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...
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| creator | Liu, Peng Robbins, Arthur H Marzahn, Melissa R McClung, Scott H Yowell, Charles A Stevens, Jr, Stanley M Dame, John B Dunn, Ben M |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0141758 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0141758</identifier><identifier>PMID: 26510189</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amino Acid Substitution ; Amino acids ; Analysis ; Animals ; Antimicrobial agents ; Aspartic Acid Endopeptidases - antagonists & inhibitors ; Aspartic Acid Endopeptidases - chemistry ; Aspartic Acid Endopeptidases - genetics ; Aspartic Acid Endopeptidases - metabolism ; Aspartic proteinase ; Biochemistry ; Biotechnology ; Blood cells ; Care and treatment ; Catalysis ; Catalytic activity ; Catalytic Domain ; Cathepsin D ; Chemical properties ; Chemotherapy ; Cloning ; Combinatorial analysis ; Combinatorial chemistry ; Complications and side effects ; Deoxyribonucleic acid ; Design ; DNA ; Drug development ; E coli ; Enzyme Activation ; Enzymes ; Erythrocytes ; Food ; Food vacuoles ; Gene Expression ; Health aspects ; Hemoglobin ; Inclusion bodies ; Infectious diseases ; Interdisciplinary aspects ; Isoforms ; Kinetics ; Malaria ; Molecular biology ; Parasites ; Pathological effects ; Peptide libraries ; Pharmaceutical sciences ; Physiological aspects ; Plasmodium ; Plasmodium (Protozoa) ; Plasmodium berghei ; Plasmodium berghei - genetics ; Plasmodium berghei - metabolism ; Plasmodium falciparum ; Proenzymes ; Protein Refolding ; Proteinase ; Proteins ; Protozoan Proteins - antagonists & inhibitors ; Protozoan Proteins - chemistry ; Protozoan Proteins - genetics ; Protozoan Proteins - metabolism ; Reaction kinetics ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Substrate inhibition ; Vaccines ; Vector-borne diseases ; Veterinary colleges ; Veterinary medicine</subject><ispartof>PloS one, 2015-10, Vol.10 (10), p.e0141758-e0141758</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Liu et al 2015 Liu et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c718t-a22d633a7cb815fce32a2e07689ea4921eea3f1cecddcd026da6da0ebd5d2e653</citedby><cites>FETCH-LOGICAL-c718t-a22d633a7cb815fce32a2e07689ea4921eea3f1cecddcd026da6da0ebd5d2e653</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624963/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624963/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26510189$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Liu, Peng</creatorcontrib><creatorcontrib>Robbins, Arthur H</creatorcontrib><creatorcontrib>Marzahn, Melissa R</creatorcontrib><creatorcontrib>McClung, Scott H</creatorcontrib><creatorcontrib>Yowell, Charles A</creatorcontrib><creatorcontrib>Stevens, Jr, Stanley M</creatorcontrib><creatorcontrib>Dame, John B</creatorcontrib><creatorcontrib>Dunn, Ben M</creatorcontrib><title>Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Amino Acid Substitution</subject><subject>Amino acids</subject><subject>Analysis</subject><subject>Animals</subject><subject>Antimicrobial agents</subject><subject>Aspartic Acid Endopeptidases - antagonists & inhibitors</subject><subject>Aspartic Acid Endopeptidases - chemistry</subject><subject>Aspartic Acid Endopeptidases - genetics</subject><subject>Aspartic Acid Endopeptidases - metabolism</subject><subject>Aspartic proteinase</subject><subject>Biochemistry</subject><subject>Biotechnology</subject><subject>Blood cells</subject><subject>Care and treatment</subject><subject>Catalysis</subject><subject>Catalytic activity</subject><subject>Catalytic Domain</subject><subject>Cathepsin D</subject><subject>Chemical properties</subject><subject>Chemotherapy</subject><subject>Cloning</subject><subject>Combinatorial analysis</subject><subject>Combinatorial chemistry</subject><subject>Complications and side effects</subject><subject>Deoxyribonucleic acid</subject><subject>Design</subject><subject>DNA</subject><subject>Drug