Chymase-directed serine protease inhibitor that reacts with a single 30-kDa granzyme and blocks NK-mediated cytotoxicity.
Cytotoxic NK and T lymphocytes kill virally infected cells within minutes without causing damage to themselves or bystander cells. One mechanism of killing involves exocytosis of granules containing serine proteases and perforin. Serine protease inhibitors block killing of target cells mediated by the cytotoxic lymphocytes. There are at least five different serine protease activities in cytolytic granules. Ten different serine protease sequences have been identified with the use of cDNA-specific clones. It is not known whether only one or several of these serine proteases are essential for cytolytic activity. In this study we show that an irreversible serine protease inhibitor, biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, selectively inhibits a chymotrypsin-like (chymase) serine protease activity of rat RNK-16 granule extracts. Under the same conditions, only one 30-kDa (reduced) band was detected on protein blots. Furthermore, only one of three chymase peaks separated by hydrophobic interaction chromatography was inhibited. When this granzyme was inhibited, granule-mediated lysis of erythrocytes was diminished. NK cell killing was completely blocked when biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 was added to cytotoxicity assays at 200 microM with rat splenocytes as effectors. By confocal fluorescence microscopy, we show that this inhibitor localizes to distinct regions within RNK-16 cells and rat NK cells. Inhibitor treatment of intact cells inactivated the chymase activity and reduced lysis found in their dense organelles. Together these data indicate that biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 inhibits a granule chymase that is essential to cytolytic activity of NK cells.
Department of Microbiology, School of Medicine, University of Nevada, Reno 89557.
S L
SL
Woodard
D S
DS
Jackson
A S
AS
Abuelyaman
J C
JC
Powers
U
U
Winkler
D
D
Hudig
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D053818:Chymases; D003594:Cytoplasmic Granules / Q000201:enzymology*; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D051381:Rats; D011916:Rats, Inbred F344; D012697:Serine Endopeptidases / Q000187:drug effects*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D015842:Serine Proteinase Inhibitors; C090494:biotinyl-6-aminocaproyl-6-aminocaproyl-phenylalanyl-leucyl-phenylalanyl-diphenylphosphonate; D001710:Biotin; D012697:Serine Endopeptidases; D053818:Chymases