Lu Junkai, Yu Zuoben, Mu Changkao, Li Ronghua, Song Weiwei, Wang Chunlin
Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, China.
Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, China.
Fish Shellfish Immunol. 2017 May;64:185-192. doi: 10.1016/j.fsi.2017.03.013. Epub 2017 Mar 10.
C-type lectins (CTLs) are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a novel C-type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus. The full-length cDNA of PtCTL1 was of 702 bp, containing a 5' untranslated region (UTR) of 91 bp, a 3' UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C-type lectin carbohydrate-recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram-positive bacterial strains and three Gram-negative bacterial strains. In addition, erythrocyte agglutination and LPS-binding activity were observed in a Ca-dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca-dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up-regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate.
C型凝集素（CTLs）是一类钙依赖性碳水化合物结合蛋白。在本研究中，从三疣梭子蟹中鉴定并表征了一种新型C型凝集素（命名为PtCTL1）。PtCTL1的全长cDNA为702 bp，包含一个91 bp的5'非翻译区（UTR）、一个带有poly（A）尾的110 bp的3'UTR以及一个501 bp的开放阅读框（ORF），编码一个由166个氨基酸组成的多肽，带有一个21个氨基酸的假定信号肽。在PtCTL1的氨基酸序列中鉴定出一个包含四个保守半胱氨酸的C型凝集素碳水化合物识别结构域（CRD）。将编码PtCTL1成熟肽的cDNA片段重组到pET-21a(+)中，C末端融合有框内六组氨酸标签，并在大肠杆菌Origami（DE3）中表达。重组PtCTL1（rPtCTL1）能够凝集所有测试细菌，包括三株革兰氏阳性菌和三株革兰氏阴性菌。此外，还观察到以钙依赖方式的红细胞凝集和LPS结合活性。EDTA抑制红细胞凝集，表明PtCTL1是钙依赖性的。PtCTL1的mRNA转录本主要在肝胰腺和血细胞组织中检测到，在溶藻弧菌攻击后，其在血细胞中的水平显著上调。这些结果表明，PtCTL1可能作为一种模式识别受体（PRR），保护三疣梭子蟹免受细菌感染。此外，这些发现也为进一步了解无脊椎动物的先天免疫提供了证据。
