N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration
Chen CY, Melo E, Jakob P, Friedlein A, Elsässer B, Goettig P, Kueppers V, Delobel F, Stucki C, Dunkley T, Fauser S, Schilling O, Iacone R.
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis.
