Leu Jiann-Horng, Tsai Chi-Hang, Yang Chia-Hsun, Chou Hsin-Yiu, Wang Hao-Ching
Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, ROC; Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan, ROC.
Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, ROC.
Dev Comp Immunol. 2021 Jul;120:104058. doi: 10.1016/j.dci.2021.104058. Epub 2021 Feb 28.
Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.
最近，L-氨基酸氧化酶（LAAOs）在几种鱼类中被鉴定为抵抗细菌感染的一线防御分子。在此，我们报告了从红斑石斑鱼（Epinephelus coioides）中克隆和鉴定一种鱼类LAAO基因EcLAAO2的过程。全长cDNA为3030 bp，其开放阅读框编码一个由511个氨基酸组成的蛋白质。EcLAAO2主要在鳍、鳃和肠道中表达。在用脂多糖（LPS）和聚肌苷酸-聚胞苷酸（poly (I:C)）刺激后，其在几个免疫器官中的表达上调。从杆状病毒表达系统中表达并纯化的重组EcLAAO2蛋白（rEcLAAO2）被确定为一种糖基化二聚体。根据过氧化氢产生试验，该重组蛋白被鉴定为具有LAAO酶活性，对L-苯丙氨酸（L-Phe）和L-色氨酸（L-Trp）有底物偏好，但不像其他已知鱼类LAAOs那样对L-赖氨酸（L-Lys）有偏好。rEcLAAO2可以有效抑制副溶血性弧菌、金黄色葡萄球菌和枯草芽孢杆菌的生长，而对大肠杆菌生长的抑制效果较差。最后，基于序列同源性构建了蛋白质模型，以预测EcLAAO2的三维结构，并解释EcLAAO2与其他已报道的鱼类LAAOs之间底物特异性的差异。总之，本研究鉴定出EcLAAO2是一种新型鱼类LAAO，其底物偏好与其他已知鱼类LAAOs不同，并揭示了它可能在抵抗入侵病原体方面发挥作用。
