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

Here, the authors investigate the impact of elevated HtrA1 levels on the retinal pigment epithelial (RPE) secretome using a polarized culture system. The authors suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. Human primary Retinal Pigmented Epithelium (RPE) cells (Sciencell, 6540) were seeded in transwells coated with Laminin 521. A model that recapitulates the structural, molecular and apical/basolateral signatures of adult RPE cells was achieved. The 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 a tube-like structure by endothelial cells.