Zhang Huan, Song Linsheng, Li Chenghua, Zhao Jianmin, Wang Hao, Qiu Limei, Ni Duojiao, Zhang Ying
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Fish Shellfish Immunol. 2008 Sep;25(3):281-9. doi: 10.1016/j.fsi.2008.06.003. Epub 2008 Jun 18.
The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a 5'-terminal untranslated region (UTR) of 62 bp and a 3' UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21(DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LPS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk.
含C1q结构域（C1qDC）的蛋白质是一类蛋白质家族，其特征在于在其C末端具有球状C1q（gC1q）结构域。它们参与脊椎动物的各种过程，并被认为是无脊椎动物先天免疫中的重要模式识别受体。在本研究中，通过表达序列标签（EST）和cDNA末端快速扩增（RACE）方法，从栉孔扇贝（Chlamys farreri）中鉴定出含C1q结构域蛋白质家族的一个新成员（命名为CfC1qDC）。CfC1qDC的全长cDNA为777 bp，由62 bp的5'末端非翻译区（UTR）和178 bp的3'UTR组成，带有聚腺苷酸化信号序列AATAAA和聚（A）尾。CfC1qDC cDNA编码一个178个氨基酸的多肽，包括一个信号肽和一个158个氨基酸的C1q结构域，理论等电点为5.19，预测分子量为17.2 kDa。CfC1qDC中的C1q结构域与软体动物唾液酸结合凝集素和高等脊椎动物的C1qDC蛋白中的结构域具有同源性。C1q结构域典型的10条β链果冻卷折叠拓扑结构以及疏水核心有效堆积所必需的残基在CfC1qDC中保存良好。通过荧光定量实时PCR，主要在肾脏、外套膜、闭壳肌和鳃中检测到CfC1qDC的mRNA转录本，在血细胞中也可少量检测到。在细菌攻击实验中，栉孔扇贝受到鳗弧菌（Listonella anguillarum）攻击后，CfC1qDC的相对表达水平显著上调，注射后6小时，mRNA表达达到最高水平，比对照栉孔扇贝高4.55倍。同样，脂多糖（LPS）刺激的血细胞混合原代培养物中CfC1qDC mRNA的表达上调，并在刺激后6小时达到最高水平，然后逐渐回落至原始水平。为了研究其功能，编码CfC1qDC成熟肽的cDNA片段在大肠杆菌BL21(DE3)中进行重组表达。重组CfC1qDC蛋白对大肠杆菌的LPS显示出显著强的结合活性，尽管未观察到对革兰氏阴性菌大肠杆菌JM109、鳗弧菌和革兰氏阳性菌藤黄微球菌有明显的抗菌或凝集活性。这些结果表明，CfC1qDC绝对是C1qDC蛋白家族的一个新成员，可能作为软体动物中的模式识别分子参与对入侵微生物的识别。
