Definition and redesign of the extended substrate specificity of granzyme B
Definition and redesign of the extended substrate specificity of granzyme B
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DOI：
10.1074/jbc.273.42.27364
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发表时间：
1998-10-16
影响因子：
4.8
通讯作者：
Craik, CS
中科院分区：
文献类型：
--
作者：
Harris, JL;Peterson, EP;Craik, CS
颗粒酶 B 是一种蛋白酶，参与诱导细胞毒性淋巴细胞快速靶细胞死亡。颗粒酶 B 的底物特异性的定义允许在此过程中鉴定体内底物。通过使用合成底物文库和底物-噬菌体展示的组合方法，跨越六个亚位点的颗粒酶 B 的最佳底物被确定为 Ile-Glu-Xaa-（Asp 向下箭头 Xaa）-Gly，同时裂解 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。精氨酸 192 突变为谷氨酸，逆转了 P3 处带负电荷的氨基酸对带正电荷的氨基酸的偏好。优选的底物序列与 caspase 3 和 caspase 7 的激活位点相匹配，因此与细胞凋亡过程中颗粒酶 B 在这些蛋白酶激活中的作用一致。在无细胞测定中，Caspase 底物聚 (ADP)-核糖聚合酶在两个位点被颗粒酶 B 切割，这两个位点类似于通过组合方法测定的颗粒酶 B 特异性。许多 caspase 底物含有颗粒酶 B 切割位点，并被提议作为潜在的颗粒酶 B 靶标，这表明某些 caspase 具有冗余功能。
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 down arrow Xaa)-Gly, with cleavage of the Asp down arrow Xaa 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.