León L, Lissi E A, Celedón G, Gonzalez G, Pazos F, Alvarez C, Lanio M E
Department of Chemistry, Chemistry and Biology Faculty, Universidad de Santiago de Chile (USACH), Santiago, Chile,
Protein J. 2014 Oct;33(5):493-501. doi: 10.1007/s10930-014-9582-x.
Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing -SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO(-)). At low ONOO(-)/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in -SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.
海葵能合成多种具有生物学意义的有毒肽和蛋白质。加勒比海葵壮丽双辐海葵（Stichodactyla helianthus）产生两种成孔毒素，刺参溶素I（St I）和刺参溶素II（St II），它们能够在细胞和脂质双层中形成寡聚孔，其氨基酸序列中典型地缺乏半胱氨酸。最近，已获得重组刺参溶素I变体（rSt I）的两个突变体，在毒素成孔活性的功能相关区域有一个半胱氨酸残基：rSt I F15C（在氨基末端序列）和rSt I R52C（在结合位点）。为了表征氧化剂对不含-SH基团的毒素（rSt I）或含有-SH基团的毒素（rSt I F15C和rSt I R52C）的影响，我们在与过氧亚硝酸根（ONOO(-)）孵育前后测量了它们的溶血活性和成孔能力。在低ONOO(-)/毒素比例下，每个添加的过氧亚硝酸根分子修饰近0.8个色氨酸基团，毒素活性降低约20%。另一方面，在含-SH的突变体中，每个过氧亚硝酸根分子仅修饰0.5个色氨酸基团，毒素活性仅降低10%。结果表明，半胱氨酸是氧化损伤的初始靶点，含半胱氨酸的毒素中的色氨酸残基比rSt I中的色氨酸残基受损程度小。色氨酸基团的这种相对保护与脂质体中溶血活性和通透能力的较小损失相关，并强调了色氨酸基团在毒素成孔能力中的相关性。
