Harris J L, Peterson E P, Hudig D, Thornberry N A, Craik C S
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.
J Biol Chem. 1998 Oct 16;273(42):27364-73. doi: 10.1074/jbc.273.42.27364.
Granzyme B is a protease involved in the induction of rapid target cell death by cytotoxic lymphocytes. Definition of the substrate specificity of granzyme B allows for the identification of in vivo substrates in this process. By using the combinatorial methods of synthetic substrate libraries and substrate-phage display, an optimal substrate for granzyme B that spans over six subsites was determined to be Ile-Glu-Xaa-(Asp downward arrowXaa)-Gly, with cleavage of the Asp downward arrowXaa peptide bond. Granzyme B proteolysis was shown to be highly dependent on the length and sequence of the substrate, supporting the role of granzyme B as a regulatory protease. Arginine 192 was identified as a determinant of P3-Glu and P1-Asp substrate specificity. Mutagenesis of arginine 192 to glutamate reversed the preference for negatively charged amino acids at P3 to positively charged amino acids. The preferred substrate sequence matches the activation sites of caspase 3 and caspase 7 and thus is consistent with the role of granzyme B in activation of these proteases during apoptosis. The caspase substrate poly(ADP)-ribose polymerase is cleaved by granzyme B in a cell-free assay at two sites that resemble the granzyme B specificity determined by the combinatorial methods. Many caspase substrates contain granzyme B cleavage sites and are proposed as potential granzyme B targets, suggesting a redundant function with certain caspases.
颗粒酶B是一种蛋白酶，参与细胞毒性淋巴细胞诱导靶细胞快速死亡的过程。确定颗粒酶B的底物特异性有助于识别该过程中的体内底物。通过使用合成底物文库和底物噬菌体展示的组合方法，确定了一种跨越六个亚位点的颗粒酶B的最佳底物为Ile-Glu-Xaa-(Asp向下箭头Xaa)-Gly，其切割Asp向下箭头Xaa肽键。颗粒酶B的蛋白水解作用高度依赖于底物的长度和序列，这支持了颗粒酶B作为调节性蛋白酶的作用。精氨酸192被确定为P3-Glu和P1-Asp底物特异性的决定因素。将精氨酸192突变为谷氨酸会使P3处对带负电荷氨基酸的偏好转变为对带正电荷氨基酸的偏好。优选的底物序列与半胱天冬酶3和半胱天冬酶7的激活位点匹配，因此与颗粒酶B在细胞凋亡过程中激活这些蛋白酶的作用一致。在无细胞测定中，半胱天冬酶底物聚（ADP）-核糖聚合酶在两个类似于通过组合方法确定的颗粒酶B特异性的位点被颗粒酶B切割。许多半胱天冬酶底物含有颗粒酶B切割位点，并被认为是潜在的颗粒酶B靶标，这表明与某些半胱天冬酶具有冗余功能。
