Qingdao University of Science and Technology, Qingdao 266042, China.
Fish Shellfish Immunol. 2010 Sep;29(3):521-9. doi: 10.1016/j.fsi.2010.05.015. Epub 2010 Jun 4.
Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes. In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Eriocheir sinensis (designated EsCystatin) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 5.48 and the predicted molecular weight of 13.39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin. Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin. But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystatin were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart. After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0.6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01) at 24 h. Afterwards, EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2.8-fold of that in blank (P < 0.01)). The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain. When the concentration of EsCystatin protein was of 300 microg mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms.
组织蛋白酶抑制剂是一种半胱氨酸蛋白酶可逆抑制剂超家族的蛋白，在一系列生理和免疫过程中发挥着重要作用。在这项研究中，通过表达序列标签（EST）分析和快速扩增 cDNA 末端（RACE）方法，从中华绒螯蟹（命名为 EsCystatin）中鉴定出组织蛋白酶抑制剂超家族的一个新成员。EsCystatin 的全长 cDNA 为 1486bp，包括 5'端非翻译区（UTR）92bp、3'UTR 1034bp 带有聚腺苷酸化信号序列 AATAAA 和聚 A 尾巴，以及开放阅读框（ORF）360bp 编码一个由 120 个氨基酸组成的多肽，理论等电点为 5.48，预测分子量为 13.39kDa。在 EsCystatin 的假定氨基酸序列中发现了一个信号 Cystatin 样结构域（Gly（25）到 Lys（112））。与其他组织蛋白酶抑制剂类似，保守的中央 Q（70）VVSG（74）基序位于 EsCystatin 的 Cystatin 样结构域中。但是 EsCystatin 缺乏信号肽和二硫键。EsCystatin 与无脊椎动物和高等脊椎动物的其他已知组织蛋白酶具有同源性，并在系统发育树中聚类为组织蛋白酶家族 1。EsCystatin 的 mRNA 转录本主要在血淋巴、鳃、肝胰腺、性腺和肌肉中表达，在心脏中也有微量表达。在鳗弧菌攻毒后，血淋巴中 EsCystatin 的相对表达水平在攻毒后 3 小时下调至 0.6 倍（P<0.05）。随后，它在 24 小时时上调至 3.0 倍（P<0.01）。之后，EsCystatin mRNA 转录本突然降至原始水平。在巴氏毕赤酵母 GS115 攻毒后，其血淋巴中的 mRNA 表达水平在 3 小时达到峰值（比空白组高 2.8 倍（P<0.01））。编码 EsCystatin 成熟肽的 cDNA 片段在大肠杆菌 Rosetta-gami（DE3）中重组表达。重组 EsCystatin 对木瓜蛋白酶表现出启动子抑制活性。当 EsCystatin 蛋白浓度为 300μg mL（-1）时，几乎 89%的木瓜蛋白酶活性可被抑制。这些结果共同表明，EsCystatin 是组织蛋白酶家族中的一个新成员，是木瓜蛋白酶的有效抑制剂，并参与了针对入侵微生物的免疫反应。
