Abstract
Purpose: :
An age-related macular degeneration (AMD) risk locus on chr10q26 spans two genes - ARMS2 and HTRA1 - both of which have been proposed to influence susceptibility to AMD. The purpose of this study was to investigate the protein interaction network of the serine protease HTRA1 (high-temperature requirement A-1) and to elucidate a possible role in AMD pathogenesis.
Methods: :
Potential interacting partners of HTRA1 were identified in a yeast two-hybrid screen performed using a placental cDNA library. Protein interactions were subsequently verified using recombinant proteins in pull-down assays. Proteins found to interact directly with HTRA1 were subjected to protease activity assays to identify substrates of the protease. In vitro assays of complement activation were carried out, including haemolysis assays, factor B cleavage and restoration of activity to complement-deficient serum.
Results: :
480 clones from the yeast two-hybrid screen were sequenced, identifying 389 interactions and 65 potential interactors. Extracellular proteins, including collagens, osteonectin and fibulin-1, were strongly represented. Complement factor D was also identified as an interactor of HTRA1. This interaction was verified by pull-down assays and CFD was shown to be a substrate for HTRA1. Investigation of the functional relevance of the interaction between HTRA1 and CFD showed that pro-CFD can be cleaved and activated by HTRA1 to form the active protease responsible for activation of factor B in the alternative complement pathway.
Conclusions: :
The identified HTRA1 interactome provides evidence for a functional role of the protease in a number of AMD disease mechanisms, including inflammation, complement activation, fibrosis and angiogenesis. We show that HTRA1 is able to activate CFD, a central component of the alternative complement pathway, which is known to be dysregulated in AMD. Thus, an HTRA1-mediated increase in alternative complement pathway activity may explain a proportion of the AMD-risk attributed to the chr10q26 locus.
Keywords: age-related macular degeneration • proteins encoded by disease genes • inflammation