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 journalArticle

149 Citations (Scopus)

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

Fingerprint

Perforin
Biosynthesis
Phospholipids
Natural Killer Cells
Cells
Membranes
Epitope Mapping
Ammonium Chloride
T-cells
Cytotoxic T-Lymphocytes
Cell membranes
Machinery
Polysaccharides
Epitopes
Assays
Cell Membrane
Calcium
Cell Line
C2 Domains
Processing

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Uellner, R., Zvelebil, M. J., Hopkins, J., Jones, J., MacDougall, L. K., Morgan, B. P., ... Griffiths, G. M. (1997). Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain. EMBO Journal, 16(24), 7287-7296. https://doi.org/10.1093/emboj/16.24.7287

Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain. / Uellner, Ruth; Zvelebil, Marketa J.; Hopkins, Jean; Jones, Jane; MacDougall, Lindsay K.; Morgan, B. Paul; Podack, Eckhard; Waterfield, Michael D.; Griffiths, Gillian M.

In: EMBO Journal, Vol. 16, No. 24, 15.12.1997, p. 7287-7296.

Research output: Contribution to journalArticle

Uellner, R, Zvelebil, MJ, Hopkins, J, Jones, J, MacDougall, LK, Morgan, BP, Podack, E, Waterfield, MD & Griffiths, GM 1997, 'Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain', EMBO Journal, vol. 16, no. 24, pp. 7287-7296. https://doi.org/10.1093/emboj/16.24.7287
Uellner, Ruth ; Zvelebil, Marketa J. ; Hopkins, Jean ; Jones, Jane ; MacDougall, Lindsay K. ; Morgan, B. Paul ; Podack, Eckhard ; Waterfield, Michael D. ; Griffiths, Gillian M. / Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain. In: EMBO Journal. 1997 ; Vol. 16, No. 24. pp. 7287-7296.
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