Hainan Key Laboratory of Tropical Aquatic Germplasm, Sanya Oceanographic Institution, Ocean University of China, Sanya, 572024, China; MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, 266003, China.
MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, 266003, China.
Fish Shellfish Immunol. 2024 Oct;153:109833. doi: 10.1016/j.fsi.2024.109833. Epub 2024 Aug 13.
C-type lectins (CTLs) are a kind of Ca-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.
C 型凝集素（CTLs）是一种 Ca2+依赖性免疫反应因子，参与病原体的识别和防御。本研究从硬壳蛤 Meretrix meretrix 中鉴定出一种新的 CTL（命名为 MmCTL4）。MmCTL4 cDNA 的全长为 608bp，编码一个假定的 19bp 信号肽和一个 131bp 的糖识别结构域（CRD）。重组 MmCTL4 蛋白（rMmCTL4）的三级结构为典型的长双环结构，具有三个保守的二硫键，并且 MmCTL4 中 Ca2+结合位点的基序为 QPN 和 WSD。SYBR Green 实时 PCR 分析表明，MmCTL4 在健康蛤的血细胞、肝胰腺和套膜中广泛表达。在灿烂弧菌刺激后，血细胞和肝胰腺中 MmCTL4 mRNA 的时间表达谱分别在 6 hpi 时增加了 7.8 倍，在 12 hpi 时增加了 3.9 倍。编码 MmCTL4 的 cDNA 片段被重组到 pET-32a（+）载体中，并转化到大肠杆菌 BL21（DE3）中。具有 Ca2+的 rMmCTL4 表现出明显的血凝活性，可凝集大肠杆菌、枯草芽孢杆菌和金黄色葡萄球菌，而仅微弱凝集副溶血弧菌和真菌巴斯德毕赤酵母。rMmCTL4 的凝集活性明显受到 D-甘露糖、D-木糖、D-乳糖、麦芽糖和脂多糖的抑制。这些结果表明，MmCTL4 作为一种典型的模式识别受体（PRRs），可以在蛤的先天免疫中保护宿主免受病原体的入侵。
