Núñez C E, Angulo Y, Lomonte B
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Toxicon. 2001 Oct;39(10):1587-94. doi: 10.1016/s0041-0101(01)00141-6.
Group II phospholipase A(2) (PLA(2)) myotoxins found in the venoms of Crotalidae snakes can be divided into 'Asp49' and 'Lys49' isoforms, the latter being considered catalytically-inactive variants. Previous studies on one Lys49 isoform, myotoxin II from Bothrops asper, indicated that its myotoxic activity is due to the presence of a short cationic/hydrophobic sequence (115-129) near its C-terminus, which displays membrane-damaging properties. Since the C-terminal region of different group II PLA(2) myotoxins presents considerable sequence variability, synthetic peptides homologous to region 115-129 of myotoxin II, but corresponding to B. asper myotoxin III (Asp49), Agkistrodon piscivorus piscivorus Asp49 PLA(2) and Lys49 PLA(2), were studied to determine the possible functional relevance of such region for the toxic activities of these proteins. Results showed that both Lys49-derived peptides (p-BaK49 and p-AppK49) were able to lyse skeletal muscle C2C12 cells in culture, and to induce edema in the mouse footpad assay. Moreover, p-AppK49, which showed a markedly stronger cytotoxic potency than p-BaK49, additionally induced skeletal muscle necrosis when injected into mice. These observations unequivocally identify the sequence 115-129 (KKYKAYFKLKCKK) of the Lys49 PLA(2) of A. p. piscivorus as containing the key structural determinants needed for myotoxicity, and represent the first report of an unmodified, PLA(2)-derived short synthetic peptide with the ability to reproduce this effect of a parent toxin in vivo. On the other hand, the two Asp49-derived peptides did not show any toxic effects in vitro or in vivo, even at high concentrations. These findings suggests that Lys49 and Asp49 group II PLA(2)s might exert their myotoxic actions through different molecular mechanisms, by implying that the latter may not utilize their C-terminal regions as main membrane-destabilizing elements.
在蝰蛇科蛇类毒液中发现的II组磷脂酶A(2)（PLA(2)）肌毒素可分为“Asp49”和“Lys49”亚型，后者被认为是催化无活性的变体。先前对一种Lys49亚型——来自矛头蝮的肌毒素II的研究表明，其肌毒性活性归因于其C末端附近存在一个短的阳离子/疏水序列（115-129），该序列具有膜损伤特性。由于不同II组PLA(2)肌毒素的C末端区域存在相当大的序列变异性，因此研究了与肌毒素II的115-129区域同源，但对应于矛头蝮肌毒素III（Asp49）、食鱼蝮Asp49 PLA(2)和Lys49 PLA(2)的合成肽，以确定该区域对这些蛋白质毒性活性的可能功能相关性。结果表明，两种源自Lys49的肽（p-BaK49和p-AppK49）都能够在培养物中裂解骨骼肌C2C12细胞，并在小鼠足垫试验中诱导水肿。此外，p-AppK49在注入小鼠体内时，除了诱导骨骼肌坏死外，还表现出比p-BaK49明显更强的细胞毒性效力。这些观察结果明确确定了食鱼蝮Lys49 PLA(2)的115-129序列（KKYKAYFKLKCKK）包含肌毒性所需的关键结构决定因素，并且代表了第一个关于未修饰的、源自PLA(2)的短合成肽能够在体内重现亲本毒素这种作用的报道。另一方面，两种源自Asp49的肽即使在高浓度下也未在体外或体内显示出任何毒性作用。这些发现表明，Lys49和Asp49 II组PLA(2)可能通过不同的分子机制发挥其肌毒性作用，这意味着后者可能不会将其C末端区域用作主要的膜不稳定元件。
