Identification of Novel Substrates for the Serine Protease HTRA1 in the Human RPE Secretome
Identification of Novel Substrates for the Serine Protease HTRA1 in the Human RPE Secretome
复制标题
DOI：
10.1167/iovs.09-4853
复制
发表时间：
2010-07-01
影响因子：
4.4
通讯作者：
Hathout, Yetrib
中科院分区：
文献类型：
--
作者：
An, Eunkyung;Sen, Supti;Hathout, Yetrib
目的.通过检测丝氨酸蛋白酶HTRA 1在原代RPE细胞外环境中的表达水平并鉴定其潜在底物，明确其在年龄相关性黄斑变性（AMD）中的作用。从人供体眼睛建立原代RPE细胞培养物，并通过使用等位基因辨别测定筛选CFH、ARMS 2和HTRA 1风险基因型。采用实时荧光定量PCR和定量蛋白质组学方法检测基因分型的RPE细胞中HTRA 1的表达。通过将RPE条件培养基与或不与人重组HTRA 1一起孵育来鉴定潜在的HTRA 1底物。选择性裂解的蛋白质定量使用差异稳定同位素标记的氨基酸在细胞培养（SILAC）的战略。HTRA 1 mRNA水平在HTRA 1启动子风险等位基因纯合子的原代RPE细胞中比在具有野生型等位基因的RPE细胞中高三倍，这转化为具有风险基因型的RPE细胞的HTRA 1分泌增加两倍。在RPE分泌组中共鉴定了196种细胞外蛋白，发现只有8种被人重组HTRA 1选择性切割。这些蛋白包括纤维调节蛋白（90%裂解）、簇蛋白（50%）、ADAM 9（54%）、玻连蛋白（54%）和α 2-巨球蛋白（55%），以及一些细胞表面蛋白包括talin-1（21%）、fascin（40%）和氯离子胞内通道蛋白1（51%）。重组HTRA 1切割参与补体途径调节的RPE分泌蛋白（簇蛋白、玻连蛋白和纤调蛋白）和淀粉样蛋白沉积（簇蛋白、α 2-巨球蛋白和ADAM 9）。这些发现表明AMD发病机制中HTRA 1、补体调节和淀粉样蛋白沉积之间存在联系。（Invest Ophthalmol维斯科学。2010; 51：3379-3386）DOI：10.1167/iovs.09-4853
PURPOSE. To define the role of the serine protease HTRA1 in age-related macular degeneration (AMD) by examining its expression level and identifying its potential substrates in the context of primary RPE cell extracellular milieu.METHODS. Primary RPE cell cultures were established from human donor eyes and screened for CFH, ARMS2, and HTRA1 risk genotypes by using an allele-discrimination assay. HTRA1 expression in genotyped RPE cells was determined by using real-time PCR and quantitative proteomics. Potential HTRA1 substrates were identified by incubating RPE-conditioned medium with or without human recombinant HTRA1. Selectively cleaved proteins were quantified by using the differential stable isotope labeling by amino acids in cell culture (SILAC) strategy.RESULTS. HTRA1 mRNA levels were threefold higher in primary RPE cells homozygous for the HTRA1 promoter risk allele than in RPE cells with the wild-type allele, which translated into a twofold increase in HTRA1 secretion by RPE cells with the risk genotype. A total of 196 extracellular proteins were identified in the RPE secretome, and only 8 were found to be selectively cleaved by the human recombinant HTRA1. These include fibromodulin with 90% cleavage, clusterin (50%), ADAM9 (54%), vitronectin (54%), and alpha 2-macroglobulin (55%), as well as some cell surface proteins including talin-1 (21%), fascin (40%), and chloride intracellular channel protein 1 (51%).CONCLUSIONS. Recombinant HTRA1 cleaves RPE-secreted proteins involved in regulation of the complement pathway (clusterin, vitronectin, and fibromodulin) and of amyloid deposition (clusterin, alpha 2-macroglobulin, and ADAM9). These findings suggest a link between HTRA1, complement regulation, and amyloid deposition in AMD pathogenesis. (Invest Ophthalmol Vis Sci. 2010; 51:3379-3386) DOI:10.1167/iovs.09-4853