Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 2010 Dec;157(4):351-6. doi: 10.1016/j.cbpb.2010.08.006. Epub 2010 Aug 20.
Fish have a complex innate defense mechanism against microbial invasion. In particular, epidermal mucus and serum in fish play important roles in innate immunity and contain a variety of bioactive substances such as complements, lectins and lysozymes, involved in host defense. Recently, L-amino acid oxidases (LAOs) with antibacterial activity were isolated from the skin and/or gill mucous secretions of rockfish, great sculpin and flounder, and were identified to be a novel type of antibacterial protein in the integument of fish. In the present study, we found LAO activity in the serum of rockfish Sebastes schlegeli. The LAO was isolated from the serum by sequential column chromatography of Con-A lectin affinity chromatography, anion exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC, and characterized. The LAO (a molecular mass of 160 kDa) comprised subunits with a molecular mass of 53 kDa and showed strict substrate specificity, catalyzing only L-lysine with Km 0.37 mM and kcat 57.1s(-1). The serum LAO exhibited a broad antibacterial activity against Gram positive and negative bacteria, most potently against Aeromonas hydrophila and Aeromonas salmonicida with a minimum inhibitory concentration of 0.078 μg/mL. This is the first report of LAO in the serum of fish and its involvement in innate immunity in the rockfish body.
鱼类具有复杂的先天防御机制来抵御微生物入侵。特别是鱼类的表皮黏液和血清在先天免疫中发挥重要作用，其中含有多种生物活性物质，如补体、凝集素和溶菌酶，参与宿主防御。最近，从石斑鱼、大杜父鱼和牙鲆的皮肤和/或鳃黏液分泌物中分离到具有抗菌活性的 L-氨基酸氧化酶（LAOs），并被鉴定为鱼类表皮中的一种新型抗菌蛋白。本研究发现，黑鲷血清中存在 LAO 活性。通过 Con-A 凝集素亲和层析、阴离子交换 HPLC、羟磷灰石 HPLC 和凝胶过滤 HPLC 顺序柱层析从血清中分离 LAO 并进行了表征。LAO（分子量为 160 kDa）由分子量为 53 kDa 的亚基组成，表现出严格的底物特异性，仅催化 L-赖氨酸，Km 为 0.37 mM，kcat 为 57.1s(-1)。血清 LAO 对革兰氏阳性和阴性菌表现出广泛的抗菌活性，对嗜水气单胞菌和鲑气单胞菌的抑制作用最强，最小抑菌浓度为 0.078 μg/mL。这是首次在鱼类血清中报道 LAO 及其在黑鲷体内先天免疫中的参与。
