Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca
Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca
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DOI：
10.1016/s0041-0101(02)00279-9
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发表时间：
2003-02-01
期刊：
影响因子：
2.8
通讯作者：
Serrano, SMT
中科院分区：
文献类型：
--
作者：
Assakura, MT;Silva, CA;Serrano, SMT
成熟的P-III型蛇毒金属蛋白酶是一种可溶的蛇毒成分，属于relysin亚家族，与哺乳动物膜结合的A Disintegrin and Metalloproteinase （ADAMs）在结构上相关。本文报道了一种具有出血和肌坏死活性的金属蛋白酶bothropasin的分子克隆。利用免疫筛选方法克隆了bothropasin前体的全长cDNA，并通过从天然bothropasin自溶样品中获得的内部片段的氨基酸序列证实了其真实性。预测的bothropasin前体由P-III毒液金属蛋白酶的信号序列、前金属蛋白酶、金属蛋白酶、解体素样结构域和富含半胱氨酸的结构域组成。在天然bothropasin的自溶过程中，崩解素样结构域和富含半胱氨酸的结构域保持完整，而金属蛋白酶结构域在不同的位点被切割。利用细菌、酵母和哺乳动物细胞表达系统获得bothropasin重组前体形式的尝试未能产生足够的量来分析酶原的激活。然而，利用细菌系统对bothropasin单个结构域的表达进行研究，结果产生了重组的亲整合素和崩解素样+富含半胱氨酸的结构域，但没有金属蛋白酶结构域。这些结果以及天然蛋白的自溶模式表明金属蛋白酶结构域在bothropasin结构稳定性中的作用。(C) 2002 Elsevier Science Ltd.版权所有。
Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like + cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin. (C) 2002 Elsevier Science Ltd. All rights reserved.