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 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.
Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Rd., Kolkata 700 032, India.
M
M
Dutta
P
P
Delhi
K M
KM
Sinha
R
R
Banerjee
A K
AK
Datta
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021983:Cyclophilins; D016572:Cyclosporine / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D006801:Humans; D015151:Immunoblotting; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007893:Leishmania donovani / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D015800:Protozoan Proteins; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
D002352:Carrier Proteins; D007166:Immunosuppressive Agents; D015800:Protozoan Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C431149:cyclosporin A-binding protein, Leishmania donovani; D016572:Cyclosporine; D021983:Cyclophilins