrid glycoforms and a minor presence of high mannose glycoforms (Figure 4D). The glycan compositions with the highest abundance were HexNAc₄Hex₇Fuc₁ (N₄H₇F₁, 20%) and HexNAc₅Hex₉Fuc₁ (N₅H₉F₁, 14%). The compositions HexNAc₃Hex₆Fuc₁ (N₃H₆F₁, 7%) and HexNAc₃Hex₇Fuc₁ (N₃H₇F1, 5%) are compatible with hybrid glycan structures. The predicted N-glycosylation sequon at N42 (Figure 1), inside the Ig-like C2-type 1 (D1) domain, was not detected.

Figure 4.

Site-specific N-glycosylation of recombinant murine Fcγ receptor IV (mFcγRIV) and human Fcγ receptor IIIa (hFcγRIIIa). (A, B) The predicted protein structures of mFcγRIV and hFcγRIII, respectively, are shown as space fill models. Asparagine (N) residues bearing N-glycans profiled in this study are labeled (blue). (C, D) mFcγRIV site N144 and N159 glycopeptide relative abundances are shown. (E, F) The relative abundances of the hFcγRIIIA glycopeptide glycoforms are shown for sites N45 and N74. Glycoform relative abundance values are shown for hFcγRIIIA V/F158 variants. (G) Comparison of the relative abundance of high mannose, hybrid, and complex type N-linked glycans at the mFcγRIV site N159 and the hFcγRIIIA site N162. (H) hFcγRIII site N162 glycopeptide glycoforms representing ≥1% relative intensity are shown. Glycoform relative abundance values are shown for hFcγRIIIA V/F158 variants. A full table of detected glycoforms can be found in Supplementary Figure S-3. (I) hFcγRIIIA site N162 glycopeptide glycoforms consistent with high mannose type N-glycans are shown, including those with relative abundance <1%. Student’s paired t test was used to compare the glycopeptide relative abundances of hFcγRIIIA V/F158 variants. N = N-acetylhexosamine, H = hexose, F = fucose, S = N-acetylneuraminic acid. T = N-glycolylneuraminic acid. *p < 0.01. **p < 0.001.

Comparison of recombinant hFcγRIIIA V158 and F158 variants revealed that the two variants display similar glycoforms across the detected N-glycosylation sites. The relative abundances of N-glycopeptides detected at sites N45, N74, and N162 are shown for both variants (Figure 4E–I, Supplemental Table S1). Site N45, within the Ig-like C2-type 1 (D1) domain, displayed eight different glycan compositions for the F158 variant and five for the V158 variant. The compositions with the highest relative abundance were HexNAc₄Hex₅Fuc₁ (N₄H₅F₁, 70% V158 and 57% F158) followed by HexNAc₄Hex₄Fuc₁ (N₄H₄F₁, 15% V158 and 10% F158). N-Glycan compositions consistent with hybrid structures were also identified, with the most abundant form, HexNAc₃Hex₄Fuc₁ (N₃H₄F₁), contributing ∼10% relative abundance in both V158 and F158 variants. HexNAc₃Hex₆Fuc₁ (N₃H₆F₁), HexNAc₄Hex₅Fuc₁NeuAc₁ (N₄H₅F₁S₁) and HexNAc₄Hex₅NeuAc₂ (N₄H₅S₂) were not detected in the hFcγRIIIA V158 variant. Also within the Ig-like C2-type 1 (D1) domain, site N74 displayed that the sialylated glycoform HexNAc₆Hex₆Fuc₁NeuAc₃ (N₆H₆F₁S₃) was detected with relative abundances of 100% and 66% for F158 and V158 variants, respectively. A similar glycan composition, HexNAc₆Hex₆Fuc₁NeuAc₂ (N₆H₆F₁S₂, 34%), with one fewer sialic acid, was detected only in the V158 variant. The V/F158 variants present very similar glycan composition patterns at the three observed glycosylation sites (Figure 4), with some exceptions such as the presence of N-glycan compositions N₄H₅F₁S₁ and N₄H₅S₂ present at N45 in the F158 variant and not in V158 variant and the glycan composition N₆H₆F₁S₂ only presents in the V158 variant at N74. Finally, while low in abundance, a single high confidence O-glycopeptide of hFcγRIIIA-F158 was detected, ²MRTEDLPKAVVF¹³ + HexNAc₁Hex₁NeuAc₂ (Supplementary Figure S-4).

