Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

A R Escalona-Montaño,I Becker,L Gutiérrez-Kobeh,J N Gómez-Sandoval,D E Vélez-Ramírez,R Mondragón-Flores,N Cabrera,M M Aguirre-García,R Pérez-Montfort

doi:10.1017/pao.2017.14

Abstract

AbstractThe main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes ofLeishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinantLmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinantLmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in differentLeishmaniaspecies; theLmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

Highlights

The parasite Leishmania major is the causative agent of cutaneous leishmaniasis in the Old World
Sporozoites of Plasmodium yoelli and Plasmodium berguei, incubated with increasing concentrations of potassium, enhanced the infectivity of the parasites and the copy number of the transcripts for phosphatase 2C (PP2C) was increased, when compared with parasites not exposed to potassium in which the expression of the enzyme was kept at basal levels (Kumar et al 2007)
Toxoplasma gondii has a protein phosphatase PP2C (TgPP2C), which is secreted from the rhoptries, a group of secretory organelles involved in host cell invasion

Summary

Introduction

The parasite Leishmania major is the causative agent of cutaneous leishmaniasis in the Old World. Different PP2C from A. thaliana participate in various signalling pathways, such as the one regulated by abscisic acid and the signalling pathways activated by mitogen protein (Mitula et al 2015). In protozoan parasites such as Plasmodium falciparum, an unusual PP2C phosphatase (PfPP2C) was identified. It has two catalytic sites, one with serine and the other with threonine, with an identity between them of about 9% (Mamoun et al 1998).

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Conclusion