Nair Dileep G, Fry Bryan G, Alewood Paul, Kumar Prakash P, Kini R Manjunatha
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543.
Biochem J. 2007 Feb 15;402(1):93-104. doi: 10.1042/BJ20060318.
We have isolated and characterized omwaprin, a 50-amino-acid cationic protein from the venom of inland taipan (Oxyuranus microlepidotus). It is a new member of the waprin family of snake venom proteins. A synthetic gene was designed and constructed for expressing the recombinant protein in Escherichia coli. Recombinant omwaprin was used for carrying out functional analyses. The protein is non-toxic to Swiss albino mice at doses of up to 10 mg/kg when administered intraperitoneally. However, it shows selective and dose-dependant antibacterial activity against Gram-positive bacteria. The minimum inhibitory doses were in the range 2-10 microg for selected species of bacteria in radial diffusion assays. The antibacterial activity is salt-tolerant up to 350 mM NaCl. However, omwaprin lost its antibacterial activity upon reduction and alkylation of its cysteine residues, or upon deletion of six N-terminal amino acid residues, four of which are positively charged. These observations indicate that the three-dimensional structure constrained by four disulfide bonds and the N-terminal residues are essential for its activity. The mechanism of action is via membrane disruption, as shown by scanning electron microscopy. Importantly, omwaprin lacks haemolytic activity on human erythrocytes. This demonstrates the specificity of omwaprin for bacterial membranes. Unlike other reported WAP (whey acidic protein) domain-containing antibacterial proteins, including elafin, EPPIN (epididymal proteinase inhibitor), SWAM1 and SWAM2 [single WAP (whey acidic protein) motif proteins 1 and 2] and SLPI (secretory leucocyte proteinase inhibitor), omwaprin shows species-specific activity on the Gram-positive bacteria tested.
我们从内陆太攀蛇（Oxyuranus microlepidotus）毒液中分离并鉴定了omwaprin，一种由50个氨基酸组成的阳离子蛋白。它是蛇毒蛋白waprin家族的新成员。设计并构建了一个合成基因，用于在大肠杆菌中表达重组蛋白。重组omwaprin用于进行功能分析。当腹腔注射时，该蛋白在剂量高达10 mg/kg时对瑞士白化小鼠无毒。然而，它对革兰氏阳性菌表现出选择性和剂量依赖性抗菌活性。在径向扩散试验中，对选定的细菌种类，最小抑菌剂量在2-10微克范围内。抗菌活性在高达350 mM NaCl的盐浓度下仍具有耐受性。然而，omwaprin在其半胱氨酸残基还原和烷基化后，或在缺失六个N端氨基酸残基（其中四个带正电荷）后，失去了抗菌活性。这些观察结果表明，由四个二硫键和N端残基所限制的三维结构对其活性至关重要。如扫描电子显微镜所示，其作用机制是通过膜破坏。重要的是，omwaprin对人红细胞缺乏溶血活性。这证明了omwaprin对细菌膜的特异性。与其他报道的含WAP（乳清酸性蛋白）结构域的抗菌蛋白不同，包括elafin、EPPIN（附睾蛋白酶抑制剂）、SWAM1和SWAM2 [单WAP（乳清酸性蛋白）基序蛋白1和2] 以及SLPI（分泌型白细胞蛋白酶抑制剂），omwaprin对所测试的革兰氏阳性菌表现出种属特异性活性。
