Lack of abundance of cytoplasmic cyclosporin A-binding protein renders free-living Leishmania donovani resistant to cyclosporin A
Lack of abundance of cytoplasmic cyclosporin A-binding protein renders free-living Leishmania donovani resistant to cyclosporin A
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DOI：
10.1074/jbc.m009379200
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发表时间：
2001-06-01
影响因子：
4.8
通讯作者：
Datta, AK
中科院分区：
文献类型：
--
作者：
Dutta, M;Delhi, P;Datta, AK
环孢菌素A（CsA）对细胞的大部分作用是由细胞质中形成的亲环蛋白A（CyPA）-CsA复合物抑制钙调神经磷酸酶（CN）的磷酸酶活性引起的。虽然CsA抑制大量寄生虫的增殖，但并非所有寄生虫都对其敏感。结构改变的CyPA与CsA亲和力较低的存在被认为是耐药性的原因。我们在这里报告了杜氏利什曼原虫（一种对 CsA 具有天然抗性的锥虫寄生虫）的高亲和力 CsA 结合蛋白 (LdCyP) 的鉴定和克隆，翻译后的 LdCyP 由 187 个氨基酸组成，具有可裂解的 α-氨基酸疏水性 NH2 末端延伸。模型研究证实，LdCyP 中负责与 CsA 相互作用的人 CyP 的所有残基在构象上顺序保守，纯化的重组蛋白显示出与人 CyP 相当的生化参数。逆转录聚合酶链反应分析证实 LdCyP 大量表达。免疫印迹实验和直接 CsA 结合研究表明，位于亚细胞器中的 LdCyP 构成了杜氏乳杆菌中存在的 CsA 结合活性的大部分，而胞质中的结合活性水平明显较低，CsA 选择性地促进培养基中 LdCyP 的分泌。基于这些结果，得出结论，杜氏乳杆菌对 CsA 不敏感可能是由于缺乏寄生虫细胞质中的 CsA 结合活性。我们认为，位于分泌途径中的 LdCyP 可能通过与膜蛋白结合而充当伴侣，而不是充当 CN 抑制的介质。
The majority of the effects of cyclosporin A (CsA) on cells is caused by the inhibition of phosphatase activity of calcineurin (CN) by the cyclophilin A (CyPA)-CsA complex formed in the cytoplasm, Although CsA inhibits the proliferation of a large number of parasites, not all are susceptible, The presence of structurally altered CyPA with lower affinity for CsA had been suggested to be the cause of resistance. We report here the identification and cloning of a high affinity CsA-binding protein (LdCyP) from Leishmania donovani, a trypanosomatid parasite that is naturally resistant to CsA, The translated LdCyP consists of 187 amino acids with a cleavable al-amino acid hydrophobic NH2-terminal extension. Modeling studies confirmed that all the residues of human CyPs responsible for interaction with CsA are sequentially and conformationally conserved in LdCyP, The purified recombinant protein displayed biochemical parameters comparable to human CyPs. Reverse transcription-polymerase chain reaction analysis confirmed that LdCyP was abundantly expressed. Immunoblot experiments and direct CsA binding studies revealed that LdCyP located in the subcellular organelles constituted the bulk of the CsA binding activity present in L, donovani, whereas the level of binding activity in the cytosol was conspicuously low, CsA selectively facilitated the secretion of LdCyP in the culture medium, Based on these results, it is concluded that the insensitivity of L. donovani to CsA is probably due to the paucity of CsA binding activity in the cytoplasm of the parasite. We suggest that LdCyP, located in the secretory pathway, may function as a chaperone by binding to membrane proteins rather than as the mediator of CN inhibition.