恶性疟原虫 DNA-3-甲基腺嘌呤糖基化酶的分子特征。

Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase.

机构信息

Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

Department of Zoology, Faculty of Science, Kasetsart University, Bangkok, Thailand.

出版信息

Malar J. 2020 Aug 6;19(1):284. doi: 10.1186/s12936-020-03355-w.

DOI:10.1186/s12936-020-03355-w

PMID:32762689

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7409487/

Abstract

BACKGROUND

The 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.

METHODS

Native 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.

RESULTS

PfMAG 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.

CONCLUSION

PfMAG 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.

摘要

背景

青蒿素耐药疟原虫的出现凸显了新型药物及其靶点的必要性。嘌呤碱基的烷基化会阻碍 DNA 复制，如果得不到解决，最终会导致细胞死亡。DNA-3-甲基腺嘌呤糖苷酶（MAG）负责修复那些烷基化的碱基。恶性疟原虫（Pf）MAG 的特性表明其有作为抗疟候选药物的潜力。

方法

从氯喹和乙胺嘧啶耐药恶性疟原虫 K1 株的粗提物中部分纯化 PfMAG，使用三种色谱程序。通过生物信息学分析，设计引物扩增、插入 pBAD202/D-TOPO 并在大肠杆菌中异源表达重组 PfMAG。对重组酶的功能和生化特性进行了表征。

结果

PfMAG 活性在疟原虫裂殖体阶段最为明显，比活为 147 U/mg（部分纯化）蛋白。与 3D7 株相比，K1 PfMAG 含有 AAT（编码天冬酰胺）的插入，与人类酶有 16%的相似性。重组 PfMAG（74 kDa）比人类酶大两倍，优先使用双链 DNA 底物，在 pH 4-9 范围内具有糖苷酶活性，最佳盐浓度为 100-200 mM NaCl，但在 250 mM NaCl 时活性降低，不需要二价阳离子，二价阳离子呈剂量依赖性抑制。

结论

PfMAG 活性随着寄生虫的发育而增加，在裂殖体阶段最高。K1 PfMAG 含有 3D7 株中不存在的插入 AAT（天冬酰胺），重组酶比人类酶大两倍。重组 PfMAG 的最适 pH 活性范围较宽，在高浓度（250 mM）NaCl 浓度以及二价阳离子存在下会受到抑制。PfMAG 的特性提供了基本数据，这应该有助于针对这一潜在寄生虫靶点开发抗疟药物。