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 minu tes without causing damage to themselves or bystander cells. One mecha nism of killing involves exocytosis of granules containing serine prot eases 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 differ ent serine protease sequences have been identified with the use of cDN A-specific clones. It is not known whether only one or several of thes e serine proteases are essential for cytolytic activity. In this study we show that an irreversible serine protease inhibitor, biotinyl-Aca- Aca-Phe-Leu-Phe(P)(OPh)(2), selectively inhibits a chymotrypsin-like ( chymase) serine protease activity of rat RNK-16 granule extracts. Unde r 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 gra nzyme was inhibited, granule-mediated lysis of erythrocytes was dimini shed. NK cell killing was completely blocked when biotinyl-Aca-Aca-Phe -Leu-Phe(P)(OPh)(2) was added to cytotoxicity assays at 200 mu M with rat splenocytes as effecters. By confocal fluorescence microscopy, we show that this inhibitor localizes to distinct regions within RNK-16 c ells and rat NK cells. Inhibitor treatment of intact cells inactivated the chymase activity and reduced lysis found in their dense organelle s. Together these data indicate that biotinyl-Aca-Aca-Phe-Leu-Phe(P)(O Ph)(2) inhibits a granule chymase that is essential to cytolytic activ ity of NK cells.