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 ly mphocytes (CTL) and natural killer (NK) cells, It is a key component o f the lytic machinery of these cells, being able to insert into the pl asma membrane of targeted cells, forming a pore which leads to their d estruction, Here we analyse the synthesis, processing and intracellula r transport of perforin in the NK cell line YT, Perforin is synthesize d 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 ac idic compartment and can be inhibited by incubation of the cells in am monium chloride, concanamycin A, leupeptin and E-64, The increased lyt ic activity of the cleaved form can be demonstrated by killing assays in which cleavage of the pro-piece is inhibited, Epitope mapping revea ls that cleavage of the pro-piece occurs at the boundary of a C2 domai n, which we show is able to bind phospholipid membranes in a calcium-d ependent manner, We propose that removal of the pro-piece, which conta ins a bulky glycan, allows the C2 domain to interact with phospholipid membranes and initiate perforin pore formation.