{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T07:46:08Z","timestamp":1785224768982,"version":"3.55.0"},"reference-count":27,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,1,16]],"date-time":"2022-01-16T00:00:00Z","timestamp":1642291200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["NRF-2015R1A2A2A01004310"],"award-info":[{"award-number":["NRF-2015R1A2A2A01004310"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2\u20134 (MP2\u20134), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2\/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2\/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2\/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.<\/jats:p>","DOI":"10.3390\/microorganisms10010193","type":"journal-article","created":{"date-parts":[[2022,1,16]],"date-time":"2022-01-16T20:45:21Z","timestamp":1642365921000},"page":"193","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4"],"prefix":"10.3390","volume":"10","author":[{"given":"H\u01b0\u01a1ng Giang","family":"L\u00ea","sequence":"first","affiliation":[{"name":"Department of Parasitology and Tropical Medicine, Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea"},{"name":"Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jung-Mi","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Tropical Medicine, Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea"},{"name":"Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tu\u1ea5n C\u01b0\u1eddng","family":"V\u00f5","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Tropical Medicine, Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea"},{"name":"Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3041-5560","authenticated-orcid":false,"given":"Won Gi","family":"Yoo","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Tropical Medicine, Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea"},{"name":"Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kon Ho","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea"},{"name":"Department of Microbiology, Gyeongsang National University College of Medicine, Jinju 52727, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6734-1673","authenticated-orcid":false,"given":"Byoung-Kuk","family":"Na","sequence":"additional","affiliation":[{"name":"Department of Parasitology and Tropical Medicine, Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea"},{"name":"Department of Convergence Medical Science, Gyeongsang National University, Jinju 52727, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1172\/JCI113766","article-title":"A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum","volume":"82","author":"Rosenthal","year":"1988","journal-title":"J. Clin. Investig."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"14961","DOI":"10.1074\/jbc.272.23.14961","article-title":"Biosynthesis and maturation of the malaria aspartic hemoglobinases plasmepsins I and II","volume":"272","author":"Francis","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1042\/bj3600481","article-title":"Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3","volume":"360","author":"Sijwali","year":"2001","journal-title":"Biochem. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"29000","DOI":"10.1074\/jbc.M004459200","article-title":"Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum","volume":"275","author":"Shenai","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1073\/pnas.98.1.271","article-title":"Malaria parasite exit from the host erythrocyte: A two-step process requiring extraerythrocytic proteolysis","volume":"98","author":"Salmon","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2002","DOI":"10.1126\/science.1077426","article-title":"A role for the protease falcipain 1 in host cell invasion by the human malaria parasite","volume":"298","author":"Greenbaum","year":"2002","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1659","DOI":"10.2174\/1381612023394197","article-title":"Cysteine Proteases of Malaria Parasites: Targets for Chemotherapy","volume":"8","author":"Rosenthal","year":"2005","journal-title":"Curr. Pharm. Des."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"346","DOI":"10.2174\/156802610790725461","article-title":"Proteases of Plasmodium falciparum as Potential Drug Targets and Inhibitors Thereof","volume":"10","author":"Gerber","year":"2010","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1111\/j.1550-7408.1992.tb04856.x","article-title":"Plasmodium falciparum: Differential Sensitivity In Vitro to E-64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)","volume":"39","author":"Bailly","year":"1992","journal-title":"J. Protozool."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1006\/expr.1995.1033","article-title":"Plasmodium falciparum: Effects of proteinase inhibitors on globin hydrolysis by cultured malaria parasites","volume":"80","author":"Rosenthal","year":"1995","journal-title":"Exp. Parasitol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"394","DOI":"10.3389\/fmicb.2019.00394","article-title":"Structural insights into key Plasmodium proteases as therapeutic drug targets","volume":"10","author":"Mishra","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"501","DOI":"10.2174\/1389450117666161221122432","article-title":"Targeting Cysteine Proteases from Plasmodium falciparum: A General Overview, Rational Drug Design and Computational Approaches for Drug Discovery","volume":"19","author":"Bekono","year":"2018","journal-title":"Curr. Drug Targets"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3049","DOI":"10.2174\/0929867311320250003","article-title":"Development of Plasmodium falciparum Protease Inhibitors in the Past Decade (2002\u20132012)","volume":"20","author":"Perez","year":"2013","journal-title":"Curr. Med. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.pt.2004.03.006","article-title":"Mixed-species malaria infections in humans","volume":"20","author":"Mayxay","year":"2004","journal-title":"Trends Parasitol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"11068","DOI":"10.1038\/s41598-020-68082-3","article-title":"Plasmodium spp. mixed infection leading to severe malaria: A systematic review and meta-analysis","volume":"10","author":"Kotepui","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1042\/bj20031487","article-title":"Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax","volume":"378","author":"Na","year":"2004","journal-title":"Biochem. J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pntd.0000849","article-title":"Biochemical properties of a novel cysteine protease of Plasmodium vivax, vivapain-4","volume":"4","author":"Na","year":"2010","journal-title":"PLoS Negl. Trop. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0051619","article-title":"Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite","volume":"7","author":"Prasad","year":"2012","journal-title":"PLoS ONE"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1056\/NEJM199802053380605","article-title":"Plasmodium malariae Infection in an Asymptomatic 74-Year-Old Greek Woman with Splenomegaly","volume":"338","author":"Vinetz","year":"1998","journal-title":"N. Engl. J. Med."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Douglas, N.M., Lampah, D.A., Kenangalem, E., Simpson, J.A., Poespoprodjo, J.R., Sugiarto, P., Anstey, N.M., and Price, R.N. (2013). Major Burden of Severe Anemia from Non-Falciparum Malaria Species in Southern Papua: A Hospital-Based Surveillance Study. PLoS Med., 10.","DOI":"10.1371\/journal.pmed.1001575"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/S0140-6736(02)09336-4","article-title":"Chloroquine-resistant Plasmodium malariae in south Sumatra, Indonesia","volume":"360","author":"Maguire","year":"2002","journal-title":"Lancet"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1006\/prep.2001.1416","article-title":"Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2","volume":"22","author":"Sijwali","year":"2001","journal-title":"Protein Expr. Purif."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1042\/bj2010189","article-title":"L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins, B, H and L","volume":"201","author":"Barrett","year":"1982","journal-title":"Biochem. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2753","DOI":"10.1016\/S0021-9258(17)40522-9","article-title":"Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin","volume":"252","author":"Bennett","year":"1977","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1007\/978-1-4419-8414-2_3","article-title":"Falcipains and other cysteine proteases of malaria parasites","volume":"712","author":"Rosenthal","year":"2011","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1042\/bj20020753","article-title":"Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites","volume":"368","author":"Singh","year":"2002","journal-title":"Biochem. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1093\/protein\/gzm009","article-title":"A chimeric cysteine protease of Plasmodium berghei engineered to resemble the Plasmodium falciparum protease falcipain-2","volume":"20","author":"Singh","year":"2007","journal-title":"Protein Eng. Sel."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/10\/1\/193\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:39:42Z","timestamp":1760362782000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/10\/1\/193"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,16]]},"references-count":27,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1]]}},"alternative-id":["microorganisms10010193"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms10010193","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,16]]}}}