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 C-Y., Melo E., Jakob P., Friedlein A., Elsässer B., Goettig P., Kueppers V., Delobel F., Stucki C., Dunkley T., Fauser S., Schilling O., Iacone R.Matrix Biology, 2018

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.