title: The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.
abstract: Serine proteases (granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at long unbranched amino acids such as Met ("Met-ase"). Here, we report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM phenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for granzyme H.
authors.affiliation: The John Connell Laboratory, The Austin Research Institute, Studley Road, Heidelberg, 3084, Australia.
authors.forename: K M
authors.initials: KM
authors.lastname: Edwards
authors.forename: C M
authors.initials: CM
authors.lastname: Kam
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
authors.forename: J A
authors.initials: JA
authors.lastname: Trapani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D053818:Chymases; D002918:Chymotrypsin / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology; D053804:Granzymes; D006801:Humans; D019169:Jurkat Cells; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C069434:GZMH protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D053818:Chymases