Lefebvre Christophe, Cocquerelle Claude, Vandenbulcke Franck, Hot David, Huot Ludovic, Lemoine Yves, Salzet Michel
Laboratoire de Neuroimmunologie des Annélides, UMR CNRS 8017, SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France.
Biochem J. 2004 Jun 15;380(Pt 3):617-25. doi: 10.1042/BJ20040478.
At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.
目前，除昆虫外，关于无脊椎动物类群（尤其是环节动物）先天免疫机制的信息很少。在本研究中，我们通过差异显示逆转录酶PCR和cDNA微阵列相结合的方法，对细菌攻击后水蛭Theromyzon tessulatum的免疫反应进行了转录组学研究。结果显示了一些已知和未知基因的相关调节。事实上，苏氨酸脱氨酶、苹果酸脱氢酶、胱抑素B、聚腺苷酸结合蛋白和α - 微管蛋白样基因在免疫刺激后上调。我们重点研究了胱抑素B（stefin B），它是脊椎动物免疫反应中半胱氨酸蛋白酶的抑制剂。我们克隆了全长cDNA，并将T. tessulatum基因命名为Tt - cysb。在Tt - cysb cDNA推导的氨基酸序列中鉴定出了胱抑素的主要结构特征；即N端的甘氨酸残基和对应于催化位点的Gln - Xaa - Val - Xaa - Gly（QXVXG）共有序列。此外，Tt - cysb是第一个在无脊椎动物中被鉴定的胱抑素B基因。我们通过原位杂交和免疫细胞化学确定Tt - cysb仅在大型体腔细胞中表达。此外，该分析证实Tt - cysb在细菌攻击后上调，并且表达增加仅发生在体腔细胞中。这些数据表明，水蛭的先天免疫反应涉及一种在蜕皮动物模型（如黑腹果蝇或秀丽隐杆线虫）中未发现的半胱氨酸蛋白酶抑制剂，因此强调了对不同无脊椎动物类群先天免疫机制信息的迫切需求。
