Molecular Characterization of Plasmodium Falciparum DNA-3-Methyladenine Glycosylase Nattapon Pinthong Mahidol University Faculty of Tropical Medicine Paviga Limudomporn Kasetsart University Faculty of Science Jitlada Vasuvat Mahidol University Faculty of Tropical Medicine Poom Adisakwattana Mahidol University Faculty of Tropical Medicine Pongruj Rattaprasert Mahidol University Faculty of Tropical Medicine Porntip Chavalitshewinkoon-Petmitr ( [email protected] ) Faculty of Tropical Medicine, Mahidol University Research Keywords: Plasmodium falciparum, DNA repair, DNA-3-methyladenine glycosylase, Malaria, glycosylase Posted Date: May 28th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-30646/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on August 6th, 2020. See the published version at https://doi.org/10.1186/s12936-020-03355-w. Page 1/21 Abstract Background The emergence of artemisinin-resistant malaria parasite highlights the need for novel drugs and their targets. Alkylation in purine bases can hinder DNA replication if remained unsolved would eventually resulting in cell death. DNA-3-methyladenine glycosylase is an enzyme responsible for the repair of those alkylated lesions. Based on the AT-rich genome of Plasmodium falciparum and unexplored Plasmodium falciparum DNA-3-methyladenine glycosylase (PfMAG), therefore PfMAG should be characterized for its potential candidate for antimalarial drug development. Methods Bioinformatics analysis of PfMAG was performed. The native PfMAG from crude extract of chloroquine and pyrimethamine resistant Plasmodium falciparum strain K1 was partially puried using three columns. The existence of PfMAG activity leads to the cloning and expression of PfMAG for further characterization. The PfMAG was amplied, cloned to expression vector (pBAD202/D-TOPO), and expressed in Escherichia coli. The molecular weight of recombinant PfMAG was analyzed by SDS-PAGE and Western blot. Both functional and biochemical properties of the recombinant enzyme were characterized. Results PfMAG activity was most prominent in schizont. Native PfMAG was partially puried with a specic activity of 147.36 units/mg protein. The DNA sequence of amplied PfMAG showed an insertion of three nucleotides coding for asparagine compared to strain 3D7 and only 16% similarity compared to the human enzyme was observed. After cloning, the 74-kDa recombinant PfMAG was expressed and identied. PfMAG showed a larger size than its human counterpart twice. PfMAG preferred a double- stranded DNA substrate. Glycosylase activity was detected in a broad range of pH 5–8. The quite narrow optimal salt concentration was observed between 100–200 mM NaCl, whereas 250 mM NaCl reduces its activity. Divalent cations are not required for its glycosylase activity; on the contrary, they inhibited glycosylase activity in a concentration-dependent manner. Conclusion PfMAG activity increases according to parasite development. Native PfMAG was partially puried, the recombinant PfMAG was successfully expressed and characterized. An insertion of AAT coding for asparagine was found compared with that of strain 3D7. PfMAG differs from the human counterpart in a twice larger size and a wide range of optimal pH. Results obtained from this study will be a benet for exploring and investigating of candidate targets toward antimalarial drug design in the future. Background Malaria is one of the major diseases threatening two-third of the world population especially those who live in tropical and subtropical regions. The disease not only brings down the quality of life but also becomes an economic burden to those countries in the region [1]. WHO reported 228 million malaria cases in 2018 with approximately 97% of the infections are caused by Plasmodium falciparum resulting in 405000 death [2]. P. falciparum causes the most severity in terms of clinical pathology and Page 2/21 complication in treatment as it develops resistance to most of antimalarial agents. The emergence of the artemisinin-resistant parasite in South-East Asia has highlighted the need for new drugs and their validated targets for a new generation of antimalarial agents [3, 4]. Though, the malaria vaccine becomes recently available, it only provided moderate protection and need years to perfect the regimen [5], thus chemotherapeutic agents still play essential roles on malaria treatment and prevention. Among several targets being studied for antimalarial development, enzymes in DNA repair pathway of P. falciparum were investigated as potentials targets for drug development including P. falciparum uracil DNA glycosylase (PfUGD) [6], P. falciparum DNA polymerase delta (PfPolδ) [7], and P. falciparum ATP- dependent DNA helicase RuvB3 (PfRuvB3) [8]. The parasite complete genome showed the absence of DNA repair enzymes in the non-homologous end joining pathway. Still, a gene of PfPolδ was observed suggesting that base excision repair mechanism in parasite