Human granzyme B is essential for DNA fragmentation of susceptible target cells.
We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YT-INDY and NK3.3, two human cytotoxic cells, were also lysed. EGTA reduced lysis by only 50%, suggesting that a perforin-independent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and 125I-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.
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B
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Department of Medicine, Indiana University School of Medicine, Indianapolis.
Bochan
M R
MR
Hanna
W L
WL
Froelich
C J
CJ
Brahmi
Z
Z
D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D002470:Cell Survival; D053818:Chymases; D003374:Coumarins / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000201:enzymology*; D003602:Cytotoxicity, Immunologic / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D053804:Granzymes; D006801:Humans; D049934:Isocoumarins; D007694:Killer Cells, Natural / Q000201:enzymology*; D051379:Mice; D012697:Serine Endopeptidases / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D003374:Coumarins; D049934:Isocoumarins; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D001312:Aurintricarboxylic Acid; C046235:3,4-dichloroisocoumarin; D004533:Egtazic Acid; D004247:DNA; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; D053818:Chymases