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

Ruth Uellner, Marketa J. Zvelebil, Jean Hopkins, Jane Jones, Lindsay K. MacDougall, B. Paul Morgan, Eckhard Podack, Michael D. Waterfield, Gillian M. Griffiths

Research output: Contribution to journalArticlepeer-review

151 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)7287-7296
Number of pages10
JournalEMBO Journal
Volume16
Issue number24
DOIs
StatePublished - Dec 15 1997

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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