RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade
RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade
复制标题
DOI：
10.1016/j.molbiopara.2007.10.009
复制
发表时间：
2008-02-01
影响因子：
1.5
通讯作者：
Brindley, Paul J.
中科院分区：
文献类型：
--
作者：
Morales, Maria E.;Rinaldi, Gabriel;Brindley, Paul J.
天冬氨酸蛋白酶组织蛋白酶D （AA族，A1家族）在血吸虫肠道中表达，在消化摄取的红细胞释放的血红蛋白中起顶端作用。在本报告中，采用RNA干扰方法研究了敲除血吸虫组织蛋白酶d的影响。将培养的曼氏血吸虫置于编码曼氏血吸虫组织蛋白酶d的cDNA特异性双链RNA （dsRNA）的方波电穿孔中，rnai介导的转录物水平降低导致表型变化，包括体外生长明显阻滞和天冬氨酸蛋白酶活性抑制。此外，在血吸虫肠道中积累的正常血红蛋白蛋白水解的终点副产物黑色素血红素在处理过的血吸虫肠道中并不明显。它们的肠道呈红色，而不是黑色，显然表明存在完整的而不是消化的宿主血红蛋白。当使用两种形式的dsRNA时，这些表型效应是明显的，一种是跨越整个目标转录物的长形式，或者是针对3'区域的短形式。脱靶效应在肠道定位的半胱氨酸蛋白酶组织蛋白酶B1的转录水平中不明显。最后，组织蛋白酶D可能是血吸虫哺乳-寄生阶段的必需酶，因为用dsRNA处理的血吸虫在转移到Balb/c小鼠后不能存活到成熟。这些和早期的研究结果表明，鉴于其在寄生虫营养中的重要功能，血吸虫组织蛋白酶D可以作为新型抗血吸虫干预措施的靶点。(c) 2007 Elsevier B.V.版权所有
The aspartic protease cathepsin D (Clan AA, Family A1) is expressed in the schistosome gut where it plays an apical role in the digestion of hemoglobin released from ingested erythrocytes. In this report, RNA interference approaches were employed to investigate the effects of knockdown of schistosome cathepsin D. Cultured schistosomules of Schistosoma mansoni were exposed by square wave electroporation to double stranded RNA (dsRNA) specific for cDNA encoding S. mansoni cathepsin D. RNAi-mediated reductions in transcript levels led to phenotypic changes including significant growth retardation in vitro and suppression of aspartic protease enzyme activity. In addition, black-pigmented heme, the end point by-product of normal hemoglobin proteolysis that accumulates in the schistosome gut, was not apparent within the guts of the treated schistosomules. Their guts appeared to be red in color, rather than black, apparently indicating the presence of intact rather than digested host hemoglobin. These phenotypic effects were apparent when either of two forms of dsRNA, a long form spanning the entire target transcript or a short form specific for the 3'-region was employed. Off-target effects were not apparent in transcript levels of the gut-localized cysteine protease cathepsin B1. Finally, cathepsin D may be an essential enzyme in the mammal-parasitic stages of schistosomes because schistosomules treated with dsRNA did not survive to maturity after transfer into Balb/c mice. These and earlier findings suggest that, given its essential function in parasite nutrition, schistosome cathepsin D could be developed as a target for novel anti-schistosomal interventions. (c) 2007 Elsevier B.V. All rights reserved.