Key Laboratory of Healthy Mariculture for the East China Sea, Minsistry of Agriculture and Rural Affairs Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-Environment, Jimei University, Xiamen 361021, China.
Int J Mol Sci. 2023 Jul 16;24(14):11539. doi: 10.3390/ijms241411539.
Galectins are proteins that play a crucial role in the innate immune response against pathogenic microorganisms. Previous studies have suggested that Galectin-3 could be a candidate gene for antibacterial immunity in the large yellow croaker . In this study, we cloned the Galectin-3 gene in the large yellow croaker, and named it LcGal-3. The deduced amino acid sequence of LcGal-3 contains a carbohydrate recognition domain with two conserved β-galactoside binding motifs. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that LcGal-3 was expressed in all the organs/tissues that were tested, with the highest expression level in the gill. In kidney cell lines, LcGal-3 protein was distributed in both the cytoplasm and nucleus. Moreover, we found that the expression of LcGal-3 was significantly upregulated upon infection with , as demonstrated by qRT-PCR analyses. We also purified the LcGal-3 protein that was expressed in prokaryotes, and found that it has the ability to agglutinate large yellow croaker red blood cells in a Ca-independent manner. The agglutination activity of LcGal-3 was inhibited by lipopolysaccharides (LPS) in a concentration-dependent manner, as shown in the sugar inhibition test. Additionally, LcGal-3 exhibited agglutination and antibacterial activities against three Gram-negative bacteria, including , , and . Furthermore, we studied the agglutination mechanism of the LcGal-3 protein using blood coagulation tests with LcGal-3 deletion and point mutation proteins. Our results indicate that LcGal-3 protein plays a critical role in the innate immunity of the large yellow croaker, providing a basis for further studies on the immune mechanism and disease-resistant breeding in and other marine fish.
半乳糖凝集素是在先天免疫反应中对抗病原微生物的关键蛋白。先前的研究表明，半乳糖凝集素-3 可能是大黄鱼抗菌免疫的候选基因。在本研究中，我们克隆了大黄鱼的半乳糖凝集素-3 基因，并将其命名为 LcGal-3。LcGal-3 的推导氨基酸序列包含一个糖识别结构域，其中含有两个保守的 β-半乳糖苷结合基序。定量 RT-PCR 分析显示，LcGal-3 在所有检测的器官/组织中均有表达，在鳃中表达水平最高。在肾细胞系中，LcGal-3 蛋白分布在细胞质和细胞核中。此外，我们发现，LcGal-3 的表达在感染 后显著上调，这是通过 qRT-PCR 分析得出的。我们还纯化了在原核细胞中表达的 LcGal-3 蛋白，并发现它具有以 Ca2+非依赖方式凝集大黄鱼红细胞的能力。凝集活性在糖抑制试验中被 LPS 以浓度依赖的方式抑制。此外，LcGal-3 对三种革兰氏阴性菌（包括 、 、 ）表现出凝集和抗菌活性。此外，我们使用 LcGal-3 缺失和点突变蛋白进行凝血试验研究了 LcGal-3 蛋白的凝集机制。我们的结果表明，LcGal-3 蛋白在大黄鱼的先天免疫中发挥着关键作用，为进一步研究其免疫机制和抗病性育种提供了基础。