Next, we sought to directly compare conserved glycosylation sites N159 (mouse) and N162 (human). Sites N159 in mFcγRIV and N162 in hFcγRIIIA, both located within Ig-like C2-type 2 (D2) domains of the respective FcγR orthologues, occupy equivalent positions (Supplementary Figure S-5) within the receptors and are located near the Fc binding interface. Given that mFcγRIV site N159 regulates IgG binding in a similar manner to hFcγRIIIA site N162 but is less well characterized, HCD and EThcD spectra are included to document unique fragment ions used to assign the MS2 spectra of the mFcγIV glycopeptide ¹⁵¹CRGLIGHNNKSSASF¹⁶⁵ + HexNAc₄Hex₆Fuc₂, precursor m/z 931.8954 [M + 4H]⁴⁺ (Figure 5).The MS2 spectra in Figure 5, including a higher-energy collisional dissociation (HCD) spectrum generated with stepped collision energy (SCE) of 15%/30%/40% (top) and a second MS2 spectrum generated via electron transfer dissociation with supplemental activation (EThcD), were selected as representative of mFcγRIV to provide evidence of glycopeptide fucosylation. Fragment ions including a Y1 ion CRGLIGHNNKSSASF+HexNAc+Fuc (m/z 996.9746, 2⁺), supporting an assignment of core fucosylation, and the oxonium ion HexNAc-Hex-Fuc (observed m/z 512.1988) indicative of the presence of outer arm fucose, support glycan composition assignment containing two fucose residues. Fragments associated with N-glycan core fucosylation, including peptide+HexNAc₁Fuc₁ (Y₁) peaks indicative of core fucose, are present in the MS2 spectra of most recombinant FcγR fucosylated glycopeptides in this study. This evidence suggests that most fucosylated glycopeptides are, at a minimum, core fucosylated in the recombinant FcγRs. Most fucosylated FcγR glycopeptides with >1 fucose display both peptide+HexNAc₁Fuc₁ and HexNAc₁Hex1Fuc₁ (m/z 512) peaks, indicative of both core and outer-arm fucosylation. Further comparison of mFcγRIV site N159 and hFcγRIIIA N162 N-glycan compositions (Figure 4G), demonstrates that complex glycans are the predominant type in the recombinant murine and human receptors, with a dramatic difference in the amount of fucosylation apparent between the two. At site N159 in mFcγRIV, the complex glycan displays 83% relative abundance followed by 17% hybrid type. On the other hand, site N162 in hFcγRIIIA shows compositions compatible with complex glycans with values of 90% and 92% for V158 and F158 variants, respectively. The remaining relative abundance is accounted for by assigned compositions of 4.2% and 2.3% of high mannose for V158 and F158 variants, respectively. Finally, 5.1% of assigned glycan compositions are consistent with hybrid type N-glycans. Glycan compositions with relative abundances of ≥1% at site N162 in the hFcγRIIIA are shown in Figure 4H. The composition profiles are similar for hFcγRIIIA F158 and V158 variants with HexNAc₄Hex₅Fuc₁ (N₄H₅F₁, 35% F and 26% V) and HexNAc₄Hex₅Fuc₁NeuAc₁ (N₄H₅F₁S₁, 34% F158and 38% V158) accounting for most of the relative abundance. In Figure 4I, only high mannose relative abundances are shown, with more processed forms including HexNAc₂Hex₅ (N₂H₅, 1.3% F158 and 1.7% V158) being the most abundant.

Figure 5.

Murine Fcγ receptor IV (mFcγRIV) site N159 glycopeptide spectra. MS2 spectra of the mFcγIV glycopeptide ¹⁵¹CRGLIGHNNKSSASF¹⁶⁵ + HexNAc₄Hex₆Fuc₂, precursor m/z 931.8954 [M + 4H]⁴⁺, including the higher collision energy dissociation (HCD) spectrum generated with stepped collision energy (SCE) of 15%/30%/40% (top) and a second MS2 spectrum generated via electron transfer dissociation with supplemental activation (EThcD) (bottom).

