Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain

Perforin is a secreted protein synthesized by activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It is a key component of the lytic machinery of these cells, being able to insert into the plasma membrane of targeted cells, forming a pore which leads to their destruction. Here we analyse the synthesis, processing and intracellular transport of perforin in the NK cell line YT. Perforin is synthesized as a 70 kDa inactive precursor which is cleaved at the C‐terminus to yield a 60 kDa active form. This proteolytic cleavage occurs in an acidic compartment and can be inhibited by incubation of the cells in ammonium chloride, concanamycin A, leupeptin and E‐64. The increased lytic activity of the cleaved form can be demonstrated by killing assays in which cleavage of the pro‐piece is inhibited. Epitope mapping reveals that cleavage of the pro‐piece occurs at the boundary of a C2 domain, which we show is able to bind phospholipid membranes in a calcium‐dependent manner. We propose that removal of the pro‐piece, which contains a bulky glycan, allows the C2 domain to interact with phospholipid membranes and initiate perforin pore formation.