Nilojan Jehanathan, Bathige S D N K, Kugapreethan Roopasingam, Thulasitha W S, Nam Bo-Hye, Lee Jehee
Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Department of Zoology, University of Jaffna, Jaffna 40000, Sri Lanka.
Fish Shellfish Immunol. 2017 Aug;67:66-77. doi: 10.1016/j.fsi.2017.05.037. Epub 2017 May 19.
Black rockfish (Sebastes schlegelii), an important aquaculture species in Korea, has been affected by bacterial diseases leading to a drastic decline in production. Goose-type lysozyme (LysG) is a key enzyme of the innate immune system to eradicate bacterial infections. In this study, two isoforms of LysG from black rockfish, designated as RfLysG1 and RfLysG2, have been identified and characterized at the molecular, transcriptional, and functional levels. The deduced amino acid sequences had the LysG family characteristics and exhibited conserved properties, including active residues and domains. The cDNA sequences of RfLysG1 and RfLysG2 were 1514 bp and 900 bp in length, respectively. The 567-bp open reading frame (ORF) of RfLysG1 encoded a protein of 188 amino acids with molecular mass 20.11 kDa, and the 600-bp ORF of RfLysG2 encoded a polypeptide with 199 amino acids and molecular mass of 22.19 kDa. Homology studies indicated that RfLysG1 showed the highest identity (84.6%) with LysG-B of Oplegnathus fasciatus, while RfLysG2 showed the highest identity (74.4%) with LysG of Siniperca chuatsi. Both sequences possessed a soluble lytic trans-glycosylase domain. Both lacked signal peptide and they were not identified as proteins secreted by non-classical pathway by the SecretomeP server. Transcriptional analysis of the two genes showed constitutive expression, where both genes were highly expressed in blood under normal physiological conditions. In response to the immune challenges lipopolysaccharide (LPS), Streptococcus iniae, and poly I:C injection, the expression of RfLysG1 and RfLysG2 was significantly upregulated in blood and spleen tissues in a time-dependent manner. Turbidimetric assays indicated that both recombinant proteins tagged with maltose-binding protein (MBP) were reactive against several Gram-positive and Gram-negative bacteria, but MBP was inactive. Optimum temperatures for the recombinant RfLysG1 and RfLysG2 were 40 °C and 50 °C, respectively, and both were highly active at pH 3.0. The results provide evidence for the vital immunological role and bacteriolytic potential of RfLysG1 and RfLysG2.
黑鲪（Sebastes schlegelii）是韩国一种重要的水产养殖品种，受到细菌性疾病的影响，导致产量急剧下降。鹅型溶菌酶（LysG）是先天性免疫系统中根除细菌感染的关键酶。在本研究中，从黑鲪中鉴定出两种LysG同工型，分别命名为RfLysG1和RfLysG2，并在分子、转录和功能水平上对其进行了表征。推导的氨基酸序列具有LysG家族特征，并表现出保守特性，包括活性残基和结构域。RfLysG1和RfLysG2的cDNA序列长度分别为1514 bp和900 bp。RfLysG1的567 bp开放阅读框（ORF）编码一个188个氨基酸的蛋白质，分子量为20.11 kDa，RfLysG2的600 bp ORF编码一个199个氨基酸的多肽，分子量为22.19 kDa。同源性研究表明，RfLysG1与条石鲷的LysG-B同一性最高（84. ~ 6%），而RfLysG2与鳜鱼的LysG同一性最高（74.~ 4%）。两个序列都具有可溶性溶菌转糖基酶结构域。两者都缺乏信号肽，并且通过SecretomeP服务器未鉴定为非经典途径分泌的蛋白质。对这两个基因的转录分析表明它们组成性表达，在正常生理条件下，两个基因在血液中均高表达。响应于免疫刺激脂多糖（LPS）、海豚链球菌和聚肌苷酸:聚胞苷酸注射，RfLysG1和RfLysG2在血液和脾脏组织中的表达以时间依赖性方式显著上调。比浊法测定表明，两种麦芽糖结合蛋白（MBP）标签的重组蛋白对几种革兰氏阳性和革兰氏阴性细菌均有反应，但MBP无活性。重组RfLysG1和RfLysG2的最适温度分别为40℃和50℃，两者在pH 3.0时均具有高活性。结果为RfLysG1和RfLysG2的重要免疫作用和溶菌潜力提供了证据。
