title: Identification of novel substrates for the serine protease HTRA1 in the human RPE secretome.
abstract: 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 alpha2-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, alpha2-macroglobulin, and ADAM9). These findings suggest a link between HTRA1, complement regulation, and amyloid deposition in AMD pathogenesis.
authors.affiliation: Center for Genetic Medicine, Children's National Medical Center, Washington, DC, USA.
authors.forename: Eunkyung
authors.initials: E
authors.lastname: An
authors.forename: Supti
authors.initials: S
authors.lastname: Sen
authors.forename: Sung Kyu
authors.initials: SK
authors.lastname: Park
authors.forename: Heather
authors.initials: H
authors.lastname: Gordish-Dressman
authors.forename: Yetrib
authors.initials: Y
authors.lastname: Hathout
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D000368:Aged; D000682:Amyloid / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D051152:Clusterin / Q000378:metabolism; D017242:Complement Factor H / Q000235:genetics; D003171:Complement Pathway, Classical / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D000071235:Fibromodulin; D005838:Genotype; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D008268:Macular Degeneration / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D011509:Proteoglycans / Q000378:metabolism; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D055213:Retinal Pigment Epithelium / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000502:physiology*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D019096:Vitronectin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: C514392:ARMS2 protein, human; D000682:Amyloid; C495242:CLU protein, human; D051152:Clusterin; D016326:Extracellular Matrix Proteins; C000604330:FMOD protein, human; D008565:Membrane Proteins; D011506:Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019096:Vitronectin; D000511:alpha-Macroglobulins; D000071235:Fibromodulin; D017242:Complement Factor H; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases; D051722:ADAM Proteins; C098339:ADAM9 protein, human