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Identification and Characterization of Entamoeba histolytica Choline Kinase

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Abstract

Purpose

Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future.

Methodology

E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes.

Results

EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35–48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner’s phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting.

Conclusions

With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

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Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information file].

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Acknowledgements

This research was funded by the Malaysia Ministry of Higher Education Fundamental Research Grant Scheme (FRGS), FRGS/1/2021/SKK0/USM/02/34 (203/PPSK/6171299). Chiat Han Chang was financially supported by National Science Fellowship from Ministry of Sciences, Technology and Innovation, Malaysia. We thank the laboratory staff of the School of Health Sciences for their technical assistance.

Funding

This work was supported by the Malaysia Ministry of Higher Education Fundamental Research Grant Scheme (FRGS), FRGS/1/2021/SKK0/USM/02/34 (203/PPSK/6171299).

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  1. School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia

    Chiat Han Chang, Wei Cun See Too, Boon Huat Lim & Ling Ling Few

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Contributions

WCST, BHL, and LLF: contributed to the study conception and design. Material preparation, data collection and analysis were performed by CHC. The first draft of the manuscript was written by CHC. All authors read and approved the final manuscript.

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Correspondence to Wei Cun See Too or Ling Ling Few.

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This study was approved by the animal ethic committee, Universiti Sains Malaysia [animal ethic approval no.: USM/Animal Ethics Approval/2008/(41)(131)].

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Chang, C.H., See Too, W.C., Lim, B.H. et al. Identification and Characterization of Entamoeba histolytica Choline Kinase. Acta Parasit. 69, 426–438 (2024). https://doi.org/10.1007/s11686-023-00763-1

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