{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T04:41:42Z","timestamp":1790829702991,"version":"4.1.0"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2016,9,28]],"date-time":"2016-09-28T00:00:00Z","timestamp":1475020800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2016,9,28]],"date-time":"2016-09-28T00:00:00Z","timestamp":1475020800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the <jats:italic>Toxoplasma gondii<\/jats:italic> genome (<jats:italic>TgAAP<\/jats:italic>), although its function remains unknown. In this study, we characterized <jats:italic>TgAAP<\/jats:italic> and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant <jats:italic>Tg<\/jats:italic>AAP (r<jats:italic>Tg<\/jats:italic>AAP) protein in <jats:italic>Escherichia coli<\/jats:italic>, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of <jats:italic>Tg<\/jats:italic>AAP using the CRISPR\/Cas9 knockout system. Western blotting demonstrated the deletion of <jats:italic>Tg<\/jats:italic>AAP in the knockout strain. Indirect immunofluorescence analysis showed that <jats:italic>Tg<\/jats:italic>AAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that <jats:italic>TgAAP<\/jats:italic> knockout inhibited the attachment\/invasion, replication, and substrate-specific activity in <jats:italic>T. gondii<\/jats:italic>. Finally, the activity of drug CID 23724194, previously described as targeting <jats:italic>Plasmodium<\/jats:italic> and malarial parasite AAP, was tested against r<jats:italic>Tg<\/jats:italic>AAP and the parasite. Overall, <jats:italic>TgAAP<\/jats:italic> knockout affected the growth of <jats:italic>T. gondii<\/jats:italic> but did not completely abolish parasite replication and growth. Therefore, <jats:italic>Tg<\/jats:italic>AAP may comprise a useful adjunct drug target of <jats:italic>T. gondii<\/jats:italic>.<\/jats:p>","DOI":"10.1038\/srep34448","type":"journal-article","created":{"date-parts":[[2016,9,28]],"date-time":"2016-09-28T09:31:34Z","timestamp":1475055094000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Characterization of aspartyl aminopeptidase from Toxoplasma gondii"],"prefix":"10.1038","volume":"6","author":[{"given":"Jun","family":"Zheng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziying","family":"Cheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Honglin","family":"Jia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yonghui","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2016,9,28]]},"reference":[{"key":"BFsrep34448_CR1","doi-asserted-by":"publisher","first-page":"793","DOI":"10.1007\/s00281-012-0339-3","volume":"34","author":"CD Dupont","year":"2012","unstructured":"Dupont, C. D., Christian, D. A. & Hunter, C. A. Immune response and immunopathology during toxoplasmosis. Seminars in Immunopathology. 34, 793\u2013813 (2012).","journal-title":"Seminars in Immunopathology"},{"key":"BFsrep34448_CR2","doi-asserted-by":"publisher","first-page":"1257","DOI":"10.1016\/j.ijpara.2008.03.007","volume":"38","author":"JP Dubey","year":"2008","unstructured":"Dubey, J. P. & Jones, J. L. Toxoplasma gondii infection in humans and animals in the United States. International Journal for Parasitology. 38, 1257\u20131278 (2008).","journal-title":"International Journal for Parasitology"},{"key":"BFsrep34448_CR3","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1093\/aje\/154.4.357","volume":"154","author":"JL Jones","year":"2001","unstructured":"Jones, J. L. et al. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. American Journal of Epidemiology. 154, 357\u2013365 (2001).","journal-title":"American Journal of Epidemiology"},{"key":"BFsrep34448_CR4","doi-asserted-by":"publisher","first-page":"e90203","DOI":"10.1371\/journal.pone.0090203","volume":"9","author":"J Flegr","year":"2014","unstructured":"Flegr, J., Prandota, J., Sovi\u010dkov\u00e1, M. & Israili, Z. H. Toxoplasmosis\u2013a global threat. Correlation of latent toxoplasmosis with specific disease burden in a set of 88 countries. Plos one 9, e90203 (2014).","journal-title":"Plos one"},{"key":"BFsrep34448_CR5","doi-asserted-by":"publisher","first-page":"150","DOI":"10.1111\/pim.12173","volume":"37","author":"N Blanchard","year":"2015","unstructured":"Blanchard, N., Dunay, I. R. & Schl\u00fcter, D. Persistence of Toxoplasma gondii in the central nervous system: a fine-tuned balance between the parasite, the brain and the immune system. Parasite Immunol. 