Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids which can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol (GPI) anchored serine protease, cleaved human PCI (hPCI) and also mouse PCI (mPCI) at their reactive sites as well as at sites close to their N-terminus. This cleavage was observed not only with testisin in solution, but also with cell membrane-anchored testisin on U937 cells. The cleavage close... More
Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids which can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol (GPI) anchored serine protease, cleaved human PCI (hPCI) and also mouse PCI (mPCI) at their reactive sites as well as at sites close to their N-terminus. This cleavage was observed not only with testisin in solution, but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N-terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of hPCI (His1-Arg11) and mPCI (Arg1-Ala18), functioned as cell penetrating peptides (CPPs). Since intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N-terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N-terminus. This may represent one of the mechanisms regulating PCI's intracellular functions.