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Published on: November 22, 2024
Plasmodium falciparum Plasmepsin V (PfPMV): Insights into recombinant expression, substrate specificity and active
Nonlawat Boonyalai1, Pichamon Sittikul2, Jirundon Yuvaniyama3
1Department of Biochemistry and Special Research Unit for Protein Engineering and Protein Bioinformatics, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan road, Lat Yao, Chatuchak, Bangkok 10900, Thailand; Center of Excellence for Innovation in Chemistry, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan road, Lat Yao, Chatuchak, Bangkok 10900, Thailand.
Abstract:
Plasmepsin V from Plasmodium falciparum (PfPMV) is responsible for the cleavage of the Plasmodium export element (PEXEL) motif at the N-terminus of several hundreds of the exported proteins. PfPMV is necessary for parasite viability and has become a novel promising target for antimalarial therapy. The first recombinant expression of soluble, active PfPMV as thioredoxin fusion proteins is reported herein. Two truncated forms of PfPMV were fused to thioredoxin (Trx) to generate Trx-PfPMVp37 and Trx-PfPMVm84. The fusion proteins were successfully purified using Ni(2+) affinity chromatography in combination with ATP treatment to eliminate Escherichia coli HSP60 contaminant. Trx-PfPMVm84 could hydrolyze the PEXEL-containing peptides more efficiently than Trx-PfPMVp37. Interestingly, both Trx-PfPMVs preferred to cleave PfEMP2 peptide over HRPII peptide. The replacement of Ser with Val or Glu at P1' position created a substrate with 75% reduction in the enzyme activity, whereas the substitution of Ile with Lys or Glu at P2 position reduced the cleavage efficiency by 30%. The activity of Trx-PfPMVm84 was inhibited by PMSF and nelfinavir but not by pepstatin A. After the removal of Trx domain, activities of both enzymes toward PfEMP2 and HRPII peptides were fitted to the Michaelis-Menten model to determine kinetic parameters. The Km values toward both peptides were apparently much lower than the previously reported data although with similar kcat values. Along with an improved PfPMV preparation protocol, these findings have provided insights into its substrate specificity at P2 and P1' positions as well as interactions among the enzyme, substrates, and inhibitors.
Insights
Plasmepsin V (PfPMV), a key Plasmodium falciparum enzyme for parasite export, was successfully expressed and purified. This research provides crucial insights into PfPMV
Area of Science:
- Malariology
- Enzymology
- Protein Biochemistry
Background:
- Plasmepsin V (PfPMV) from Plasmodium falciparum cleaves the Plasmodium export element (PEXEL) motif, essential for exporting hundreds of parasite proteins.
- PfPMV is vital for parasite survival, making it a significant target for antimalarial drug development.
Purpose of the Study:
- To achieve the first recombinant expression of soluble, active PfPMV.
- To characterize the substrate specificity and kinetic properties of PfPMV.
Main Methods:
- Two truncated PfPMV forms were fused to thioredoxin (Trx) and expressed as Trx-PfPMVp37 and Trx-PfPMVm84.
- Purification involved Ni(2+) affinity chromatography and ATP treatment to remove contaminants.
- Enzyme activity assays were performed using PEXEL-containing peptides (PfEMP2 and HRPII), and kinetic parameters were determined using the Michaelis-Menten model.
Main Results:
- Both Trx-PfPMV fusion proteins were successfully purified and shown to be active.
- Trx-PfPMVm84 exhibited higher hydrolytic activity than Trx-PfPMVp37, with a preference for PfEMP2 over HRPII peptide.
- Substitutions at the P1' and P2 positions significantly affected enzyme activity, indicating specific substrate recognition.
- PfPMV activity was inhibited by PMSF and nelfinavir, but not pepstatin A.
- Kinetic analysis revealed lower Km values for PfPMV compared to previous reports.
Conclusions:
- The study reports the first successful recombinant expression and purification of active Plasmepsin V.
- Characterization revealed insights into PfPMV's substrate specificity, particularly at the P2 and P1' positions.
- Findings contribute to understanding enzyme-substrate interactions and inform antimalarial drug target strategies.
