Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.
Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.
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DOI：
10.1016/j.fsi.2011.12.006
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发表时间：
2012-03
影响因子：
4.7
通讯作者：
Yeqing Qian;Ye Li;Fan Yang;Yan-Qin Yu;Jin-Shu Yang;Wei-Jun Yang
中科院分区：
文献类型：
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作者：
Yeqing Qian;Ye Li;Fan Yang;Yan-Qin Yu;Jin-Shu Yang;Wei-Jun Yang
Kazal-type inhibitors（KPIs）在血液凝固、免疫应答和生殖等许多生物和生理过程中发挥重要作用。在本研究中，两个雄性生殖道KPI，称为Man-KPI和Ers-KPI，分别在日本沼虾和中华绒螯蟹。测定了重组Man-KPI和Ers-KPI对糜蛋白酶、弹性蛋白酶、胰蛋白酶和凝血酶的抑制活性。结果表明，它们对胰凝乳蛋白酶和弹性蛋白酶有较强的抑制作用。动力学研究，以阐明其抑制机制。此外，还表达了单个结构域，以进一步了解哪个结构域有助于完整KPI的抑制活性。只有Man-KPI_结构域3在胰凝乳蛋白酶和弹性蛋白酶的抑制中具有活性。同时，Ers-KPI_结构域2和3负责胰凝乳蛋白酶的抑制，而Ers-KPI_结构域2、3和4负责弹性蛋白酶的抑制。同时，将这两种关键肽抑制剂对罗氏沼虾、日本沼虾和中华绒螯蟹精子的抑制活性与前期研究中发现的罗氏沼虾生殖道中的Kazal型肽酶抑制剂（MRPINK）进行了比较。结果表明，KPI能完全抑制同种精子蛋白酶的明胶分解活性，但与不同种属精子蛋白酶之间存在不同程度的交叉抑制作用。这些结果可能为进一步阐明雄性生殖系统中KPI-proteins相互作用的机制提供新的视角。
Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system.