State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Fish Shellfish Immunol. 2010 Sep;29(3):388-98. doi: 10.1016/j.fsi.2010.04.002. Epub 2010 May 6.
Granzyme (Gzm) is an important member of serine protease family, and key component in the specific and non-specific cell-mediated cytotoxicity. Partial GzmA/K cDNA sequence of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp GzmA/K was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp GzmA/K was 1053 bp, consisting of a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 214 bp, and an open reading frame of 774 bp. Amino acid sequence analysis indicated the existence of a signal peptide, eight consensus cysteine residues, one conserved IIGG motif and three conserved residues as central elements of the GzmA/K active site. Carp GzmA/K shared 36% and 39% amino acid identity to human GzmA and K, respectively, and was phylogenetically related to the granzyme A and K subgroups. Then, a genomic DNA, which covers the promoter region and entire coding region of carp GzmA/K, was obtained by PCR. In the 5.4 k-long genomic sequence, five exons and four introns were identified. Real-time RT-PCR analysis showed that carp GzmA/K transcript was predominantly detected in the immune-related tissues, and after SVCV infection, was up-regulated in most immune-related tissues in a time-dependent manner. Real-time RT-PCR results also showed that carp GzmA/K transcript was up-regulated in thymus tissue of GH transgenic carp. These results will help to understand the molecular characterization and the potential role of teleost GzmA/K, a cytotoxic cell granule-associated serine protease.
颗粒酶（Gzm）是丝氨酸蛋白酶家族的重要成员，是特异性和非特异性细胞介导的细胞毒性的关键组成部分。本研究通过抑制性消减杂交（SSH）方法从鲤鱼（Cyprinus carpio L.）胸腺 cDNA 文库中分离出部分 GzmA/K cDNA 序列，随后通过 3' RACE 和 5' RACE 分别获得了鲤鱼 GzmA/K 的全长 cDNA。鲤鱼 GzmA/K 的全长 cDNA 为 1053bp，包括 5'-非翻译区（UTR）65bp、3'-UTR214bp 和开放阅读框 774bp。氨基酸序列分析表明存在信号肽、8 个保守半胱氨酸残基、一个保守的 IIGG 基序和三个保守残基作为 GzmA/K 活性位点的中心元件。鲤鱼 GzmA/K 与人类 GzmA 和 K 的氨基酸序列分别具有 36%和 39%的同源性，与颗粒酶 A 和 K 亚群在系统进化上相关。然后，通过 PCR 获得了覆盖鲤鱼 GzmA/K 启动子区和整个编码区的基因组 DNA。在 5.4kb 的基因组序列中，鉴定出 5 个外显子和 4 个内含子。实时 RT-PCR 分析显示，鲤鱼 GzmA/K 转录本主要在免疫相关组织中检测到，在 SVCV 感染后，大多数免疫相关组织中的转录本呈时间依赖性上调。实时 RT-PCR 结果还显示，GH 转基因鲤鱼的胸腺组织中 GzmA/K 转录本上调。这些结果有助于了解鱼类 GzmA/K 作为细胞毒性细胞颗粒相关丝氨酸蛋白酶的分子特征和潜在作用。
