Assakura Marina T, Silva Carlos A, Mentele Reinhard, Camargo Antonio C M, Serrano Solange M T
Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brasil 1500, CEP 05503-900, São Paulo, SP, Brazil.
Toxicon. 2003 Feb;41(2):217-27. doi: 10.1016/s0041-0101(02)00279-9.
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.
成熟的P-III型蛇金属蛋白酶是可溶性毒液成分，属于解聚素亚家族，在结构上与哺乳动物的膜结合型解聚素和金属蛋白酶（ADAMs）相关。在此，我们展示了从巴西矛头蝮毒液中分离出的具有出血和肌坏死活性的金属蛋白酶——矛头蝮蛋白酶的分子克隆。通过免疫筛选克隆了编码矛头蝮蛋白酶前体的全长cDNA，并通过从天然矛头蝮蛋白酶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。预测的矛头蝮蛋白酶前体由P-III型毒液金属蛋白酶的元件组成：信号序列、前肽、金属蛋白酶、解聚素样和富含半胱氨酸的结构域。在天然矛头蝮蛋白酶的自溶过程中，解聚素样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位点被切割。使用细菌、酵母和哺乳动物细胞表达系统获得重组矛头蝮蛋白酶前体形式的尝试未能产生足够量的产物以分析酶原的激活。然而，使用细菌系统对矛头蝮蛋白酶各个结构域的表达研究产生了重组前肽和解聚素样+富含半胱氨酸的结构域，但没有产生金属蛋白酶结构域。这些结果以及天然蛋白质的自溶模式表明金属蛋白酶结构域在矛头蝮蛋白酶的结构稳定性中起作用。
