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

由于鱼类的附着和微生物的侵害在第一行防御机制中，鱼类覆盖了PR事件。 。基于确定的部分氨基酸序列……更多的CE，由3'种族和RT-PCR方法克隆了全长的cDNA（2037bp）。肽（58个残基）和成熟的蛋白质（496个残基）与L-氨基酸酶（LAO）家族的蛋白质相似。用抗鱼类病原体的裂解液体，salmonicida，msleae subsp.piscicida，peromonas hydrophila和virahayticus具有0.078、0.16、0.31、0.31和0.63μg/ml，SSAP抗细菌效应，并具有0.078、0.16、0.31和0.63μg/ml的核酸盐效应。 A.在存在过氧化氢酶的情况下，SSAP生成的H_2O_2是细胞死亡的反应，SSAP的h_2o_2在存在的情况下，SSAP的h_2o_2几乎是竞争性的。首先揭示了较少的鱼皮中老挝活动的发明