Lv Changhuan, Zhang Dongling, Wang Zhiyong
Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen, 361021, China.
Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen, 361021, China.
Fish Shellfish Immunol. 2016 Mar;50:231-41. doi: 10.1016/j.fsi.2016.01.032. Epub 2016 Jan 30.
C-type lectins (CTLs) are generally recognized as a superfamily of Ca(2+)-dependent carbohydrate-binding proteins, which serve as pattern recognition receptors (PRRs) in innate immunity of vertebrates. In this study, the molecular characterization and immune roles of a novel CTL from Larimichthys crocea (designated as LcNTC) were investigated. LcNTC is a novel protein that shared 33%-49% homology with other teleosts CTLs. The full-length cDNA of LcNTC was composed of 859 bp with a 465 bp open reading frame encoding a putative protein of 154 residues. LcNTC contained a single CRD with four conserved disulfide-bonded cysteine residues (Cys(57)-Cys(148), Cys(126)-Cys(140)) and EPN/AND motifs instead of invariant EPN/WND motifs required for carbohydrate-binding specificity and constructing Ca(2+)-binding sites. LcNTC mRNA was detected in all examined tissues with the most abundant in the gill. After challenged with poly I:C and Vibrio parahaemolyticus, the temporal expression of LcNTC was significantly up-regulated in the liver, spleen and head-kidney. LcNTC transcripts were also induced in the gill, skin, spleen and head-kidney post-infection with Cryptocaryon irritans. The recombinant LcNTC (rLcNTC) purified from Escherichia coli BL21 (DE3) exhibited strong agglutination activity against erythrocytes from human, rabbit and large yellow croaker in a Ca(2+)-dependent manner, and the agglutination could be inhibited by D-Mannose, D-Glucose, D-Fructose, α-Lactose, D-Maltose and LPS. Positive microbial agglutination activities of rLcNTC were observed against all tested bacteria in the presence of Ca(2+), including Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus lysoleikticus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). These findings collectively indicated that LcNTC might be involved in the innate immunity of L. crocea as a PRR.
C型凝集素（CTLs）通常被认为是一类依赖钙离子的碳水化合物结合蛋白超家族，它们在脊椎动物的固有免疫中作为模式识别受体（PRRs）发挥作用。在本研究中，对大黄鱼中一种新型CTL（命名为LcNTC）的分子特征和免疫作用进行了研究。LcNTC是一种新型蛋白质，与其他硬骨鱼的CTLs具有33%-49%的同源性。LcNTC的全长cDNA由859个碱基对组成，其中有一个465个碱基对的开放阅读框，编码一个154个氨基酸残基的假定蛋白。LcNTC包含一个单一的CRD结构域，有四个保守的二硫键连接的半胱氨酸残基（Cys(57)-Cys(148)，Cys(126)-Cys(140)）以及EPN/AND基序，而不是碳水化合物结合特异性和构建钙离子结合位点所需的不变的EPN/WND基序。在所有检测的组织中都检测到了LcNTC mRNA，在鳃中含量最为丰富。用聚肌胞苷酸（poly I:C）和副溶血性弧菌攻击后，LcNTC在肝脏、脾脏和头肾中的时间表达显著上调。感染刺激隐核虫后，鳃、皮肤、脾脏和头肾中也诱导产生了LcNTC转录本。从大肠杆菌BL21(DE3)中纯化得到的重组LcNTC（rLcNTC）以钙离子依赖的方式对人、兔和大黄鱼的红细胞表现出强烈的凝集活性，并且凝集作用可被D-甘露糖、D-葡萄糖、D-果糖、α-乳糖、D-麦芽糖和脂多糖抑制。在有钙离子存在的情况下，观察到rLcNTC对所有测试细菌都有阳性的微生物凝集活性，包括革兰氏阳性菌（枯草芽孢杆菌、金黄色葡萄球菌和溶壁微球菌）和革兰氏阴性菌（大肠杆菌、副溶血性弧菌、溶藻弧菌和气单胞菌）。这些发现共同表明，LcNTC可能作为一种PRR参与大黄鱼的固有免疫。
