Recognition of sphingomyelin by lysenin and lysenin-related proteins
Recognition of sphingomyelin by lysenin and lysenin-related proteins
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DOI：
10.1021/bi049561j
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发表时间：
2004-08-03
期刊：
影响因子：
2.9
通讯作者：
Kobayashi, T
中科院分区：
文献类型：
--
作者：
Kiyokawa, E;Makino, A;Kobayashi, T
Lysenin是一种鞘磷脂（SM）特异性毒素，分离自蚯蚓体腔液。胞溶素包括与胞溶素相关蛋白I（LRP-1，胞溶素2）和LRP-2（胞溶素3）一起的蛋白质家族。在本研究中，我们的特点LRP-1和LRP-2与溶素一起使用麦芽糖结合蛋白标记的重组蛋白。LRP-2特异性结合SM并诱导溶血。相反，LRP-1的结合和溶血活性比LRP-2和胞溶素低10倍。Lysenin和LRP-2共有30个芳香族氨基酸位点。其中，只有一个位置，苯丙氨酸210，取代异亮氨酸在LRP-1。通过将异亮氨酸210引入苯丙氨酸的单个氨基酸取代，LRP-1的活性显著增加，表明该芳香族氨基酸在胞溶素和LRP的生物活性中的重要性。芳香族氨基酸的重要性进一步表明了系统的色氨酸丙氨酸突变的胞溶素。Lysenin含有六个色氨酸残基，其中五个在LRP-1和-2中是保守的。我们发现，在SM的识别和溶血素的溶血活性中，保守的而不是非保守的Rehans是必需的。我们的研究结果表明，色氨酸在毒素功能中的重要性可能是由于直接识别SM或维持蛋白质结构。
Lysenin is a sphingomyelin (SM)-specific toxin isolated from the coelomic fluid of the earthworm Eisenia foetida. Lysenin comprises a family of proteins together with lysenin-related protein I (LRP-1, lysenin 2) and LRP-2 (lysenin 3). In the present study, we characterized LRP-1 and LRP-2 together with lysenin using maltose-binding-protein-tagged recombinant proteins. LRP-2 specifically bound SM and induced hemolysis like lysenin. In contrast the binding and hemolytic activities of LRP-1 were 10 times less than those of lysenin and LRP-2. Lysenin and LRP-2 share 30 common sites of aromatic amino acids. Among them, only one position, phenylalanine 210, is substituted for isoleucine in LRP-1. The activity of LRP-1 was dramatically increased by introducing a single amino acid substitution of isoleucine 210 to phenylalanine, suggesting the importance of this aromatic amino acid in biological activities of lysenin and LRPs. The importance of aromatic amino acids was further indicated by a systematic tryptophan to alanine mutation of lysenin. Lysenin contains six tryptophan residues of which five are conserved in LRP-1 and -2. We showed that the conserved tryptophans but not the nonconserved one were required both in the recognition of SM and in the hemolytic activity of lysenin. Our results suggest the importance of tryptophan in the toxin function likely due to a direct recognition of SM or in maintaining the protein structure.