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

• Project/Area Number: 16580167
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Fisheries chemistry
• Research Institution: Tokyo University of Marine Science and Technology
• Principal Investigator: NAGASHIMA Yuji Tokyo University of Marine Science and Technology, Department of Food Science and Technology, Professor, 海洋科学部, 教授 (40180484)
• Co-Investigator(Kenkyū-buntansha): NAGASHIMA Yuji Tokyo University of Marine Science and Technology, Department of Food Science and Technology, Professor (40180484)
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,800,000 (Direct Cost: ¥3,800,000); Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2004: ¥2,700,000 (Direct Cost: ¥2,700,000)
• Keywords: 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

Research Abstract

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 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.