Selvapandiyan A, Duncan R, Debrabant A, Bertholet S, Sreenivas G, Negi N S, Salotra P, Nakhasi H L
Laboratory of Bacterial, Parasitic, and Unconventional Agents, Division of Emerging and Transfusion Transmitted Disease, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
J Biol Chem. 2001 Nov 16;276(46):43253-61. doi: 10.1074/jbc.M106806200. Epub 2001 Sep 5.
Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.
杜氏利什曼原虫是一种原生动物寄生虫，可导致人类患内脏疾病。为了鉴定控制其生长的基因，我们首次在动质体目生物中从杜氏利什曼原虫中分离出一个编码中心蛋白的基因。中心蛋白是一种钙结合细胞骨架蛋白，对中心体复制或分离至关重要。蛋白质序列相似性和免疫反应性证实，利什曼原虫中心蛋白是人类中心蛋白2的同源物。免疫荧光分析将该蛋白定位在基体中。钙结合分析表明，其C端钙结合结构域结合的钙比N端结构域多16倍。用乙二醇双四乙酸（EGTA）处理后中心蛋白的电泳迁移率发生改变，而在缺乏钙结合位点的突变体中这种迁移率改变消失，这表明钙与这些区域的结合可能在蛋白质构象中起作用。在培养物中，寄生虫指数生长期间中心蛋白mRNA和蛋白质的水平较高，而在稳定期则降至低水平。在寄生虫中表达N端缺失的中心蛋白会显著降低其生长速率，并且发现与对照细胞相比，更多的细胞停滞在G2/M期。这些研究表明，中心蛋白可能在利什曼原虫的生长中发挥功能作用。