Comparison of Global N-Glycosylation Patterns across Human and Murine FcγRs

Following the characterization of human and murine FcγR N-glycosylation, we compared patterns of glycosylation across the receptors (Figure 6A–D). Notable patterns include the high level of fucosylation and sialylation of complex glycans on hFcγRs produced in CHO cells, largely consistent with previous studies.²⁵ Recombinant mouse FcγRs produced in NS0 cells displayed very high levels of complex N-glycan compositions modified with fucose (Figure 6A,D), with mFcγRIII displaying the highest level of sialylation among the three (Figure 6C). mFcγRI has two predicted N-glycosylation sequons in domain D1, at N4 and N24, that are absent in the human receptor (Supplementary Figure S-6). In this analysis, mFcγRI site N24 glycopeptides were detected, with a large proportion of detected glycoforms consistent with complex glycans (64%), while about one-third (36%) of glycoforms were consistent with high mannose type N-glycans. Sialylated glycans were not detected at this position. Site N45 of mFcγRI (comparable to hFcγRI N35) displayed 12% and 13% high mannose and hybrid glycans, respectively, while site N24 has the highest abundance of high mannose structures (36%). In the human receptor hFcγRI, compositions consistent with high mannose glycans were detected with 100% relative abundance at site N35 within domain D1. This is consistent with the observation that this site has a very low solvent accessibility, which may prevent N-glycan processing. Similarly, hFcγRIIIA site N45 shares high local amino acid identity, which is proposed to enable intramolecular contacts with the polypeptide chain, resulting in a decrease in the movement of the glycan chain and decreased accessibility for N-glycan processing (remodelling) enzymes.³⁸

Figure 6.

Summary of recombinant murine and human Fcγ receptor N-glycosylation. Schematics illustrating the relative abundance of complex type (A), high mannose type (B), sialic acid-bearing (C), and fucose-bearing (D) N-glycans were used for mFcγRI, mFcγRIII, mFcγRIV, hFcγRI, and hFcγRIIIA (F158). N-Glycosylation sites are labeled according to position. Below each position, the relative abundance (%) of glycans in the designated category is shown.

In our analyses of recombinant hFcγRIIIA produced in CHO cells, site N162 N-glycan compositions consistent with the presence of high mannose type N-glycans were very low, while high levels of complex glycans were present (Figure 6A,B). Site N162 high mannose type N-glycans play a critical role in hFcγRIIIA by increasing the affinity of the receptor for IgG immune complexes. However, given that the recombinant proteins were expressed to maximize protein yield, and likely without consideration of glycosylation, it is not surprising that the glycan profile does not mirror that of endogenous hFcγRIIIA produced in native immune cell types. Similarly, in the mFcγRIV, the mouse orthologue of human FcγRIIIA, site N159 glycopeptides consistent with the presence of high mannose N-linked glycans were not detected (Figure 6B). At mFcγRIV sites N144 and N159, the two most abundant glycan compositions were N₄H₇F₁ and N₅H₉F₁. N-Glycans displayed at mFcγRIV site N159, are required for high affinity interaction with the Fc domain of IgG,³ analogous to hFcγRIIIA site N162.¹¹ In contrast to the human receptor, higher levels of compositions consistent with hybrid type N-glycans (17%) were detected at this site. By comparing the sialic acid content between the recombinant mFcγRIV and hFcγRIIIA in this study, it is apparent that it is quite low on the mouse receptor (∼0–5%) compared to its human counterpart (50–100%).

Surprisingly, mFcγRIV site N42 glycopeptides in domain D1 were not detected. This residue corresponds to the hFcγRIIIA site N45, which was detected. This may be due to the low yield of the peptides generated in this region, as is the case for hFcγRIIIA. In such a scenario, it may be that the glycopeptides fell below the limit of detection. Unoccupied (nonglycosylated) peptides were not detected at this position either. In the present analyses, the glycosylation at hFcγRIIIA site N45 displays compositions consistent with ∼15–20% of hybrid type (not shown in Figure 6) and 80–85% of complex type glycans. Previous studies of hFcγRIIIA derived from human cells or plasma have shown an elevated proportion of high mannose and hybrid glycans at hFcγRIIIA site N45 in certain immune cell types, particularly in NK cells.²⁸ This represents the highest proportion of high mannose or hybrid glycan content within the three detected hFcγRIIIA sites in this study. Consistent with this observation, site N45 also has a low solvent accessibility.

