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

Abstract

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.

Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

“…Finally, it has a C-terminal domain, this region is able to make catenary's interactions of calciumdependent membrane binding (63). The second domain of the latter region is essential for the binding between cell membrane and perforin; at the end of the 20 amino acid residues, there is a signal sequence for N-glycosydic linkage and a breaking site considered important for perforin activation (64).…”

Perforin deficiency and susceptibility to cancer

“…It has been hypothesized that a putative protease cleaves the extreme C-terminal peptide together with its N-glycosylation moiety in the SGs, thereby activating PRF. 4 However, a protease and direct evidence for the essential role of PRF cleavage are yet to be shown. Irrespective of the mechanism, the acidic environment of SGs prevents the PRF C2-domain from binding Ca 2 þ .…”

“…3 Following exocytic release, PRF and the granzymes are exposed to the neutral pH and calciumrich environment of the immune synapse. 4 After binding calcium through their C2 domains, PRF monomers acquire the ability to bind generic lipids in the target cell membrane, 5 and then coalesce into large transmembrane pores that permit the granzymes to access key death substrates in the cytosol. [6][7][8] Although the diverse apoptotic pathways triggered by granzymes have been extensively studied and are now understood in considerable detail, it is only of late that insights into the molecular and cellular functions of PRF have been even partly addressed.…”

Nitric Oxide Synthase-2 and Expression of Perforin in Uterine NK Cells

“…Perforin is known to contain two potential glycosylation sites (17), and the occurrence of multiple bands is likely due to alternative glycosylation and/or proteolytic cleavage. These alternative forms may represent different stages of perforin biosynthesis and maturation (18). Analysis by Northern blot hybridization demonstrated that reduction of perforin protein was accompanied by a reduction in steady-state levels of perforin mRNA, thus demonstrating a link between NOS-2 and availability of perforin mRNA for processing into protein.…”