National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316022, PR China.
National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316022, PR China.
Fish Shellfish Immunol. 2017 Dec;71:411-422. doi: 10.1016/j.fsi.2017.09.067. Epub 2017 Sep 28.
Glutathione peroxidases, a vital family of antioxidant enzymes in oxybiotic organisms, are involved in anti-pathogen immune response. In this study, two complete selenium-dependent glutathione peroxidase 1 cDNAs (designated as LcGPx1a and LcGPx1b) were obtained from the large yellow croaker Larimichthys crocea by rapid amplification of cDNA ends. The full-length sequence of LcGPx1a was 917 bp with a 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 289 bp, and an open reading frame of 576 bp encoding 191 amino acid (aa) polypeptides. The cDNA of LcGPx1b was composed of 884 bp with a 5'-UTR of 59 bp, a 3'-UTR of 258 bp, and an open reading frame of 567 bp encoding 188 aa polypeptides. The conserved selenocysteine insertion sequence was detected in the 3'-UTR of both isoforms, which can classify types I and II. Protein sequence analysis revealed that both isoforms included a selenocysteine encoded by an opal codon (TGA) and formed the functioning tetrad site with glutamine, tryptophan, and asparagine. Three conservative motifs, including one active site motif ("GKVVLIENVASLUGTT") and two signature site motifs ("LVILGVPCNQFGHQENC" and "V(A/S)WNFEKFLI"), were conserved both in sequence and location. Multiple alignments revealed that they exhibited a high level of identities with GPx1 from other organisms, especially in the abovementioned conserved amino acid sequence motifs. Tissue expression analysis indicated that LcGPx1a and LcGPx1b had a wide distribution in nine tissues with various abundances. The transcript level of LcGPx1a was not significantly different among the nine tissues, whereas that of LcGPx1b was higher in the kidney and head kidney than in the other tissues. After Vibrio parahaemolyticus stimulation, the expression levels of LcGPx1a and LcGPx1b were unanimously altered in the liver, spleen, kidney, and head kidney but with different magnitudes and response time. LcGPx1a and LcGPx1b showed distinct expression trends in the liver, where LcGPx1b was induced and LcGPx1a was depressed in response to pathogen infection. These results indicate that LcGPx1a and LcGPx1b display functional diversities and play crucial roles in mediating the immune response of fish.
谷胱甘肽过氧化物酶是需氧生物中抗氧化酶的重要家族，参与抗病原体免疫反应。本研究通过快速扩增 cDNA 末端，从大黄鱼（Larimichthys crocea）中获得了两个完整的硒依赖谷胱甘肽过氧化物酶 1 cDNA（分别命名为 LcGPx1a 和 LcGPx1b）。LcGPx1a 的全长序列为 917 bp，包括 5'-UTR 为 52 bp，3'-UTR 为 289 bp，开放阅读框为 576 bp，编码 191 个氨基酸（aa）多肽。LcGPx1b 的 cDNA 由 884 bp 组成，包括 5'-UTR 为 59 bp，3'-UTR 为 258 bp，开放阅读框为 567 bp，编码 188 个 aa 多肽。两个同工型的 3'-UTR 都检测到了保守的硒代半胱氨酸插入序列，可以将其归类为 I 型和 II 型。蛋白序列分析表明，两个同工型都包含一个由终止密码子（TGA）编码的硒代半胱氨酸，并与谷氨酰胺、色氨酸和天冬酰胺形成功能四联体位点。三个保守基序，包括一个活性位点基序（“GKVVLIENVASLUGTT”）和两个特征位点基序（“LVILGVPCNQFGHQENC”和“V（A/S）WNFEKFLI”），在序列和位置上都保守。多重比对表明，它们与其他生物的 GPx1 具有高度的同一性，特别是在上述保守的氨基酸序列基序中。组织表达分析表明，LcGPx1a 和 LcGPx1b 在九个组织中广泛分布，丰度各不相同。LcGPx1a 在九个组织中的转录水平没有显著差异，而 LcGPx1b 在肾脏和头肾中的转录水平高于其他组织。在副溶血弧菌刺激后，LcGPx1a 和 LcGPx1b 的表达水平在肝脏、脾脏、肾脏和头肾中均发生一致改变，但幅度和反应时间不同。LcGPx1a 和 LcGPx1b 在肝脏中的表达趋势明显不同，其中 LcGPx1b 被诱导，而 LcGPx1a 则在病原体感染时被抑制。这些结果表明，LcGPx1a 和 LcGPx1b 表现出功能多样性，在介导鱼类免疫反应中发挥着重要作用。
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