MOE Key Laboratory of Aquatic Product Safety/State Key Laboratory for Biocontrol, School of Marine Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou, PR China.
Dev Comp Immunol. 2012 Sep;38(1):66-77. doi: 10.1016/j.dci.2012.04.005. Epub 2012 Apr 30.
The inositol-requiring enzyme-1 (IRE1)-X-box binding protein 1 (IRE1-XBP1) pathway is the key branch of the unfolded protein response (UPR). To investigate the role of the IRE1-XBP1 pathway in reducing environmental stress and increasing anti-viral immunity in Litopenaeus vannamei, homologues of IRE1 (designated as LvIRE1) and XBP1 (designated as LvXBP1) were identified and characterized. The full-length cDNA of LvIRE1 is 4908bp long, with an open reading frame (ORF) that encodies a putative 1174 amino acid protein. The full-length cDNA of LvXBP1 is 1746bp long. It contains two ORFs that encode putative 278 amino acid and 157 amino acid proteins, respectively. LvXBP1 mRNA has the predicted IRE1 splicing motifs CNG'CNGN located within the loop regions of two short hairpins. Sequencing of the splicing fragment induced by endoplasmic reticulum (ER)-stress showed a 3bp or 4bp frame shift from the predicted sites. The spliced form LvXBP1 (LvXBP1s) contained an ORF encodes a putative 463 amino acid protein. The reporter gene assays indicated that LvXBP1s activates the promoter of L. vannamei immunoglobulin heavy chain binding protein (LvBip), an important UPR effector. RT-PCR showed that LvXBP1 was spliced during the experiments. For heat shock treatment, the total LvXBP1 expression was increased and peaked at about 36h, whereas the percentages of the two isoforms were relatively stable. For the WSSV challenge, LvXBP1 was upregulated during the experiment and the percentage of the spliced form continuously declined after 18h of infection. Knock-down of LvXBP1 by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Furthermore, the expression profiles of LvIRE1 and LvXBP1 in the gills, hemocytes, intestines, and hepatopancreas of the WSSV-challenged shrimp were detected using real-time RT-PCR. Taken together, these results confirm that the IRE1-XBP1 pathway is important for L. vannamei environmental stress resistance, suggest that L. vannamei IRE1-XBP1 may activated by WSSV and be annexed to serve the virus.
肌醇需求酶 1（IRE1）-X 盒结合蛋白 1（IRE1-XBP1）途径是未折叠蛋白反应（UPR）的关键分支。为了研究 IRE1-XBP1 途径在降低凡纳滨对虾环境应激和增强抗病毒免疫中的作用，鉴定并表征了 IRE1（命名为 LvIRE1）和 XBP1（命名为 LvXBP1）的同源物。LvIRE1 的全长 cDNA 长 4908bp，含有一个开放阅读框（ORF），编码一个假定的 1174 个氨基酸蛋白。LvXBP1 的全长 cDNA 长 1746bp，包含两个 ORF，分别编码假定的 278 个氨基酸和 157 个氨基酸蛋白。LvXBP1 mRNA 在两个短发夹环区具有预测的 IRE1 剪接基序 CNG'CNGN。内质网（ER）应激诱导的剪接片段测序显示，从预测的位点发生了 3bp 或 4bp 的移码。剪接形式的 LvXBP1（LvXBP1s）包含一个 ORF，编码一个假定的 463 个氨基酸蛋白。报告基因分析表明，LvXBP1s 激活了凡纳滨对虾免疫球蛋白重链结合蛋白（LvBip）的启动子，这是一种重要的 UPR 效应物。RT-PCR 显示，LvXBP1 在实验过程中被剪接。对于热休克处理，总 LvXBP1 表达增加，并在约 36h 时达到峰值，而两个同工型的比例相对稳定。对于 WSSV 攻毒，LvXBP1 在实验过程中上调，感染后 18h 剪接形式的比例持续下降。通过 RNA 干扰敲低 LvXBP1 导致凡纳滨对虾在 WSSV 感染下的累积死亡率降低。此外，通过实时 RT-PCR 检测了 WSSV 攻毒虾的鳃、血细胞、肠和肝胰腺中 LvIRE1 和 LvXBP1 的表达谱。综上所述，这些结果证实了 IRE1-XBP1 途径对凡纳滨对虾环境应激抗性很重要，表明凡纳滨对虾 IRE1-XBP1 可能被 WSSV 激活并被附加以服务于病毒。
