Caputo A, Garner R S, Winkler U, Hudig D, Bleackley R C
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1993 Aug 25;268(24):17672-5.
Murine cytotoxic cell proteinase-1 (granzyme B) is a member of a family of novel serine proteinases that have been implicated to participate in destruction of target cells by cytotoxic T lymphocytes. Comparison of the sequence of the cDNA with the sequence of the protein isolated from cytoplasmic granules of cytotoxic T lymphocytes suggested that this protein may be synthesized as a preproenzyme containing an amino-terminal activation dipeptide. Here we show that this activation dipeptide regulates the activity of the enzyme in hydrolysis of its preferred substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester. Lysates of COS cells transfected with a vector expressing the unmodified cytotoxic cell proteinase-1 cDNA were unable to hydrolyze this substrate, whereas lysates of cells transfected with a construct in which the activation dipeptide codons has been deleted were able to hydrolyze the substrate. In each case Western blotting of the lysates revealed a form of the proteinase with an apparent molecular weight of 27,000. We conclude that the activation dipeptide regulates activity of the enzyme. This is the first report of production of an enzymatically active recombinant cytotoxic T cell serine proteinase. The strategy for successful expression of an activated form of cytotoxic cell proteinase-1 may be applicable to other members of this proteinase family.
小鼠细胞毒性细胞蛋白酶-1（颗粒酶B）是一类新型丝氨酸蛋白酶家族的成员，该家族成员被认为参与细胞毒性T淋巴细胞对靶细胞的破坏作用。将细胞毒性T淋巴细胞胞质颗粒中分离出的蛋白质序列与cDNA序列进行比较，结果表明该蛋白质可能以前体酶原的形式合成，前体酶原含有一个氨基末端激活二肽。在此我们表明，这种激活二肽在其首选底物叔丁氧羰基-丙氨酸-丙氨酸-天冬氨酸-硫代苄酯的水解过程中调节该酶的活性。用表达未修饰的细胞毒性细胞蛋白酶-1 cDNA的载体转染的COS细胞裂解物不能水解该底物，而用缺失激活二肽密码子的构建体转染的细胞裂解物能够水解该底物。在每种情况下，裂解物的蛋白质印迹分析都显示出一种表观分子量为27,000的蛋白酶形式。我们得出结论，激活二肽调节该酶的活性。这是关于产生具有酶活性的重组细胞毒性T细胞丝氨酸蛋白酶的首次报道。成功表达细胞毒性细胞蛋白酶-1激活形式的策略可能适用于该蛋白酶家族的其他成员。