development</subject><subject>E coli</subject><subject>Enzyme Activation</subject><subject>Enzymes</subject><subject>Erythrocytes</subject><subject>Food</subject><subject>Food vacuoles</subject><subject>Gene Expression</subject><subject>Health aspects</subject><subject>Hemoglobin</subject><subject>Inclusion bodies</subject><subject>Infectious diseases</subject><subject>Interdisciplinary aspects</subject><subject>Isoforms</subject><subject>Kinetics</subject><subject>Malaria</subject><subject>Molecular biology</subject><subject>Parasites</subject><subject>Pathological effects</subject><subject>Peptide libraries</subject><subject>Pharmaceutical sciences</subject><subject>Physiological aspects</subject><subject>Plasmodium</subject><subject>Plasmodium (Protozoa)</subject><subject>Plasmodium berghei</subject><subject>Plasmodium berghei - genetics</subject><subject>Plasmodium berghei - metabolism</subject><subject>Plasmodium falciparum</subject><subject>Proenzymes</subject><subject>Protein Refolding</subject><subject>Proteinase</subject><subject>Proteins</subject><subject>Protozoan Proteins - antagonists & inhibitors</subject><subject>Protozoan Proteins - chemistry</subject><subject>Protozoan Proteins - genetics</subject><subject>Protozoan Proteins - metabolism</subject><subject>Reaction kinetics</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Substrate inhibition</subject><subject>Vaccines</subject><subject>Vector-borne diseases</subject><subject>Veterinary colleges</subject><subject>Veterinary medicine</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11v0zAUhiMEYqPwDxBEQkJw0eKPxElukKZqg0pDmwrs1jqxT1pXSdzZCbD9etw1mxq0C2RLTuznvPZ57RNFrymZUZ7RTxvbuxbq2da2OCM0oVmaP4mOacHZVDDCnx58H0UvvN8QkvJciOfRERMpJTQvjqPfp-3tTQOdUfF8DQ5Uh87chn_bxraKl6hsU5oW2i4-s1bHV6B6W2N8WYNvcOtNGydx5WwTd2uMl1ZjIL9BDc5AfBkEvekG2mrTN3GJbrVG8zJ6VkHt8dUwTqKfZ6c_5l-n5xdfFvOT86nKaN5NgTEtOIdMlTlNK4WcAUOSibxASApGEYFXVKHSWmnChIbQCZY61QxFyifR273utrZeDp55STOWFYUQKQnEYk9oCxu5daYBdyMtGHk3Yd1Kggv-1CiZxryAlNIy5UnJMU90rmiVYFmkWCENWp-H3fqyQa2CGQ7qkeh4pTVrubK_ZCJYUoREJ9GHQcDZ6x59JxvjFdY1tGj7u3PnIhOcZAF99w_6eHYDtYKQgGkrG_ZVO1F5knBWCMITFqjZI1RoGhujwgOrTJgfBXwcBQSmwz_dCnrv5eL78v_Zi6sx-_6AXSPU3drbut-9Rz8Gkz2onPXeYfVgMiVyVx_3bshdfcihPkLYm8MLegi6Lwj-F9nXDR4</recordid><startdate>20151028</startdate><enddate>20151028</enddate><creator>Liu, Peng</creator><creator>Robbins, Arthur H</creator><creator>Marzahn, Melissa R</creator><creator>McClung, Scott H</creator><creator>Yowell, Charles A</creator><creator>Stevens, Jr, Stanley M</creator><creator>Dame, John B</creator><creator>Dunn, Ben M</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20151028</creationdate><title>Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei</title><author>Liu, Peng ; Robbins, Arthur H ; Marzahn, Melissa R ; McClung, Scott H ; Yowell, Charles A ; Stevens, Jr, Stanley M ; Dame, John B ; Dunn, Ben M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c718t-a22d633a7cb815fce32a2e07689ea4921eea3f1cecddcd026da6da0ebd5d2e653</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amino Acid Substitution</topic><topic>Amino acids</topic><topic>Analysis</topic><topic>Animals</topic><topic>Antimicrobial agents</topic><topic>Aspartic Acid Endopeptidases - antagonists & inhibitors</topic><topic>Aspartic Acid Endopeptidases - chemistry</topic><topic>Aspartic Acid Endopeptidases - genetics</topic><topic>Aspartic Acid Endopeptidases - metabolism</topic><topic>Aspartic proteinase</topic><topic>Biochemistry</topic><topic>Biotechnology</topic><topic>Blood cells</topic><topic>Care and treatment</topic><topic>Catalysis</topic><topic>Catalytic activity</topic><topic>Catalytic Domain</topic><topic>Cathepsin D</topic><topic>Chemical properties</topic><topic>Chemotherapy</topic><topic>Cloning</topic><topic>Combinatorial analysis</topic><topic>Combinatorial chemistry</topic><topic>Complications and side effects</topic><topic>Deoxyribonucleic acid</topic><topic>Design</topic><topic>DNA</topic><topic>Drug