Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences
Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences
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DOI：
10.1074/jbc.m602372200
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发表时间：
2006-09-01
影响因子：
4.8
通讯作者：
Goettig, Peter
中科院分区：
文献类型：
--
作者：
Debela, Mekdes;Magdolen, Viktor;Goettig, Peter
人组织激肽释放酶（hKs）形成15个密切相关的（chymo）胰蛋白酶样丝氨酸蛋白酶家族。这些组织激肽释放酶在广泛的组织中表达，包括中枢神经系统、唾液腺和内分泌调节的组织，如前列腺、乳腺或睾丸，并且可能具有多种生理功能。对于几种组织激肽释放酶，表达与不同类型的癌症之间已经建立了明显的相关性。例如，前列腺特异性抗原（PSA或hK 3）作为肿瘤标志物，用于监测治疗反应。使用一种新的策略，我们已经克隆，在大肠杆菌或昆虫细胞中表达，重折叠，激活，并纯化七个人组织激肽释放酶hK 3/PSA，hK 4，hK 5，hK 6，hK 7，hK 10和hK 11。此外，我们已经确定了他们的扩展底物特异性的nonprime侧使用四肽底物的位置扫描组合库。hK 3/PSA和hK 7表现出胰凝乳蛋白酶样的特异性，更喜欢在P1位置的大的疏水性或极性残基。相比之下，hK 4，hK 5，和不太严格的hK 6显示胰蛋白酶样特异性与强烈的偏好P1-Arg，而hK 10和hK 11显示矛盾的特异性，接受基本和大的脂肪族P1残基。扩展的底物特异性谱与已知的底物裂解位点非常一致，但也与实验解决的（hK 4、hK 6和hK 7）或建模结构雅阁。特异性配置文件可能会导致更好地了解人体组织激肽释放酶的功能，并协助确定其生理蛋白质底物，以及在设计更具选择性的抑制剂。
Human tissue kallikreins (hKs) form a family of 15 closely related (chymo) trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved ( hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.