Xue Qinggang, Beguel Jean-Phillipe, Gauthier Julie, La Peyre Jerome
Zhejiang Key Laboratory of Aquatic Germplasm Resources and College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, Zhejiang 315100, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China; School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
Fish Shellfish Immunol. 2017 Mar;62:332-340. doi: 10.1016/j.fsi.2017.01.040. Epub 2017 Jan 31.
Protease inhibitors are an extremely diverse group of proteins that control the proteolytic activities of proteases and play a crucial role in biological processes including host defenses. The I84 family of protease inhibitors in the MEROPS database currently consists of cvSI-1 and cvSI-2, two novel serine protease inhibitors purified and characterized from the eastern oyster Crassostrea virginica plasma and believed to play a role in host defense and disease resistance. In the present study, a third member of I84 family, named cvSI-3, was identified from C. virginica by cDNA cloning and sequencing. The full cvSI-3 cDNA was composed of 342 bp including a 255 bp open reading frame (ORF) that encodes an 84-amino acid peptide. The mature cvSI-3 molecule was predicted to have 68 amino acid residues after removal of a 16-amino acid signal peptide, with a calculated molecular mass of 7724.5 Da and a theoretical isoelectric point (pI) of 6.28. CvSI-3 amino acid sequence shared 41% identity with cvSI-2 and 37% identity with cvSI-1, which included 12 conserved cysteines. Quantitative real-time PCR determined that cvSI-3 gene expressed primarily in oyster digestive glands. Real-time PCR also detected that cvSI-1, cvSI-2 and cvSI-3 expression levels in digestive glands varied significantly, with cvSI-2 showing the highest expression level and cvSI-3 the lowest. Additionally, a significant correlation was detected between cvSI-2 and cvSI-3 mRNAs levels. Searches into sequence databases using cvSI-1, cvSI-2 and cvSI-3 as queries retrieved ESTs suggesting the possible existence of at least 9 more I84 family members in eastern oysters and of I84 family protease inhibitors in various bivalve and gastropod species. Moreover, orthologs of all C. virginica I84 family members or potential member genes were found to be present in the C. gigas genome, and their distributions among species provided important information about the evolution of the I84 family of protease inhibitors. It appears that the I84 family of protease inhibitors is widely distributed and actively evolving in the Phylum Mollusca.
蛋白酶抑制剂是一类极其多样的蛋白质，它们控制蛋白酶的蛋白水解活性，并在包括宿主防御在内的生物过程中发挥关键作用。MEROPS数据库中的I84蛋白酶抑制剂家族目前由cvSI - 1和cvSI - 2组成，这是两种从美国东海岸牡蛎（Crassostrea virginica）血浆中纯化并鉴定的新型丝氨酸蛋白酶抑制剂，被认为在宿主防御和抗病性中发挥作用。在本研究中，通过cDNA克隆和测序从美国东海岸牡蛎中鉴定出I84家族的第三个成员，命名为cvSI - 3。完整的cvSI - 3 cDNA由342 bp组成，包括一个255 bp的开放阅读框（ORF），编码一个84个氨基酸的肽段。预测成熟的cvSI - 3分子在去除16个氨基酸的信号肽后有68个氨基酸残基，计算分子量为7724.5 Da，理论等电点（pI）为6.28。CvSI - 3氨基酸序列与cvSI - 2的一致性为41%，与cvSI - 1的一致性为37%，其中包括12个保守的半胱氨酸。定量实时PCR确定cvSI - 3基因主要在牡蛎消化腺中表达。实时PCR还检测到消化腺中cvSI - 1、cvSI - 2和cvSI - 3的表达水平差异显著，cvSI - 2表达水平最高，cvSI - 3最低。此外，还检测到cvSI - 2和cvSI - 3 mRNA水平之间存在显著相关性。以cvSI - 1、cvSI - 2和cvSI - 3为查询序列在序列数据库中搜索，检索到的ESTs表明美国东海岸牡蛎中可能至少还存在9个I84家族成员，以及各种双壳类和腹足类物种中存在I84家族蛋白酶抑制剂。此外，发现巨蛎（C. gigas）基因组中存在所有美国东海岸牡蛎I84家族成员或潜在成员基因的直系同源物，它们在物种间的分布为I84蛋白酶抑制剂家族的进化提供了重要信息。看来I84蛋白酶抑制剂家族在软体动物门中广泛分布且在积极进化。
