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Roles of P-glycoprotein genes in drug resistance and intracellular parasitism in Leishmania

P-糖蛋白基因在利什曼原虫耐药和细胞内寄生中的作用

基本信息

批准号：
10670241
负责人：
KATAKURA Ken
金额：
$ 1.98万
依托单位：
Gunma University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

We isolated and characterized two different P-glycoprotein genes in a parasitic protozoan, Leishmania amazonensis. The LaMDR1 gene encodes 1341 amino acids for a protein consisting of two similar halves, each containing one ATP-binding and six transmembrane domains. Transfection and over-expression of the LaMDR1 gene in wild-type promastigotes of L. amazonensis conferred resistance to vinblastine, doxorubicin and actinomycin D, indicating that the LaMDR1 gene is a homologue of human multidrung resistance MDR1 gene. The LaMDR2 gene encodes 1267 amino acids, revealing only 47% amino acid identity to the LaMDR1 protein. Transfectants with the LaMDR2 gene showed resistance to 5-fluorouracil (5-FU), a fluoropyrimidine used for cancer chemotherapy, but not to other anti-cancer drugs described above. A slightly higher intracellular accumulation of [ィイD13ィエD1H] vinblastine was observed in the LaMDR1-transfected cells compared with control transfectant cells. In transfectants with the LaMDR2 gene, an increased accumulation of [ィイD13ィエD1H]5-FU was measured in the first 30 min after drug exposure, but the drug accumulation was gradually decreased and reached to 20-30% of the control level at 120 min after the exposure. This 5-FU accumulation kinetics in transfectants with the LaMDR2 gene suggests that the LaMDR2 protein may locate on the membrane of cytoplasmic vesicles and transport 5-FU inside the vesicles during the initial phase, and then drugs were secreted by exocytosis during the later phase. These results revealed that the LaMDR2 gene is a new member of the ATP-binding cassette (ABC) family of protein with the P-glycoprotein structure and play a novel role in survival of the parasites in the host cells. Subcellular localization of the LaMDR2 protein using anti-LaMDR2 antibodies is under study. Functional analysis of the LaMDR2 gene by gene-disruption technology is also in process.

我们在寄生原生动物亚马逊利什曼原虫中分离并鉴定了两种不同的 P-糖蛋白基因。 LaMDR1 基因编码 1341 个氨基酸的蛋白质，由两个相似的半部分组成，每个半部分包含 1 个 ATP 结合域和 6 个跨膜结构域。 LaMDR1基因在亚马逊野生型前鞭毛体中的转染和过表达赋予了对长春花碱、阿霉素和放线菌素D的抗性，表明LaMDR1基因是人类多药耐药性MDR1基因的同源物。 LaMDR2 基因编码 1267 个氨基酸，与 LaMDR1 蛋白仅具有 47% 的氨基酸同一性。带有 LaMDR2 基因的转染子对 5-氟尿嘧啶 (5-FU)（一种用于癌症化疗的氟嘧啶）表现出耐药性，但对上述其他抗癌药物没有耐药性。与对照转染细胞相比，在 LaMDR1 转染细胞中观察到[ィイD13ィエD1H]长春花碱的细胞内积累略高。在LaMDR2基因的转染子中，在药物暴露后的前30分钟内测量到[ィイD13ィエD1H]5-FU的积累增加，但药物积累逐渐减少，并在暴露后120分钟达到对照水平的20-30%。 LaMDR2基因转染子中5-FU积累动力学表明，LaMDR2蛋白可能位于细胞质囊泡膜上，并在初始阶段将5-FU转运到囊泡内，然后在后期通过胞吐作用分泌药物。这些结果表明，LaMDR2基因是具有P-糖蛋白结构的ATP结合盒（ABC）蛋白家族的新成员，在宿主细胞中寄生虫的生存中发挥着新的作用。使用抗 LaMDR2 抗体对 LaMDR2 蛋白进行亚细胞定位正在研究中。通过基因破坏技术对 LaMDR2 基因进行功能分析也在进行中。