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 α2-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, α2-macroglobulin, and ADAM9). These findings suggest a link between HTRA1, complement regulation, and amyloid deposition in AMD pathogenesis.