Shimokawa K, Jia L G, Shannon J D, Fox J W
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA.
Arch Biochem Biophys. 1998 Jun 15;354(2):239-46. doi: 10.1006/abbi.1998.0698.
Crotalid snake venom metalloproteinases often have associated with them nonproteinase domains that may be processed from the mature proteinases. Nascent atrolysin E, from the western diamondback rattlesnake, Crotalus atrox, has a metalloproteinasedomain and a non-RGD disintegrin domain that is lacking in the mature metalloproteinase. In this studywe report on the isolation, sequence analysis, andbiological activity of the 7.4-kDa atrolysin E disintegrin domain (atrolysin E/D). Atrolysin E/D represents approximately 0.2% of the total protein fromthe crude venom. The protein begins with a glycinyl residue found in the latter part of the spacer region. The sequence of atrolysin E/D is identical to thatof the non-RGD disintegrin domain of atrolysin E. The structure is termed a non-RGD disintegrin sincein lieu of the characteristic RGD sequence, a Met-Val-Asp (MVD) is found instead. Nevertheless, the protein is a potent inhibitor of both collagen- and ADP-stimulated platelet aggregation with IC50 values of 4 and 8 nM, respectively. A cyclized synthetic peptide, Ac-CRVSMVDRNDDTC-NH2, which represents the sequence of the atrolysin E/D non-RGD loop, was demonstrated to be an effective inhibitor of platelet aggregation. Therefore, this region of atrolysin E/D's structure, as in the disintegrins proper, is important for the biological activity of the protein. Thus, like the non-RGD disintegrin barbourin from Sistrurus miliarius barbouri, a RGD sequence in the context of the disintegrin protein backbone is not an absolute requirement for platelet aggregation inhibitory activity. These data underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of the precursor metalloproteinases and exemplify how the processed fragments may contribute to the observed pathological effects of the venom.
响尾蛇科蛇毒金属蛋白酶通常与其可能从成熟蛋白酶加工而来的非蛋白酶结构域相关联。来自西部菱斑响尾蛇（Crotalus atrox）的新生阿特罗溶素E具有一个金属蛋白酶结构域和一个成熟金属蛋白酶中不存在的非RGD整合素结构域。在本研究中，我们报道了7.4 kDa阿特罗溶素E整合素结构域（阿特罗溶素E/D）的分离、序列分析及生物学活性。阿特罗溶素E/D约占粗毒液总蛋白的0.2%。该蛋白起始于间隔区后半部分的一个甘氨酰残基。阿特罗溶素E/D的序列与阿特罗溶素E的非RGD整合素结构域的序列相同。该结构被称为非RGD整合素，因为代替特征性的RGD序列，发现的是甲硫氨酸-缬氨酸-天冬氨酸（MVD）。然而，该蛋白是胶原蛋白和ADP刺激的血小板聚集的有效抑制剂，IC50值分别为4 nM和8 nM。一种环化合成肽Ac-CRVSMVDRNDDTC-NH2，代表阿特罗溶素E/D非RGD环的序列，被证明是血小板聚集的有效抑制剂。因此，与真正的整合素一样，阿特罗溶素E/D结构的这一区域对该蛋白的生物学活性很重要。因此，就像来自巴氏猪鼻蛇（Sistrurus miliarius barbouri）的非RGD整合素巴布林一样，整合素蛋白主链中的RGD序列并非血小板聚集抑制活性的绝对必要条件。这些数据强调了由于前体金属蛋白酶的不同蛋白水解加工，响尾蛇科蛇毒的生化和功能复杂性，并举例说明了加工片段如何可能导致毒液所观察到的病理效应。