development</topic><topic>E coli</topic><topic>Enzyme Activation</topic><topic>Enzymes</topic><topic>Erythrocytes</topic><topic>Food</topic><topic>Food vacuoles</topic><topic>Gene Expression</topic><topic>Health aspects</topic><topic>Hemoglobin</topic><topic>Inclusion bodies</topic><topic>Infectious diseases</topic><topic>Interdisciplinary aspects</topic><topic>Isoforms</topic><topic>Kinetics</topic><topic>Malaria</topic><topic>Molecular biology</topic><topic>Parasites</topic><topic>Pathological effects</topic><topic>Peptide libraries</topic><topic>Pharmaceutical sciences</topic><topic>Physiological aspects</topic><topic>Plasmodium</topic><topic>Plasmodium (Protozoa)</topic><topic>Plasmodium berghei</topic><topic>Plasmodium berghei - genetics</topic><topic>Plasmodium berghei - metabolism</topic><topic>Plasmodium falciparum</topic><topic>Proenzymes</topic><topic>Protein Refolding</topic><topic>Proteinase</topic><topic>Proteins</topic><topic>Protozoan Proteins - antagonists & inhibitors</topic><topic>Protozoan Proteins - chemistry</topic><topic>Protozoan Proteins - genetics</topic><topic>Protozoan Proteins - metabolism</topic><topic>Reaction kinetics</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Substrate inhibition</topic><topic>Vaccines</topic><topic>Vector-borne diseases</topic><topic>Veterinary colleges</topic><topic>Veterinary medicine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Liu, Peng</creatorcontrib><creatorcontrib>Robbins, Arthur H</creatorcontrib><creatorcontrib>Marzahn, Melissa R</creatorcontrib><creatorcontrib>McClung, Scott H</creatorcontrib><creatorcontrib>Yowell, Charles A</creatorcontrib><creatorcontrib>Stevens, Jr, Stanley M</creatorcontrib><creatorcontrib>Dame, John B</creatorcontrib><creatorcontrib>Dunn, Ben M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Liu, Peng</au><au>Robbins, Arthur H</au><au>Marzahn, Melissa R</au><au>McClung, Scott H</au><au>Yowell, Charles A</au><au>Stevens, Jr, Stanley M</au><au>Dame, John B</au><au>Dunn, Ben M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-10-28</date><risdate>2015</risdate><volume>10</volume><issue>10</issue><spage>e0141758</spage><epage>e0141758</epage><pages>e0141758-e0141758</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26510189</pmid><doi>10.1371/journal.pone.0141758</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2015-10, Vol.10 (10), p.e0141758-e0141758 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1727996650 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Amino Acid Substitution Amino acids Analysis Animals Antimicrobial agents Aspartic Acid Endopeptidases - antagonists & inhibitors Aspartic Acid Endopeptidases - chemistry Aspartic Acid Endopeptidases - genetics Aspartic Acid Endopeptidases - metabolism Aspartic proteinase Biochemistry Biotechnology Blood cells Care and treatment Catalysis Catalytic activity Catalytic Domain Cathepsin D Chemical properties Chemotherapy Cloning Combinatorial analysis Combinatorial chemistry Complications and side effects Deoxyribonucleic acid Design DNA Drug development E coli Enzyme Activation Enzymes Erythrocytes Food Food vacuoles Gene Expression Health aspects Hemoglobin Inclusion bodies Infectious diseases Interdisciplinary aspects Isoforms Kinetics Malaria Molecular biology Parasites Pathological effects Peptide libraries Pharmaceutical sciences Physiological aspects Plasmodium Plasmodium (Protozoa) Plasmodium berghei Plasmodium berghei - genetics Plasmodium berghei - metabolism Plasmodium falciparum Proenzymes Protein Refolding Proteinase Proteins Protozoan Proteins - antagonists & inhibitors Protozoan Proteins - chemistry Protozoan Proteins - genetics Protozoan Proteins - metabolism Reaction kinetics Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Substrate inhibition Vaccines Vector-borne diseases Veterinary colleges Veterinary medicine |
| title | Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei |
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