title: Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death.
abstract: Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Pieter J A
authors.initials: PJ
authors.lastname: de Koning
authors.forename: J Alain
authors.initials: JA
authors.lastname: Kummer
authors.forename: Stefanie A H
authors.initials: SA
authors.lastname: de Poot
authors.forename: Razi
authors.initials: R
authors.lastname: Quadir
authors.forename: Roel
authors.initials: R
authors.lastname: Broekhuizen
authors.forename: Anne F
authors.initials: AF
authors.lastname: McGettrick
authors.forename: Wayne J
authors.initials: WJ
authors.lastname: Higgins
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: D Margaret
authors.initials: DM
authors.lastname: Worrall
authors.forename: Niels
authors.initials: N
authors.lastname: Bovenschen
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D053804:Granzymes / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D042541:Intracellular Space / Q000201:enzymology*; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D000951:Antigens, Neoplasm; D011994:Recombinant Proteins; D015843:Serpins; C051072:squamous cell carcinoma-related antigen; C509218:GZMM protein, human; D053804:Granzymes