37, 150\u2013158 (2015).","journal-title":"Parasite Immunol"},{"key":"BFsrep34448_CR6","doi-asserted-by":"publisher","first-page":"1741","DOI":"10.1074\/jbc.M508955200","volume":"281","author":"DL Gardiner","year":"2005","unstructured":"Gardiner, D. L. et al. Overexpression of leucyl aminopeptidase in Plasmodium falciparum parasites. Target for the antimalarial activity of bestatin. J Biol Chem. 281, 1741\u20131745 (2005).","journal-title":"J Biol Chem"},{"key":"BFsrep34448_CR7","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1111\/pim.12157","volume":"37","author":"A Parlog","year":"2015","unstructured":"Parlog, A., Schl\u00fcter, D. & Dunay, I. R. Toxoplasma gondii-induced neuronal alterations. Parasite Immunol. 37, 159\u2013170 (2015).","journal-title":"Parasite Immunol"},{"key":"BFsrep34448_CR8","doi-asserted-by":"publisher","first-page":"1385","DOI":"10.1016\/j.ijpara.2009.04.003","volume":"39","author":"G Pappas","year":"2009","unstructured":"Pappas, G., Roussos, N. & Falagas, M. E. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. International Journal for Parasitology. 39, 1385\u20131394 (2009).","journal-title":"International Journal for Parasitology"},{"key":"BFsrep34448_CR9","doi-asserted-by":"publisher","first-page":"33","DOI":"10.7326\/0003-4819-116-1-33","volume":"116","author":"B Dannemann","year":"1992","unstructured":"Dannemann, B. et al. Treatment of toxoplasmic encephalitis in patients with AIDS-a randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulfadiazine. Ann. Intern. Med. 116, 33\u201334 (1992).","journal-title":"Ann. Intern. Med."},{"key":"BFsrep34448_CR10","doi-asserted-by":"publisher","first-page":"1360","DOI":"10.1128\/AAC.40.6.1360","volume":"40","author":"JM Jacobson","year":"1996","unstructured":"Jacobson, J. M. et al. Pyrimethamine pharmacokinetics in human immunodeficiency virus-positive patients seropositive for Yoxoplasma gondii. Antimicrob. Agents Chemother. 40, 1360\u20131365 (1996).","journal-title":"Agents Chemother."},{"key":"BFsrep34448_CR11","doi-asserted-by":"crossref","unstructured":"Hooper, N. M. & Lendeckel, U. Aminopeptidases in Biology and Disease (Proteases in Biology and Disease). Springer, New York (2004).","DOI":"10.1007\/978-1-4419-8869-0"},{"key":"BFsrep34448_CR12","doi-asserted-by":"publisher","first-page":"30817","DOI":"10.1074\/jbc.M704938200","volume":"282","author":"F Teuscher","year":"2007","unstructured":"Teuscher, F. et al. The M18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum. J. Biol. Chem. 282, 30817\u201330826 (2007).","journal-title":"J. Biol. Chem."},{"key":"BFsrep34448_CR13","doi-asserted-by":"crossref","first-page":"6979","DOI":"10.1016\/S0021-9258(18)83527-X","volume":"264","author":"YH Chang","year":"1989","unstructured":"Chang, Y. H. & Smith, J. A. Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae. J. Biol. Chem. 264, 6979\u20136983 (1989).","journal-title":"J. Biol. Chem."},{"key":"BFsrep34448_CR14","doi-asserted-by":"publisher","first-page":"15961","DOI":"10.1074\/jbc.273.26.15961","volume":"273","author":"S Wilk","year":"1998","unstructured":"Wilk, S., Wilk, E. & Magnusson, R. P. Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase. J. Biol. Chem. 273, 15961\u201315970 (1998).","journal-title":"J. Biol. Chem."},{"key":"BFsrep34448_CR15","doi-asserted-by":"publisher","first-page":"176","DOI":"10.1016\/S0003-9861(02)00494-0","volume":"407","author":"S Wilk","year":"2002","unstructured":"Wilk, S., Wilk, E. & Magnusson, R. P. Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase. Arch. Biochem. Biophys. 407, 176\u2013183 (2002).","journal-title":"Arch. Biochem. Biophys."},{"key":"BFsrep34448_CR16","doi-asserted-by":"publisher","first-page":"13356","DOI":"10.1074\/jbc.M112.347518","volume":"287","author":"Y Chen","year":"2012","unstructured":"Chen, Y. et al. Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase. J. Biol. Chem. 287, 13356\u201313370 (2012).","journal-title":"J. Biol. Chem."},{"key":"BFsrep34448_CR17","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1016\/j.bbrc.2014.03.109","volume":"447","author":"DD Nguyen","year":"2014","unstructured":"Nguyen, D. D. et al. Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa . Biochem Biophys Res Commun. 447, 101\u2013107 (2014).","journal-title":"Biochem Biophys Res Commun"},{"key":"BFsrep34448_CR18","doi-asserted-by":"publisher","first-page":"35978","DOI":"10.1074\/jbc.M703643200","volume":"282","author":"S Dalal","year":"2007","unstructured":"Dalal, S. & Klemba, M. Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum. J. Biol. Chem. 282, 35978\u201335987 (2007).","journal-title":"J. Biol. Chem."},{"key":"BFsrep34448_CR19","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1111\/j.1365-2958.2009.06600.x","volume":"72","author":"MA Dura","year":"2009","unstructured":"Dura, M. A. et al. The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea. Mol. Microbiol. 72, 26\u201340 (2009).","journal-title":"Mol. Microbiol."},{"key":"BFsrep34448_CR20","doi-asserted-by":"publisher","first-page":"682","DOI":"10.1017\/S0031182011000199","volume":"138","author":"JM Kang","year":"2011","unstructured":"Kang, J. M., Ju, H. L., Sohn, W. M. & Na, B. K. Molecular cloning and characterization of a M17 leucine aminopeptidase of Cryptosporidium parvum. Parasitology. 138, 682\u2013690 (2011).","journal-title":"Parasitology"},{"key":"BFsrep34448_CR21","doi-asserted-by":"publisher","first-page":"1107","DOI":"10.1177\/1087057114525852","volume":"19","author":"T Spicer","year":"2014","unstructured":"Spicer, T. et al. Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening. J. Biomol. Screen. 19, 1107\u20131115 (2014).","journal-title":"J. Biomol. Screen."},{"key":"BFsrep34448_CR22","first-page":"e01188","volume":"15","author":"Z Dou","year":"2014","unstructured":"Dou, Z., McGovern, O. L., Di, Cristina, M. & Carruthers, V. B. Toxoplasma gondii ingests and digests host cytosolic proteins. MBio. 15, e01188\u201314 (2014).","journal-title":"MBio"},{"key":"BFsrep34448_CR23","doi-asserted-by":"publisher","first-page":"495","DOI":"10.1016\/j.jmb.2012.06.006","volume":"422","author":"KK Sivaraman","year":"2012","unstructured":"Sivaraman, K. K. et al. X-ray crystal structure and specificity of the Plasmodium falciparum malaria aminopeptidase PfM18AAP. J. Mol. Biol. 422, 495\u2013507 (2012).","journal-title":"J. Mol. Biol."},{"key":"BFsrep34448_CR24","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1186\/1472-6807-12-14","volume":"12","author":"A Chaikuad","year":"2012","unstructured":"Chaikuad, A. et al. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family. BMC. Struct. Biol. 12, 14 (2012).","journal-title":"BMC. Struct. Biol."},{"key":"BFsrep34448_CR25","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.molbiopara.2009.11.005","volume":"170","author":"H Jia","year":"2010","unstructured":"Jia, H. et al. Characterization of a leucine aminopeptidase from Toxoplasma gondii. Mol. Biochem. Parasitol. 170, 1\u20136 (2010).","journal-title":"Mol. Biochem. Parasitol."},{"key":"BFsrep34448_CR26","doi-asserted-by":"publisher","first-page":"1659","DOI":"10.2174\/1381612023394197","volume":"8","author":"PJ Rosenthal","year":"2002","unstructured":"Rosenthal, P. J., Sijwali, P. S., Singh, A. & Shenai, B. R. Cysteine proteases of malaria parasites: targets for chemotherapy. Curr Pharm Des. 8, 1659\u20131672 (2002).","journal-title":"Curr Pharm Des"},{"key":"BFsrep34448_CR27","doi-asserted-by":"publisher","first-page":"690","DOI":"10.1038\/nrd3053","volume":"9","author":"M Drag","year":"2010","unstructured":"Drag, M. & Salvesen, G. S. Emerging principles in protease-based drug discovery. Nat. Rev. Drug Discov. 9, 690\u2013701 (2010).","journal-title":"Nat. Rev. Drug Discov."