Structure and action mode of Gram-negative bacteria-selective antibacterial protein from skin mucus of the rockfish Sebastes schlegeli

石斑鱼皮肤粘液革兰氏阴性菌选择性抗菌蛋白的结构及作用模式

• 批准号: 16580167
• 负责人: NAGASHIMA Yuji
• 金额: $ 2.43万
• 依托单位: Tokyo University of Marine Science and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16580167/
• 关键词: antibacterial protein; rockfish Sebastes schlegeli; skin mucus; L-amino acid oxidase; hydrogen peroxide; Gram-negative bacterium; electron microscopy; innate immunity; 抗菌作用; 構造解析; フラボプロテイン; 糖タンパク質

Since fish are in intimate contact with their environment containing a high concentration of pathogenic microbes, their skin is covered with mucous layer to prevent from attachment and invasion of microbes. Fish skin mucus has a variety of bioactive substances including antimicrobials that appear to play an important role in the first line defense mechanism of fish. Although a number of antibacterial substances have been found in skin mucus of various fish, antibacterial proteins have been poorly understood. Recently, the skin mucus of rockfish Sebastes schlegeli has been reported to possess a potent antibacterial protein, designated as SSAP (Sebastes schlegeli antibacterial protein). This study was undertaken to elucidate the structure and the mode of antibacterial action of SSAP. SSAP was extracted from the rockfish skin mucus and isolated by a combination of lectin affinity chromatography, anion exchange HPLC and hydroxyapatite HPLC. Based on the determined partial amino acid sequen … More ce, a full-length cDNA (2037bp) encoding SSAP was cloned by 3' RACE, 5'RACE and RT-PCR methods. The precursor of SSAP (554 residues) was assumed to comprise a signal peptide (58 residues) and a mature protein (496 residues). The sequence alignment analysis revealed that SSAP is structurally similar to the proteins of L-amino acid oxidase (LAO) family. In accordance with this, SSAP was demonstrated to have LAO activity by H_2O_2 generation assay. The LAO activity of SSAP was featured by strict substrate specificity, catalyzing exclusively L-Lys with a Km value of 0.19 mM. SSAP showed antibacterial activity against fish pathogens such as Aeromonas salmonicida, Photobacterium damselae subsp.piscicida, Aeromonas hydrophila and Vibrio parahaemolyticus with MIC of 0.078, 0.16, 0.31 and 0.63 μg/ml, respectively. SSAP was found to exert bactericidal effect against A.salmonicida and V.parahaemolyticus while bacteriostatic effect against A.hydrophila and P.damselae subsp.piscicida by microbiological experiments and scanning electron microscopic analysis. However, the antibacterial activity of SSAP was almost completely lost in the presence of catalase, confirming that H_2O_2 generated by SSAP is responsible for cell death. It is unlikely that bacterium-selectivity of SSAP correlated with the binding ability to bacterial cell wall. The present study is the first to reveal the involvement of LAO activity in innate immunity of fish skin. Less

由于鱼类与含有高浓度病原微生物的环境密切接触，因此它们的皮肤覆盖着粘液层，以防止微生物的附着和入侵。鱼皮粘液中含有多种生物活性物质，包括抗菌物质，在鱼类的第一道防线中发挥着重要作用。虽然在各种鱼类的皮肤粘液中发现了许多抗菌物质，但对抗菌蛋白的了解很少。近年来，有报道称许氏平鲉的皮肤粘液中含有一种有效的抗菌蛋白，命名为SSAP（许氏平鲉抗菌蛋白）。本研究旨在阐明SSAP的结构和抗菌作用方式。从石斑鱼皮肤粘液中提取SSAP，并通过凝集素亲和层析、阴离子交换HPLC和羟基磷灰石HPLC相结合进行分离。根据测定的部分氨基酸序列， ...更多信息 ce，采用3' RACE、5' RACE和RT-PCR方法克隆了SSAP的全长cDNA（2037 bp）。SSAP的前体（554个残基）被认为包括信号肽（58个残基）和成熟蛋白（496个残基）。序列比对分析表明，SSAP在结构上与L-氨基酸氧化酶（LAO）家族蛋白相似。H_2O_2生成实验表明SSAP具有抗LAO活性。SSAP对杀鲑气单胞菌（Aeromonassalmonicida）、杀鱼发光杆菌（Photobacteriumdamselaesubsp.piscicida）、嗜水气单胞菌（Aeromonasjaponica）和副溶血性弧菌（Vibrioparahaemolyticus）均有抗菌活性，MIC分别为0.078、0.16、0.31和0.63 μg/ml。通过微生物学实验和扫描电镜分析，发现SSAP对杀鲑性放线菌和副溶血性弧菌有杀菌作用，对杀鱼性放线菌和杀鱼性紫菜单胞菌有抑菌作用。而在过氧化氢酶存在下，SSAP的抗菌活性几乎完全丧失，证实了SSAP产生的H_2O_2是导致细胞死亡的原因。SSAP对细菌的选择性与其对细菌细胞壁的结合能力无关。本研究首次揭示了LAO活性参与鱼皮天然免疫的机制。少