title: Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L.
abstract: The pore-forming protein perforin is synthesized as an inactive precursor in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and becomes active when a short C-terminal peptide is cleaved within acidic lysosome-like cytotoxic granules. Although it was shown more than a decade ago that this cleavage is pH dependent and can be inhibited by the generic cysteine cathepsin inhibitor E-64d, no protease capable of processing the perforin C terminus has been identified. Neither is it known whether a single protease is responsible or the processing has inbuilt redundancy. Here, we show that incubation of human NK cells and primary antigen-restricted mouse CTLs with the cathepsin L (CatL) inhibitor L1 resulted in a marked inhibition of perforin-dependent target cell death and reduced perforin processing. In vitro, CatL preferentially cleaved a site on full-length recombinant perforin close to its C terminus. The NK cells of mice deficient in CatL showed a reduction but not a complete absence of processed perforin, indicating that cysteine proteases other than CatL are also able to process perforin. We conclude that granule-bound cathepsins are essential for processing perforin to its active form, and that CatL is an important, but not exclusive, participant in this process.
authors.affiliation: Department of Biochemistry, Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Spela
authors.initials: S
authors.lastname: Konjar
authors.forename: Vivien R
authors.initials: VR
authors.lastname: Sutton
authors.forename: Sabine
authors.initials: S
authors.lastname: Hoves
authors.forename: Urška
authors.initials: U
authors.lastname: Repnik
authors.forename: Hideo
authors.initials: H
authors.lastname: Yagita
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinheckel
authors.forename: Christoph
authors.initials: C
authors.lastname: Peters
authors.forename: Vito
authors.initials: V
authors.lastname: Turk
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
authors.forename: Nataša
authors.initials: N
authors.lastname: Kopitar-Jerala
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D056668:Cathepsin L / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D004533:Egtazic Acid / Q000494:pharmacology; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010047:Ovalbumin / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology
chemical_list: D015853:Cysteine Proteinase Inhibitors; C110366:OVA-8; C510468:PRF1 protein, human; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C510467:perforin 1, mouse; D054353:Perforin; D004533:Egtazic Acid; D010047:Ovalbumin; D010447:Peptide Hydrolases; D053804:Granzymes; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D007930:Leucine; C108192:aloxistatin