The Biochemistry and Pathophysiological Function of Testisin

Abstract

Testisin is a unique trypsin-like serine protease that is localized to the extracellular membrane of cells through a glycophosphatidylinositol (GPI)-anchor. Testisin is expressed in sperm, eosinophils, and microvascular endothelial cells, and is overexpressed in ovarian cancers. Little is known about the regulation of testisin activity, proteolytic substrate specificity, or the physiological role of testisin. We generated recombinant human testisin using two expression systems to study its biochemical properties. We found that human testisin may be activated by recombinant hepsin, a transmembrane serine protease. Active human recombinant testisin forms an SDS-stable inhibitory complex with the serpin, protein C inhibitor (PCI). Examination of testisin substrate specificity using commercially available recombinant mouse testisin serine protease domain revealed that testisin prefers to cleave peptide substrates after Arg amino acid residues, and efficiently cleaves a peptide substrate derived from the N-terminal activation domain of the G-protein coupled receptor, Protease-Activated Receptor-2 (PAR2). When PAR2 was expressed in cell lines, we found that recombinant testisin cleaved and activated PAR2 to induce transient release of intracellular calcium and the phosphorylation of ERK1/2. When full-length human testisin and PAR2 are co-expressed on the cell surface, testisin is able to cleave PAR2, causing constitutive activation of several intracellular signaling pathways, the induction of cytokine expression, and internalization and loss of PAR2 from the cell surface. These data provide new insight into the biochemical properties of testisin, and its ability to activate PAR2, a critical signaling receptor important in inflammation, wound healing, and cancer. While testisin is not found in mature vasculature, it is expressed by microvascular endothelial cells in vascular beds associated with active angiogenesis. Testisin-deficient mice display delayed and aberrant corpus luteal angiogenesis. We found that capillary outgrowth from aortic rings isolated from testisin-deficient mice is reduced substantially. Silencing of testisin expression by siRNA knockdown in primary microvascular endothelial cells plated on Matrigel basement membranes suggests that capillary-like tubule formation is also decreased. These data demonstrate a functional role for testisin in processes required by microvascular endothelial cells for capillary formation, and may suggest a potential pathway involving testisin activation of PAR2 on the surface of microvascular endothelial cells.