Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus
Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus
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DOI：
10.1111/j.1742-4658.2009.07497.x
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发表时间：
2010-01-01
期刊：
影响因子：
5.4
通讯作者：
Miura, Tomisato
中科院分区：
文献类型：
--
作者：
Kasai, Kosuke;Ishikawa, Takashi;Miura, Tomisato
鱼产生粘液物质，作为防御环境外来生物和捕食者的外部屏障。最近，我们在牙鲆粘液层中发现了一种生物活性蛋白，它对表皮葡萄球菌、金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌具有抗菌活性。在本研究中，我们通过一系列柱层析步骤从星状拟青霉的粘液成分中分离和鉴定了抗菌蛋白。然后，我们进行了凝胶电泳和cDNA克隆，以确定该蛋白的特性。粘液中的抗菌蛋白分子量约为52 kDa，等电点为5.3，测序结果表明该蛋白与鳃抗菌蛋白的多肽序列完全一致。BLAST搜索表明，该基因编码的抗菌蛋白与一些L氨基酸氧化酶(LAAO)具有相同的同源性，并且在黄素蛋白中具有几个保守的基序。用特异的引物进行RT-PCR，用抗LAAO抗体进行免疫组织化学分析，证实了该病毒在鳃中的组织特异性表达和定位。此外，抗LAAO抗体能够中和该蛋白对耐甲氧西林金黄色葡萄球菌的抗菌活性。因此，我们证明了该抗菌蛋白是一种新的具有抗菌活性的LAAO样蛋白，类似于蛇的LAAO，从星形拟青霉的表皮粘液中鉴定出来。
Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.