Characterization of differentially regulated carboxypeptidase (metallopeptidase M32) protein in Miltefosine resistant Leishmania donovani parasites

Carboxypeptidase, a member of the metallopeptidase M32 family, catalyses the C-terminal hydrolysis of a variety of peptides and proteins in the presence of metal ions. To characterize Leishmania donovani carboxypeptidase (LdCP) in miltefosine (MIL) drug-resistant parasites. We performed the MTT assay and cell cycle analysis to confirm the MIL resistance of clinical isolates. LdCP gene was cloned and expressed in E. coli Artic strain. The purified LdCP protein was used for antibody generation and biochemical characterization. MTT assay and cell cycle analysis revealed that all three isolates exhibit MIL resistance. LdCP constitutively expressed in both promastigote and amastigote stages of parasites, and its activity increased 2–3 fold in MIL-resistant parasites. LdCP has high α-helical content at physiological pH and temperature. The protein is quite thermostable with a Tm of 63 °C and susceptible to chemical denaturation, with 50 % unfolding induced by 3.59 M urea or 0.31 M guanidine hydrochloride (GdmCl). LC-MS/MS study reveals that LdCP interacts with membrane-associated proteins that have ATP binding sites and involved in protein phosphorylation. To our knowledge, this is the first study to characterize the carboxypeptidase of L. donovani that appears to contribute to the development of MIL resistance parasites.