Discussion

Here, we defined the glycan compositions across the N-glycosylation sites of three murine activating Fcγ receptors, mFcγRI, mFcγRIII, and mFcγRIV in a site-specific manner and compared the glycosylation of these murine receptors to that of their better-defined human orthologues hFcγRI and hFcγRIIIA. We documented site-specific glycoforms of mFcγRI at sites N24, N45, and N144, mFcγRIII at sites N34, N61, N135, and N142, and mFcγRIV at sites N144 and N159. The nLC-MS/MS methods utilized here can be applied directly to murine models to empower the study of how N-glycosylation regulates immunity in health and disease. These data indicate that recombinant mouse FcγRs produced in NS0 cells displayed very high levels of complex N-glycan compositions modified with fucose (Figure 6A,D), with mFcγRIII displaying the highest level of sialylation among the three (Figure 6C). mFcγRI has two predicted N-glycosylation sequons in domain D1, at N4 and N24, that are absent in the human receptor. Of these, we report that a large proportion of mFcγRI site N24 glycopeptides were consistent with complex glycans, while about one-third of glycoforms at this site were consistent with high mannose type N-linked glycans. Surprisingly, we found high levels of complex N-linked glycans displayed on all mFcγRs across all sites analyzed in this study. This is attributed in part to the specific properties of the NS0 cell line, in which the murine receptors were expressed. In addition to cell-specific glycosyltransferase expression, structural factors also impact N-glycan processing,³⁹ but our analyses suggest that local factors including solvent accessibility surface area do not explain all of our results. Additional factors that influence glycosylation, including activated monosaccharide substrate levels and Golgi residence time, may provide the missing link to explain differences in glycan type relative abundance.⁴⁰

Of relevance, we defined 33 unique N-linked glycan compositions associated with mFcγRIV site N159. This site is of particular interest because it represents a conserved N-glycosylation site shared between the orthologues murine FcγRIV and human FcγRIIIA. It is located within the region of mFcγRIV known to interact with the Fc region of IgG. In murine FcγRIV, expression of high mannose type N-linked glycans at site N159 is required for high affinity interaction with the Fc domain of IgG.³ It is known that cell-type specific glycosylation is particularly influential in the glycosylation of the analogous conserved human FcγRIIIA site N162.^25,41 Display of hybrid or high mannose type N-linked glycans at site N162 of hFcγRIIIA converts the receptor into a high affinity binder of IgG immune complexes, with mannose residues in the chitobiose core and the extended branch of a hybrid/high mannose glycan contributing to this interaction.^11,14 Of the 33 unique mFcγRIV site N159 N-glycan compositions reported here, glycan compositions consistent with the presence of high mannose type N-linked glycans accounted for <1% of the relative composition at mFcγRIV site N159, while 17% of compositions were consistent with hybrid type glycans, and the remaining 83% were consistent with complex type N-linked glycans. Therefore, our analyses captured biologically relevant glycoforms at mFcγRIV site N159. Given that recombinant mFcγRIV used in this study was expressed in mouse NS0 cells, its glycosylation is likely not representative of mFcγRIV natively expressed in immune cells. Therefore, caution must be taken in interpreting the glycosylation data reported here as reflective of glycoforms or glycoform abundances present in immune cells. While it was not the primary focus of our analyses, our analyses also captured the site-specific glycosylation of human FcγRIIIA F/V158 variants. The detection of these glycopeptides could be critical in the future to study changes in glycosylation within and between patient populations with these disease-associated variants.⁴²

As previously discussed, N-glycosylation is known to regulate the affinity and function of IgGs and their receptors, the Fcγ receptors,^43,44 with potentially broad implications for the role of glycosylation in the regulation of immune response in health and disease. As early as 1989, it was reported that enzymatic cleavage (removal) of high mannose and hybrid type N-linked glycans from FcyRIIIA-B altered the affinity of the receptor for IgG.⁴⁵ Shortly after, it was reported that FcγRIIIA isolated from monocytes and NK cells displayed N-linked glycans with different structural characteristics, which the authors suggested may account for functional differences in the receptor.⁴¹ Since then, multiple studies have elaborated the outsized effect that high mannose and hybrid type N-glycans have on FcγR function.^{9,11,12,24,46,47} Ferrara et al. (2011) found that glycans on human FcγRIIIA site N162 and IgG Fc site N247 directly interact to favor optimal binding. In particular, one mannose (Mannose 3) in the N-glycan core of the human FcγRIIIA site N162 glycan and a second mannose (Mannose 5) outside of the N-glycan core (found in either a hybrid or high mannose type N-glycan structure) interact with the IgG Fc region, specifically with Fc-Gln295, and Fc-Tyr296.¹² In the future, a deeper understanding of how these modifications change in specific immune diseases will be needed for this knowledge to be translated into a clinical impact.

