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

Krishan Kumar; Sneha Banerjee; Hemalatha Sanivarapu; Jalaja Veronica; Madhulika Namdeo; Anjali Anand; Shyam Sundar; Musti J Swamy; Radheshyam Maurya

doi:10.1016/j.ijbiomac.2024.138648

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

Int J Biol Macromol. 2024 Dec 12:289:138648. doi: 10.1016/j.ijbiomac.2024.138648. Online ahead of print.

Authors

Krishan Kumar¹, Sneha Banerjee², Hemalatha Sanivarapu³, Jalaja Veronica¹, Madhulika Namdeo¹, Anjali Anand¹, Shyam Sundar⁴, Musti J Swamy², Radheshyam Maurya⁵

Affiliations

¹ Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
² School of Chemistry, University of Hyderabad, Hyderabad 500046, India.
³ International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad 502324, India.
⁴ Department of Medicine, Institute of Medical Sciences (IMS), Banaras Hindu University, Varanasi 221005, India.
⁵ Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India. Electronic address: rmusl@uohyd.ac.in.

PMID: 39674486
DOI: 10.1016/j.ijbiomac.2024.138648

Abstract

Introduction: 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.

Objective: To characterize Leishmania donovani carboxypeptidase (LdCP) in miltefosine (MIL) drug-resistant parasites.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Carboxypeptidase (metallopeptidase M32); Circular dichroism, mass spectrometry; Miltefosine resistance, Leishmania donovani.