Lack of abundance of cytoplasmic cyclosporin A-binding protein renders free-living Leishmania donovani resistant to cyclosporin A

The majority of the effects of cyclosporin A (CsA) on cells is caused by th e inhibition of phosphatase activity of calcineurin (CN) by the cyclophilin A (CyPA)-CsA complex formed in the cytoplasm, Although CsA inhibits the pr oliferation of a large number of parasites, not all are susceptible, The pr esence of structurally altered CyPA with lower affinity for CsA had been su ggested to be the cause of resistance. We report here the identification an d cloning of a high affinity CsA-binding protein (LdCyP) from Leishmania do novani, a trypanosomatid parasite that is naturally resistant to CsA, The t ranslated 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 sequential ly and conformationally conserved in LdCyP, The purified recombinant protei n displayed biochemical parameters comparable to human CyPs. Reverse transc ription-polymerase chain reaction analysis confirmed that LdCyP was abundan tly expressed. Immunoblot experiments and direct CsA binding studies reveal ed that LdCyP located in the subcellular organelles constituted the bulk of the CsA binding activity present in L, donovani, whereas the level of bind ing activity in the cytosol was conspicuously low, CsA selectively facilita ted the secretion of LdCyP in the culture medium, Based on these results, i t 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. W e suggest that LdCyP, located in the secretory pathway, may function as a c haperone by binding to membrane proteins rather than as the mediator of CN inhibition.