Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% of α-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as permeabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family.

肌动孔蛋白构成在海葵中发现的一类独特的成孔毒素，它们以可溶性单体的形式分泌并能够结合并透化膜，从而导致细胞死亡。由于它们具有高的细胞毒性，可以适当地用于设计针对肿瘤细胞的免疫毒素和针对细胞溶质的抗原释放系统，因此人们对这些蛋白质的兴趣日益浓厚。在这项工作中，我们描述了由中美洲太平洋发现的海葵Anthopleura nigrescens生产的新型放线菌素。在这里，我们报告了放线菌素的氨基酸序列，该序列是从从全身RNA获得的cDNA推导出来的。在BL21 Star（DE3）大肠杆菌中表达编码一种溶细胞素变体的合成DNA序列并通过CM Sephadex C-25色谱纯化的蛋白质具有97％的同质性，通过MS-MS和HPLC分析证实。该肌动孔蛋白包含179个氨基酸残基，与其观察到的同位素平均分子量为19 661 Da相符。该毒素缺乏Cys，可以很容易地渗透红细胞以及L1210细胞。CD光谱显示其二级结构主要由β结构（58.5％）和5.5％的α-螺旋以及35％的无规结构主导。此外，与脂质单层和脂质体的结合实验以及在囊泡中的通透性研究表明，该毒素对含有鞘磷脂的膜的亲和力与sticholysin II（StII）非常相似。通过光谱技术的比较和建模的黑素（Ng）的三维结构显示与StII的高度同源性，但也可以检测到一些差异。综上所述，这些结果强化了以下观点：Ng是肌动孔蛋白成孔毒素（PFT）家族的新成员，其HA高达StII（迄今为止描述的最有效的肌动孔蛋白），但具有独特的结构特征扩展了对该蛋白家族中结构-功能关系的理解。