PLoS ONE. 2015 October;10(10):e0141758

Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei


Peng Liu, Arthur H. Robbins, Melissa R. Marzahn, Scott H. McClung, Charles A. Yowell, Stanley M. Stevens, Jr., John B. Dame, Ben M. Dunn


Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida, United States of America.

Protein Core, Interdisciplinary Center for Biotechnology Research, University of Florida, College of Medicine, Gainesville, Florida, United States of America.

Department of Infectious Diseases and Pathology, University of Florida, College of Veterinary Medicine, Gainesville, Florida, United States of America

Current address: Department of Neurology and N. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, Minnesota, United States of America.

Current address: Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.

Current address: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, United States of America.



The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria.  Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites.  Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells.  In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA.  We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding.  Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5.  Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment.  We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases.  Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’.  Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD).  We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD.  Data from this study further our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.

PMID: 26510189



Malaria, a devastating infectious disease, afflicts approximately half of the world’s population and causes nearly half a million deaths worldwide in 2015 [4].  To date, the rapid development and spread of anti-malarial drug resistance of the parasite urges identifying new molecular targets for effective drug design.  Plasmepsins are potential targets for anti-malarial therapy.


Plasmepsins belong to the aspartic proteinase super-family.  The name arises from a combination of Plasmodium, the malaria-causing protozoa, and pepsin, a principle proteinase involved in food digestion.  Plasmepsins are categorized into those residing and functioning inside the food vacuole (FV), a parasite organelle of acidic pH, and those outside the FV (i.e., non-FV plasmepsins).


Early studies showed that inhibitors of FV plasmepsins bear anti-parasitic activities [5-7]; however, recent research using pharmacological and genetic manipulation has indicated that FV plasmepsins may be dispensable for parasite development, and that non-FV plasmepsins (e.g., plasmepsins 5 and 10) play a pivotal role in malaria pathogenesis [8-13].  Nonetheless, the functions of neither FV nor non-FV plasmepsins have been fully understood yet; as such, the enzymatic features of plasmepsins still warrant characterization.


In this study, we focused on characterizing PbPM4, which is thus far the only known FV plasmepsin of Plasmodium berghei, one of the four Plasmodium spp. that infect rodents [14].  We first cloned, expressed, and enzymatically characterized a recombinant zymogen form of PbPM4. We found that PbPM4 exhibits catalytic activity in the presence of the pro-segment despite that the pro-segment markedly disrupts enzyme recognition of substrate, a novel phenomenon found in none of the other characterized plasmepsins to our knowledge. The lower catalytic activity shown by PbPM4 zymogen compared to the mature enzyme is potentially caused by a competitive binding of the pro-segment to the active site cleft, or by a pro-segment-mediated structural alteration of the active site towards inappropriate accommodation of substrates.


In contrast to a recent report of the incapability of the PbPM4 zymogen to perform auto-maturation [15], we found that auto-maturation to mature PbPM4 occurs in both pH- and time-dependent manners.  We then identified the optimal conditions for the auto-maturation process, and used thus obtained mature PbPM4 to study enzyme kinetics on substrates and inhibitors of aspartic proteinases.


The active site cleft of a proteinase can be subdivided into several pockets, each of which is comprised of a set of spatially close amino acids of the enzyme (Figure 1).  In the second half of this study, we analyzed the active site preferences of PbPM4 at subsites S3, S2, S1, S1’, S2’ and S3’ using two sets of combinatorial chemistry-based peptide  substrate libraries previously employed for characterization of other FV plasmepsins [16, 17].  We first determined the primary subsite preferences at S1 by analyzing the initial cleavage velocities of peptide pools differing only at the amino acid accommodated at S1. We chose the three pools that gave rise to the highest cleavage velocities. Similarly, we determined the primary subsite preferences at S1’.  We then determined the secondary subsite preferences at S3, S2, S2’and S3’ from the selected peptide pools by measuring the relative abundances of penta- and tri-peptides produced from PbPM4 digestion using liquid chromatography-mass spectrometry.




Figure 1  Schematic diagram of a peptide substrate fitting to the active site cleft of an endopeptidase.  The subsite pockets are nominated S4-S3’ and the side chains that are accommodated in these subsites are nominated P4-P3’. The enzyme catalyzed hydrolysis occurs at the peptide bond between P1 and P1’ [2].  In the case of PbPM4, the amino acid residues that comprise subsites S3-S3’ are listed.


Next, by comparing the active site preferences of PbPM4 to those of a homologous human aspartic proteinase cathepsin D (hcatD) that were previously determined [18], we designed and synthesized a selective peptidomimetic compound (KPYEFψRQF, where ψ = -CH2-NH-) of PbPM4.  We then analyzed the inhibitory effects of this compound on PbPM4, seven FV plasmepsins functioning in human malaria parasites, and hcatD.  We found that this compound exhibits inhibition of PbPM4 in micromolar magnitude and a more than 10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 enzymes versus hcatD.  Interesting, a compound (KPLEFψYRV) previously designed using the same strategy for plasmepsin 4 of the human malaria parasite P. ovalae shows a sub-nanomolar inhibition of PbPM4 and a more than 70-fold selectivity to PbPM4 versus hcatD, thus an inhibitor of tighter binding affinity and higher selectivity to PbPM4.  The potential interaction of compound KPLEFψYRV and PbPM4 was proposed in Figure 2 following docking the compound into the active site cleft of the enzyme using molecular modeling.




Figure 2  Molecular modeling of PbPM4 in complex with the compound (KPLEFψYRV, where ψ = -CH2-NH-).  Modeling of the interaction between PbPM4 and the compound was performed using Sybyl7.1 [1] and the figure was generated using PyMOL [3]. The surface of the active site cleft is illustrated based on the electrostatic potential (blue: positively charged areas; red: negatively charged areas, grey: electrostatically neutral areas) . Hydrogen bonding interactions are highlighted in cyan dashed lines. The subsites and the enzyme residues that maintain hydrogen bonding interactions with the inhibitors are labeled.  Amino Acid residues are numbered based on the sequence of human pepsin A.


Importance of the study – The P. berghei culture model and P. berghei-infected mouse models have been extensively used for anti-malarial drug screening and development.  Genetic ablation of PbPM4 results in attenuated virulence and induces protective immunity in the parasite-infected host [19-21], indicating that PbPM4 plays an essential role in rodent malaria pathogenesis.  A comprehensive characterization of PbPM4 not only broadens and deepens our knowledge on the plasmepsin family but also provides fresh leads for anti-malarial drug design.



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This work is supported by National Institutes of Health ( grant AI39211 to BMD and JBD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.



Peng Liu, Ph.D.

Department of Neurology and N. Bud Grossman Center for Memory Research and

Care, University of Minnesota, Minneapolis, Minnesota 55455, United States of America

Ben M. Dunn, Ph.D.

Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville,

Florida 32610, United States of America




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