},{"key":"BFsrep34448_CR28","doi-asserted-by":"publisher","first-page":"e0115859","DOI":"10.1371\/journal.pone.0115859","volume":"10","author":"A Paiardini","year":"2015","unstructured":"Paiardini, A. et al. & McGowan, S. Screening the Medicines for Malaria Venture \u201cMalaria Box\u201d against the Plasmodium falciparum Aminopeptidases, M1, M17 and M18. PLoS One. 10, e0115859 (2015).","journal-title":"PLoS One"},{"key":"BFsrep34448_CR29","doi-asserted-by":"publisher","first-page":"2069","DOI":"10.1074\/jbc.M609251200","volume":"282","author":"CM Stack","year":"2007","unstructured":"Stack, C. M. et al. Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. J. Biol. Chem. 282, 2069\u20132080 (2007).","journal-title":"J. Biol. Chem."},{"key":"BFsrep34448_CR30","doi-asserted-by":"publisher","first-page":"828","DOI":"10.1016\/j.sbi.2013.07.015","volume":"23","author":"S McGowan","year":"2013","unstructured":"McGowan, S. Working in concert: the metalloaminopeptidases from Plasmodium falciparum. Current Opinion in Structural Biology. 23, 828\u2013835 (2013).","journal-title":"Current Opinion in Structural Biology"},{"key":"BFsrep34448_CR31","first-page":"e01114","volume":"5","author":"B Shen","year":"2014","unstructured":"Shen, B., Brown, K. M., Lee, T. D. & Sibley, L. D. Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR\/CAS9. MBio. 5, e01114\u2013e01114 (2014).","journal-title":"MBio"},{"key":"BFsrep34448_CR32","doi-asserted-by":"publisher","first-page":"e100450","DOI":"10.1371\/journal.pone.0100450","volume":"9","author":"SM Sidik","year":"2014","unstructured":"Sidik, S. M. et al. Efficient genome engineering of Toxoplasma gondii using CRISPR\/Cas9. PLoS One. 9, e100450 (2014).","journal-title":"PLoS One"},{"key":"BFsrep34448_CR33","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1016\/j.ijpara.2014.09.003","volume":"45","author":"J Zheng","year":"2015","unstructured":"Zheng, J., Jia, H. L. & Zheng, Y. H. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR\/Cas9. Int. J. Parasitol. 45, 141\u2013148 (2015).","journal-title":"Int. J. Parasitol."},{"key":"BFsrep34448_CR34","doi-asserted-by":"publisher","first-page":"e62906","DOI":"10.1371\/journal.pone.0062906","volume":"8","author":"T Spangenberg","year":"2013","unstructured":"Spangenberg, T. et al. The open access malaria box: a drug discovery catalyst for neglected diseases. PLoS One 8, e62906 (2013).","journal-title":"PLoS One"},{"key":"BFsrep34448_CR35","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1016\/S0091-679X(01)65014-3","volume":"65","author":"H Sch\u00e4gger","year":"2001","unstructured":"Sch\u00e4gger, H. Blue Native Gels to Isolate Protein Complexes from Mitochondria. Meth. Cell Biol. 65, 231\u2013244 (2001).","journal-title":"Meth. Cell Biol"},{"key":"BFsrep34448_CR36","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1126\/science.8469986","volume":"260","author":"D Soldati","year":"1993","unstructured":"Soldati, D. & Boothroyd, J. C. Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science. 260, 349\u2013352 (1993).","journal-title":"Science"},{"key":"BFsrep34448_CR37","doi-asserted-by":"publisher","first-page":"1271","DOI":"10.1128\/AAC.45.4.1271-1277.2001","volume":"45","author":"MG Reynolds","year":"2001","unstructured":"Reynolds, M. G., Oh, J. & Roos, D. S. In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase. Antimicrob. Agents Chemother. 45, 1271\u20131277 (2001).","journal-title":"Antimicrob. Agents Chemother."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/srep34448.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep34448","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep34448.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,5]],"date-time":"2023-01-05T06:02:56Z","timestamp":1672898576000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/srep34448"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,9,28]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2016,10,31]]}},"alternative-id":["BFsrep34448"],"URL":"https:\/\/doi.org\/10.1038\/srep34448","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,9,28]]},"assertion":[{"value":"16 May 2016","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 September 2016","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 September 2016","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"34448"}}