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

由于鱼类与含有高浓度致病微生物的环境密切接触，因此它们的皮肤上覆盖着一层粘膜，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，其中包括抗菌素，这些物质在鱼类的一线防御机制中起着重要作用。尽管在各种鱼类的皮肤粘液中发现了许多抗菌物质，但人们对抗菌蛋白的了解却很少。近年来，有报道称石斑鱼的皮肤黏液中含有一种强效抗菌蛋白，称为SSAP （sebases schlegeli antimicrobial protein）。本研究旨在阐明SSAP的结构和抑菌作用模式。从岩鱼皮肤黏液中提取SSAP，采用凝集素亲和层析、阴离子交换高效液相色谱和羟基磷灰石高效液相色谱相结合的方法分离SSAP。通过3′RACE、5′RACE和RT-PCR方法，分别克隆了SSAP全长cDNA （2037bp）。假设SSAP的前体（554个残基）包含一个信号肽（58个残基）和一个成熟蛋白（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）家族相似。据此，通过H_2O_2生成实验证明SSAP具有LAO活性。SSAP的LAO活性具有严格的底物特异性，仅催化l -赖氨酸，Km值为0.19 mM。SSAP对鱼类病原菌如沙门气单胞菌、豆色光杆菌等具有抗菌活性。MIC分别为0.078、0.16、0.31和0.63 μg/ml。结果表明，SSAP对沙门氏菌和副溶血性假单胞菌均有杀菌作用，对嗜水假单胞菌和豆selae假单胞菌均有抑菌作用。通过微生物学实验和扫描电镜分析。然而，在过氧化氢酶存在下，SSAP的抑菌活性几乎完全丧失，这证实了SSAP产生的H_2O_2是导致细胞死亡的原因。SSAP的细菌选择性不太可能与细菌细胞壁的结合能力相关。本研究首次揭示了LAO活性在鱼皮肤先天免疫中的作用。少

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