Jia Airong, Zhang Xiao-Hua
Department of Marine Biology, Ocean University of China, Qingdao 266003, China.
Fish Shellfish Immunol. 2009 Apr;26(4):606-13. doi: 10.1016/j.fsi.2008.09.011. Epub 2008 Oct 4.
Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. The EST sequence of turbot (Scophthalmus maximus L.) cathepsin D was obtained from a subtractive cDNA library. In the present study, 5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 91 bp 5'-UTR, a 1191 bp open reading frame encoding 396 amino acids, and a 329 bp 3'-UTR. The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramundi (Lates calcarifer B., 89% aa similarity). Quantitative real-time PCR (q PCR) demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with Vibrio harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. This result was different from the expression of MHCII of which the expression lever was increased upon challenge. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.
组织蛋白酶D是一种溶酶体内切蛋白水解天冬氨酸蛋白酶，在巨噬细胞的内体中也有发现。它被认为在动物的发育和生理过程中发挥关键作用。大菱鲆（Scophthalmus maximus L.）组织蛋白酶D的EST序列是从一个消减cDNA文库中获得的。在本研究中，进行了5'-RACE和3'-RACE以获得大菱鲆组织蛋白酶D的完整cDNA序列，该序列包含一个91 bp的5'-UTR、一个1191 bp的开放阅读框，编码396个氨基酸，以及一个329 bp的3'-UTR。推导的组织蛋白酶D氨基酸序列由一个18个氨基酸的信号肽、一个延伸43个氨基酸的前导肽和一个335个氨基酸的成熟肽组成。BLAST分析表明，大菱鲆组织蛋白酶D与其他已知的组织蛋白酶D具有高度相似性，并且与尖吻鲈（Lates calcarifer B.）的组织蛋白酶D具有显著同源性（氨基酸相似性为89%）。实时定量PCR（qPCR）表明，大菱鲆组织蛋白酶D在肝脏中的表达水平最高。在用哈维氏弧菌攻击大菱鲆后，在8小时时检测到肝脏、脾脏和头肾中组织蛋白酶D的表达水平最低。这一结果与MHCII的表达不同，MHCII的表达水平在受到攻击后会升高。8小时后，肝脏和头肾中组织蛋白酶D的表达水平逐渐升高，并在24小时后超过背景水平。在脾脏中，8小时后表达水平增强，并在12小时后保持在高于原始水平的状态。结果表明，组织蛋白酶D可能处理抗原以呈递给免疫系统，并在注射后12小时内与凋亡途径具有协同作用。
