Sun J, Bird C H, Sutton V, McDonald L, Coughlin P B, De Jong T A, Trapani J A, Bird P I
Department of Medicine, Monash Medical School, Box Hill Hospital, Box Hill 3128, Australia.
J Biol Chem. 1996 Nov 1;271(44):27802-9. doi: 10.1074/jbc.271.44.27802.
Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.
我们采用聚合酶链反应策略，在人骨髓中鉴定出一种丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），它与细胞丝氨酸蛋白酶抑制剂6（PI - 6）和病毒丝氨酸蛋白酶抑制剂细胞因子反应调节因子A（CrmA）相关。这种丝氨酸蛋白酶抑制剂，即蛋白酶抑制剂9（PI - 9），具有不寻常的反应中心P1（Glu）-P1'（Cys），这表明它抑制在酸性残基后切割的丝氨酸蛋白酶。唯一已知具有这种特异性的丝氨酸蛋白酶是颗粒酶B，一种由细胞毒性淋巴细胞产生的颗粒细胞毒素。为了测试PI - 9与颗粒酶B的相互作用，我们在酵母表达系统中制备了重组六组氨酸标记的PI - 9。将重组蛋白添加到天然颗粒酶B中，形成了丝氨酸蛋白酶抑制剂 - 丝氨酸蛋白酶相互作用典型的抗SDS复合物。进一步分析表明，复合物形成遵循双分子动力学，二级速率常数为1.7±0.3×10⁶ M⁻¹ s⁻¹，这处于生理上显著的丝氨酸蛋白酶抑制剂 - 蛋白酶相互作用范围内。重组PI - 9在体外也完全消除了颗粒酶B和穿孔素介导的细胞毒性。对PI - 9 mRNA分布的检查表明它在免疫组织中表达，主要在淋巴细胞中。在自然杀伤细胞白血病细胞系和白细胞介素 - 2刺激的外周血单个核细胞中观察到最高水平的PI - 9 mRNA和蛋白质，这些细胞也产生颗粒酶B。与PI - 6一样，PI - 9被证明是一种不分泌的胞质蛋白。对自然杀伤细胞和刺激的外周血单个核细胞进行分级分离表明，PI - 9与颗粒酶B处于不同的亚细胞区室。这些结果表明，PI - 9用于在细胞毒性细胞脱颗粒后使错误导向的颗粒酶B失活。这可能解释了为什么细胞毒性细胞在破坏异常细胞时不会被自身的颗粒酶B损伤。
