Lack of Abundance of Cytoplasmic Cyclosporin A-binding Protein Renders Free-living Leishmania donovaniResistant to Cyclosporin A

Madhuri Dutta,Praveen Delhi,Krishna M Sinha,Rahul Banerjee,Alok K Datta

doi:10.1074/jbc.m009379200

Abstract

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 21-amino acid hydrophobic NH(2)-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.

Highlights

Studies on mechanisms of drug action and drug resistance have often led to the discovery of new biological phenomena
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
Primary structure analysis of LdCyP and its modeling revealed that the entire set of 13 residues in human CyPA known to interact with CsA are sequentially and conformationally conserved in LdCyP, making it quite capable of equivalent interactions with the ligand molecule (Figs. 2 and 3) [1]

Summary

Introduction

Studies on mechanisms of drug action and drug resistance have often led to the discovery of new biological phenomena. The CyP1⁄7CsA complex formed in the cytoplasm binds to calcineurin and inhibits its serine-threonine protein phosphatase activity, which leads to immunosuppression [2] The formation of such a binary complex between CyP and CsA is the first crucial step for CsA action. Leishmania donovani, a dimorphic parasitic protozoan, is the causative agent of kala azar in humans This protozoan exists as a flagellated promastigote (extracellular form) in the sand fly vector and is transformed into amastigote (intracellular form) in the mammalian macrophages. The cause of resistance in some of these parasites has been attributed to the absence of the concensus CsA binding motif in their CyPs. Despite normal uptake of the drug, the in vivo and in vitro effects of CsA on various Leishmania parasites are conflicting. CsA distinctly exacerbated intracellular L. donovani (amastigote) parasite load in murine cells, suggesting possible differences in the status of CyPs in these two species of Leishmania [13]

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Conclusion