Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.
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 furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.
Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
Peng
P
Liu
Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
Arthur H
AH
Robbins
Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
Melissa R
MR
Marzahn
Protein Core, Interdisciplinary Center for Biotechnology Research, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
Scott H
SH
McClung
Department of Infectious Diseases and Pathology, University of Florida, College of Veterinary Medicine, Gainesville, Florida, United States of America.
Charles A
CA
Yowell
Protein Core, Interdisciplinary Center for Biotechnology Research, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
Stanley M
SM
Stevens
Department of Infectious Diseases and Pathology, University of Florida, College of Veterinary Medicine, Gainesville, Florida, United States of America.
John B
JB
Dame
Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, Florida, United States of America.
Ben M
BM
Dunn
D019943:Amino Acid Substitution; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D015870:Gene Expression; D007700:Kinetics; D010962:Plasmodium berghei / Q000235:genetics / Q000378:metabolism*; D058849:Protein Refolding; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D015800:Protozoan Proteins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin