白细胞上的Fcγ受体(FcγRs)结合免疫球蛋白G(IgG)免疫复合物以介导效应子功能。FcγR介导的过程失调有助于多种炎性疾病,包括类风湿性关节炎,狼疮,和免疫性血小板减少症。严重的,FcγR和IgG上的免疫调节N-聚糖修饰改变FcγR-IgG结合亲和力。需要快速表征跨多个Fcγ受体的N-聚糖的方法来推动对FcγRN-聚糖的疾病特异性贡献的研究。这里,我们利用纳米液体色谱串联质谱(nLC-MS/MS)来表征FcγR糖基化,并报告了重组人FcγRI的定量和位点特异性N-聚糖表征,来自CHO细胞和鼠FcγRI的FcγRIIIAV158和FcγRIIIAF158,FcγRIII,和来自NS0细胞的FcγRIV。数据可通过具有标识符PXD043966的ProteomeXchange获得。在小鼠FcγRIV位点N159和人FcγRIIIA位点N162(进化上保守的位点)观察到广泛的糖型分布(≥30)。Further,检测到跨越所有四个预测的N-糖基化序列的小鼠FcγRIIIN-糖肽。据报道,hFcγRIIIAV/F158多态性变体的糖型相对丰度,证明了该工作流程在测量常见人类FcγRIIIA等位基因变体与疾病相关结局之间的糖基化差异方面的临床潜力。此处报道的多Fcγ受体糖蛋白质组学工作流程将使研究集中在FcγRN-糖基化在自身免疫性疾病中的作用。
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.