The establishment of sensitive, rapid, and high-throughput methods that can analyze these modulatory glycoforms is essential, given that multiple groups have reported that FcγRIIIA high mannose and hybrid type glycoforms have a higher affinity for IgG Fc compared to FcγRIIIA with complex type N-linked glycans. Nanoliquid chromatography tandem mass spectrometry excels at the rapid and sensitive characterization of protein glycoforms. In the analytical realm, Zeck et al. (2011) initially characterized FcγRIIIA in HEK and CHO cells utilizing an nLC-MS/MS-based approach and reported 25 N-glycan compositions associated with site N162 on FcγRIIIA derived from HEK cells.²⁵ However, none of the reported compositions were consistent with high mannose type N-linked glycans, likely due to the low expression of these glycoforms in these cell lines. Since this time, several research groups have applied nLC-MS/MS to the analysis of FcγR N-glycosylation.²⁶⁻³⁰ Patel et al. reported that CD16a from NK cells displays high levels of hybrid (>20%) and high mannose type N-glycans (>20%), though the N-glycosylation site(s) bearing these high-mannose structures were not reported.¹⁴ Site-directed mutagenesis of CD16a N-glycosylation sites, performed in HEK293 cells, was subsequently utilized to infer that high-mannose and hybrid N-linked glycans at site N162 increase the receptor affinity for IgG1. In the current study, <5% of N-glycan compositions associated with site N162 were consistent with the presence of high mannose and hybrid type N-glycan structures on both recombinantly expressed FcγRIIIA (CD16a) variants(V/F158). This observation is consistent with other studies that have demonstrated that the N-glycan structures displayed at FcγRIIIA site N162 vary depending on cell type.^5,6,25 Therefore, while recombinant receptors may be essential to the optimization of analytical workflows such as the one documented here, it is also important to diligently design analytical workflows with biologically relevant glycoforms in mind and to select appropriate models that closely match the cell type and biological context as closely as possible.

To the best of our knowledge, the results reported here match or exceed all other known results in terms of the depth of glycoforms reported for murine mFcγRIV site N159 and two common variants (F/V158) of its human orthologue hFcγRIIIA, with ≥30 glycoforms reported for each. Given the functional significance of the N159 glycan, the high N-glycan heterogeneity observed here could reflect the plasticity of N-glycans at this site, with cues originating within immune cells ultimately shaping the balance of glycoforms.

Conclusions

The methods and results reported here will serve as a foundation for future pan-FcγR analyses of mouse and human FcγRs. Monitoring FcγR glycosylation using this nLC-MS/MS workflow has the potential to reveal new aspects of FcγR function and is of high significance in the realm of autoimmune diseases for diagnostic and biomarker applications, including in the form of “companion biomarkers” to prospectively monitor the effectiveness of treatments to predict response or toxicity.⁴⁸ We anticipate that efforts will intensify to establish sensitive, rapid, and high-throughput methods to monitor these modulatory glycoforms in laboratory and clinical settings. It is our hope that these methods will contribute to the development of a deeper understanding of how N-linked glycan modifications change in specific immune diseases, knowledge that is needed to translate prior FcγR glycobiology findings into clinical impact.

Glossary

Abbreviations

FcγR

Fc γ (gamma) receptor

IgG

immunoglobulin G

ADCC

antibody-dependent cellular cytotoxicity

ADCP

antibody-dependent cellular phagocytosis

MHC

major histocompatibility complex

Fc

the crystallizable fragment region of an IgG

FcγRI (CD64)

Fc γ (gamma) receptor I

FcγRIIA-C (CD32a-c)

Fc γ (gamma) receptor IIA-C

FcγRIIIA-B (CD16a-b)

Fc γ (gamma) receptor IIIA-B

ITAM

immunoreceptor tyrosine-based activation motifs

ITIM

immunoreceptor tyrosine-based inhibitory motif

Data Availability 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.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00835.

• Supplemental Figure S1: mFcγRI and mFcγRIII extracellular domains. Supplemental Figure S2: Relative abundance of mFcγRIII Asn64 chymotryptic/Glu-C peptides. Supplemental Figure S3: hFcγRIIIA site Asn162 glycan compositions. Supplemental Figure S4: HCD MS2 spectrum of hFcγRIIIF O-glycopeptide. Supplemental Figure S5: Comparison of mFcγRIV and hFcγRIIIF protein structure, D1 and D2 domains. Supplemental Figure S6: Protein sequence alignment of mFcγRI and hFcγRI. (PDF)

• Supplemental Table S1: Human FcgR glycopeptide assignments. (XLSX)

• Supplemental Table S2: Mouse FcγR glycopeptide assignments and areas. (XLSX)

The authors declare no competing financial interest.