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引用本文:黄萌萌,雷宇桐,王高杨,康婷,许学伟.三疣梭子蟹(Portunus trituberculatus)含新型关键识别基序DPY/WTD单结构域C型凝集素PtCTL-6的免疫功能研究.海洋与湖沼,2019,50(6):1302-1308.
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三疣梭子蟹(Portunus trituberculatus)含新型关键识别基序DPY/WTD单结构域C型凝集素PtCTL-6的免疫功能研究
黄萌萌1,2, 雷宇桐2, 王高杨2, 康婷2, 许学伟1
1.自然资源部第二海洋研究所 国家海洋局海洋生态系统与生物地球化学重点实验室 杭州 310012;2.浙江理工大学 生命科学与医药学院 杭州 310018
摘要:
C型凝集素是一类依赖于钙离子的糖识别蛋白超家族,该家族蛋白在海洋无脊椎动物先天免疫系统中的非己识别和对病原微生物的清除过程中起重要作用。本文以三疣梭子蟹(Portunus trituberculatus)含单个糖识别域(CRD)的C型凝集素PtCTL-6为研究对象展开研究。研究表明PtCTL-6的开放阅读框(ORF)编码一条由信号肽、LDLa和CRD组成的多肽链。该C型凝集素的识别关键基序为DPY/WTD(Asp-Pro-Tyr/Trp-Thr-Asp),未见于前人研究。病原相关分子模式(PAMPs)结合活性检测结果表明:PtCTL-6重组蛋白(rPtCTL-6)能结合脂多糖(LPS)、肽聚糖(PGN)和β-葡聚糖(β-glucan),并能结合7种微生物,包括革兰氏阴性菌、革兰氏阳性菌和真菌。凝菌活性实验证明,PtCTL-6能选择性地凝集真菌毕赤酵母(Pichia pastoris)与酿酒酵母(Saccharomyces cerevisiae)。以上结果证实PtCTL-6能作为模式识别受体参与机体对微生物的识别,并且该分子具备较广泛的免疫识别谱。最后,本文初步探索了该分子对"非己"物质的识别机制,发现PtCTL-6通过特异性的结合D-甘露糖和D-岩藻糖对"非己"物质进行识别,是一种具备新型糖结合模式的C型凝集素。
关键词:  C型凝集素  三疣梭子蟹  先天免疫  海洋无脊椎动物  非己识别
DOI:10.11693/hyhz20190700131
分类号:Q789
基金项目:国家自然科学基金项目,31702375号;浙江省自然科学基金,LQ17D060007号;自然资源部第二海洋研究所基本科研业务费,JB1701号;中国博士后科学基金,2017M620234号。
CHARACTERIZATION OF SINGLE CRD CONTAINING C-TYPE LECTIN WITH NOVEL MOTIF DPY/WTD FROM PORTUNUS TRITUBERCULATUS
HUANG Meng-Meng1,2, LEI Yu-Tong2, WANG Gao-Yang2, KANG Ting2, XU Xue-Wei1
1.Key Laboratory of Marine Ecosystem and Biogeochemistry, State Oceanic Administration&Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou 310012, China;2.College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China
Abstract:
C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including non-self-recognition and pathogen elimination. In this study, a single-CRD containing C-type lectin was identified from swimming crab Portunus trituberculatus (designated as PtCTL-6). The open reading frame (ORF) of PtCTL-6 encoded polypeptides of 326 amino acids with a signal peptide, an LDLa domain, and a single carbohydrate-recognition domain (CRD). The key motif determining carbohydrate binding specificity in PtCTL-6 was DPY/WTD (Asp -Pro-Tyr/Trp-Thr-Asp). DPY/WTD is a motif identified for the first time. The binding of different PAMPs of the recombinant proteins PtCTL-6 (rPtCTL-6) has been observed in our experiments. The rPtCTL-6 could bind three pathogen-associated molecular patterns (PAMPs) in relatively high affinity, including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN). Moreover, rPtCTL-6 could bind seven kinds of microbes in microbe binding assay. In addition, rPtCTL-6 exhibited agglutination activity against fungi in agglutination assay. All these results illustrate that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) in a broad non-self-recognition spectrum involved in the immune defense against invaders. In addition, the results of carbohydrate binding specificity show that PtCTL-2 with novel key motif had broad carbohydrate binding specificity.
Key words:  C-type lectin  Portunus trituberculatus  innate immunity  marine invertebrates  non-self-recognition
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