might rely mainly on a long patch pathway [9]. The sequencing further revealed its 23 megabases consisted of approximately 80 percent of A-T content and as high as 90 percent in the intergenic region [10]. This implies the potential of targeting this A-T rich region by using alkylating agents and also enzymes repairing alkylated bases lesion [11]. DNA-3-methyladenine DNA glycosylase (MAG), a single subunit monofunctional DNA repair enzyme, belongs to AAG structural superfamily characterized by an antiparallel β-sheet and anked by α-helices [12]. The enzyme is capable of removal of 3-methyladenine (m3A) as well as other cyclic adducts formed in DNA including εA, 3,N4-ethenocytosine (εC), N2,3-ethenoguaine (N2,3-εG), and l,N2-ethenoguanine (1,N2-εG) [13]. MAG orthologs were previously observed in Escherichia coli, Saccharomyces cerevisiae, rodents, humans, and plants [14, 15]. It is also known as N-methylpurine DNA glycosylase (MPG) and alkyladenine DNA glycosylase (AAG) due to its versatility of substrate recognized in the active site [16]. MAG knockdown in the animal model and cell culture showed that there was a modulation of sensitivity to alkylating agents in MAG decient cells [17, 18]. Moreover, 3-methyladenine and 1,N6-ethenoadenine are able to inhibit DNA replication fork progression where the adducts inhibit DNA replication process [19–21]. Since, DNA-3-methyladenine glycosylase plays an important role in DNA repair and little is known about PfMAG; therefore these provide an opportunity to study and exploit the advantages of A-T rich content of the parasites as a potential target for antimalarial drug development [22]. In this study, native PfMAG was partially puried from parasite crude extract. Cloning and expression of PfMAG were performed and the recombinant enzyme was further characterized for its biochemical and functional properties. Materials And Methods Screening of PfMAG activity on P. falciparum asexual stages P. falciparum K1 strain, a chloroquine and pyrimethamine resistant strain from Thailand [23], was cultivated in RPMI 1640 media (Invitrogen™, CA, USA) supplemented with 10% human serum and human Page 3/21 red blood cell (RBC) at 37oC in candle jar [24]. The media was changed daily along with morphology and parasitemia observation was made under a microscope using thin blood lm stained with Giemsa stain. Parasite culture was started with 2% initial parasitemia of ring form after synchronization by sorbitol treatment [25]. The ring form, trophozoite and schizont stages of parasites were separately harvested when parasitemia reached 20–30%. The pellet of each parasite stage was prepared by incubation of packed infected red blood cells with an equal volume of phosphate-buffered saline (PBS), pH 7.6, containing 0.15% (w/v) saponin at 37 °C for 20 min. The cell suspension was washed twice with PBS by centrifugation at 700xg at 25 °C for 10 min, and the parasite pellet was kept at -80oC until used. Approximately 0.5 ml of each stage of parasite pellet was resuspended in 4 volumes of extraction buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 2 mM DTT, 0.01% NP40 and 1 mM PMSF) and cells were broken by Dounce homogenizer. Sample was then added with equal volume of dilution buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 2 mM DTT, 20% (w/v) sucrose, 0.01% NP40 and 1 mM PMSF). A 3 M KCl was slowly added into the sample to a nal concentration of 0.5 M KCl while stirring and keep stirring on ice for 30 min. The sample was centrifuged at 100000xg at 4°C for 45 min. The supernatant was dialysed at 4 °C overnight against buffer A containing 25 mM Tris-HCl pH 8.5, 1 mM EDTA, 1 mM PMSF, 1 mM DTT, 5% sucrose, 20% glycerol, 0.01% NP40. Parasite extract of each stage was examined for its PfMAG activity. Preparation of parasite crude extract for partial purication of native PfMAG Parasite culture for partial purication of native PfMAG was performed by using a large-scale culture method [26]. P. falciparum cultures containing mostly trophozoite and schizont were harvested after more than 20% parasitemia was obtained by centrifugation at 500xg for 10 min at 25oC. Approximately 2 ml of parasite pellet was resuspended, homogenized and nuclear protein was extracted and prepared as described above. Parasite extract was then used for purication of native PfMAG. Partial purication of native PfMAG from crude extract After centrifugation and dialysis against buffer A at 4°C overnight, the protein was then loaded onto HiTrap Q column (GE Healthcare, USA) equilibrated with buffer A. The column was washed with 10 ml of buffer A and eluted using 10 ml of linear gradient from 0–1 M KCl in buffer A. Fractions of 250 µl were collected and tested for glycosylase activity. The active fractions were dialyzed against buffer B (50 mM Tris pH 8.0, 1 mM PMSF, 2 mM DTT, 1 mM EDTA, 5% sucrose, 20% glycerol, 0.01% NP40) and then loaded onto HiTrap Capto S column equilibrated with buffer B.