title: 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.
authors.affiliation: MRC Laboratory of Molecular Biology, University College London, Gower St, London WC1E 6BT, UK.
authors.forename: R
authors.initials: R
authors.lastname: Uellner
authors.forename: M J
authors.initials: MJ
authors.lastname: Zvelebil
authors.forename: J
authors.initials: J
authors.lastname: Hopkins
authors.forename: J
authors.initials: J
authors.lastname: Jones
authors.forename: L K
authors.initials: LK
authors.lastname: MacDougall
authors.forename: B P
authors.initials: BP
authors.lastname: Morgan
authors.forename: E
authors.initials: E
authors.lastname: Podack
authors.forename: M D
authors.initials: MD
authors.lastname: Waterfield
authors.forename: G M
authors.initials: GM
authors.lastname: Griffiths
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D007694:Killer Cells, Natural / Q000276:immunology*; D008081:Liposomes; D008213:Lymphocyte Activation; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054353:Perforin; D054803:Phospholipase C delta; D010743:Phospholipids / Q000378:metabolism*; D052899:Pore Forming Cytotoxic Proteins; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry
chemical_list: D007527:Isoenzymes; D008081:Liposomes; D008562:Membrane Glycoproteins; D010743:Phospholipids; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D054353:Perforin; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D006596:Hexosaminidases