Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools
Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools
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DOI：
10.1111/j.1432-1033.1997.t01-1-00171.x
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发表时间：
1997-10-01
期刊：
影响因子：
--
通讯作者：
Rammensee, HG
中科院分区：
文献类型：
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作者：
Arnold, D;Keilholz, W;Rammensee, HG
细胞蛋白酶对蛋白抗原的降解是T细胞介导的免疫应答启动的关键步骤。但是对于负责处理抗原的酶，包括它们的特异性，仍然知之甚少。在本文中，我们表明，结合自动化N-末端测序与新开发的方法，由天冬氨酸蛋白酶组织蛋白酶D和E产生的肽池的C-末端测序是一种快速简便的方法，以获得这些内肽酶的底物特异性的详细信息。使用15个残基的合成肽库和天然蛋白质作为底物，我们确认和扩展的知识有关的切割基序的组织蛋白酶E的位置P1和P1'的基板必须完全由疏水性氨基酸与芳香族或脂肪族侧链占据。然而，P1位不允许有瓦尔和Ile残基。位置P2'接受广泛的氨基酸，包括带电的和极性的氨基酸。关于底物位置P3'和P4'的其他要求也通过合并测序来定义。此外，用天冬氨酸蛋白酶组织蛋白酶D和E分析蜂毒肽酶提供了两种酶共享相同切割基序的证据，与来自肽文库和天然蛋白的切割基序相同。因此，池测序分析是确定任何内肽酶的底物特异性的有价值且快速的工具。
Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible fur the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1' of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2' accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3' and P4' were also defined by pool sequencing. Furthermore, analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.