IgA肾病尿液糖蛋白质组学构成的研究

Study of Urinary Glycoproteomic Composition of IgA Nephropathy

目的应用尿糖蛋白组学寻找尿液中糖基化修饰蛋白质可能和IgA肾病(IgA nephropathy,IgAN)相关的潜在生物学信息,尿蛋白组学可能提示IgAN诊断的生物标志物。方法采用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)在数据依赖性采集(data-dependent acquisition,DDA)模式下检测并比较IgAN患者(n=3)和健康对照者(n=3)的尿液糖蛋白质组。通过基因本体(gene ontology,GO)分析、京都基因、基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)对IgAN患者尿液中异表达的糖基化修饰蛋白质进行鉴定并通过网络互作图分析其相互关联性。最后结合目前研究现状,探索差异糖蛋白对IgAN诊断和预后可能存在的生物标志物作用。结果(1)糖蛋白质质谱结果显示,与健康对照组相比,IgAN患者尿液中上调蛋白共39个,且其中包含有11个特异性表达上调的蛋白;差异下调蛋白共149个,其中特异性表达下调蛋白有22个;(2)PPI分析显示140个差异表达蛋白有直接或间接相互作用关系。对各个差异蛋白进行GO功能富集分析显示差异糖蛋白与细胞成分组织、细胞生长的调节、蛋白质结合、O-酰基转移酶活性、多糖结合、胰岛素样生长因子结合及锚定相关。其中有富集的包括细胞黏附分子和代谢相关细胞功能(P<0.05)。参与这些信号通路的差异表达蛋白主要分布在细胞外基质和细胞膜中。KEGG富集显示差异糖蛋白参与生物路径主要包括细胞黏附分子、脂肪细胞因子信号通路、mTOR信号通路、胆固醇代谢、ECM-受体相互作用;(3)差异表达蛋白CCL25、PD-L1、HLA-DRB1、IL7R、CP、AFM和WDR82在PPI互作分析图中连结度较大,这些尿液差异表达蛋白质参与了KEGG所记录的如脂肪细胞因子信号通路,mTOR信号通路,胆固醇代谢通路,细胞黏附分子,P53信号通路,Notch信号通路,Hedgehog信号通路,ECM-受体交互作用通路。结论(1)IgAN患者与健康人群尿液中的糖蛋白表达水平存在明显差异;(2)GO和KEGG分析结果揭示IgAN患者尿液差异糖蛋白涉及与IgAN相关的多种生物功能和途径,基于UHPLC-MS/MS质谱分析的尿糖蛋白质组学可能是诊断IgAN的另一种选择;(3)IgAN诊断判断的潜在生物标志物可能包括但不限于CCL25、PD-L1、HLA-DRB1、IL7R、CP、AFM和WDR82。

Objective To explore the potential biological information of urinary glycated proteins that may be related to IgA nephropathy(IgAN)by using urinary glycoproteomics,which may provide biomarkers for the diagnosis of IgAN.Methods Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to detect and compare the urinary glycoproteome of IgAN patients(n=3)and healthy controls(n=3)in data dependent acquisition(DDA)mode.The differentially expressed glycosylation modified proteins in the urine of IgAN patients were identified and analyzed by gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG),and their mutual correlation was analyzed by network interaction map.Finally,combined with the current research status,the possible role of differential glycoproteins in the diagnosis and prognosis of IgAN was explored.Results(1)Compared with the healthy control group,39 proteins were up-regulated in the urine of IgAN patients,including 11 specifically up-regulated proteins;a total of 149 differentially down-regulated proteins were identified,including 22 specifically down-regulated proteins;(2)PPI analysis showed that 140 differently expressed proteins had direct or indirect interactions.GO functional enrichment analysis of each differential protein showed that the differential glycoproteins were related to cell component organization,regulation of cell growth,protein binding,O-acyltransferase activity,polysaccharide binding,insulin-like growth factor binding and anchoring.Among them,cell adhesion molecules and metabolism-related cell functions were significantly enriched(P<0.05).The differently expressed proteins involved in these signaling pathways were mainly distributed in the extracellular matrix and cell membrane.KEGG enrichment showed that differential glycoproteins were involved in the biological pathways,including cell adhesion molecules,adipokine signaling pathway,mTOR signaling pathway,cholesterol metabolism and ECM-receptor interaction;(3)the differently expressed proteins CCL25,PD-L1,HLA-DRB1,IL7R,CP,AFM and WDR82 were highly connected in PPI interaction analysis map.These urine differentially expressed proteins were involved in KEGG record such as adipokine signaling pathway,mTOR signaling pathway and cholesterol metabolism pathway,cell adhesion molecules,P53 signaling pathway,Notch signaling pathway,Hedgehog signaling pathway,and ECM-receptor interaction pathway.Conclusion(1)There is a significant difference in the expression level of urinary glycoprotein between IgA nephropathy patients and healthy people;(2)GO and KEGG analysis revealed that urinary differential glycoproteins in patients with IgA nephropathy were involved in a variety of biological functions and pathways related to IgA nephropathy.Urinary glycoproteomics based on UHPLC-MS/MS mass spectrometry may be another choice for the diagnosis of IgA nephropathy;(3)potential biomarkers for IgA nephropathy diagnosis may include,but are not limited to,CCL25,PD-L1,HLA-DRB1,IL7R,CP,AFM,and WDR82.