Abstract
BackgroundThe emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate.MethodsNative PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized.ResultsPfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4–9, optimal salt concentration of 100–200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner.ConclusionPfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.
Highlights
The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets
Partial purification of native PfMAG Native PfMAG activity was monitored according to P. falciparum developmental stages
Crude extract of P. falciparum trophozoite and schizont stages from synchronized culture were subjected to purification of PfMAG employing sequential anion exchange, cation exchange and heparin affinity chromatography
Summary
The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum causes most severity in terms of clinical pathology and complication in treatment as it readily develops resistance to all existing anti-malarial agents, including most recently the artemisinins [3, 4], highlighting the urgent need for identification of new parasite targets and development of safe and effective novel drugs targeting them. The high A–T content of the malaria parasite genome implies the potential of these regions being modified (alkylated), thereby the need of a parasite repair enzyme. MAG orthologues are present in Escherichia coli, Saccharomyces cerevisiae, rodents, humans, and plants [12, 13] It is known as N-methylpurine DNA glycosylase (MPG) due to its versatility in accommodating a variety of substrates in the active site [14]. A gene encoding PfMAG was found located on chromosome 14 of chloroquine- and pyrimethamine-sensitive P. falciparum 3D7 strain comprising of 1506 nucleotides coding 501 amino acids (PlasmoDB: PF3D7_1467100)
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