Skip to main content

Advertisement

SpringerLink
Log in

Localization and enzyme kinetics of aminopeptidase N3 from Toxoplasma gondii

  • Protozoology - Short Communication
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Aminopeptidase N is an important metalloenzyme from the M1 zinc metallopeptidase family, which is present in numerous apicomplexan parasites, including Plasmodium, Eimeria, and Cryptosporidium. Aminopeptidase N is a potential drug target, and hence, its properties have been widely investigated. In the current study, the cellular localization and enzyme characteristics of Toxoplasma gondii aminopeptidase N3 (TgAPN3) were evaluated in vitro. Cellular localization analysis revealed that TgAPN3 and GRA protein were co-located in the organelle and parasitophorous vacuole of T. gondii. The secretion assay showed that TgAPN3 could be co-secreted from the tachyzoites with GRA protein. A functional recombinant Toxoplasma aminopeptidase N3 (rTgAPN3) was produced in Escherichia coli. The enzyme activity was first determined using a fluorogenic H-Ala-MCA substrate. Some activity of rTgAPN3 was observed between pH 3.0 and 8.0, with a peak at pH 7.0. The activity was significantly enhanced in the presence of Co2+ ions. Substrate specificity of rTgAPN3 was then evaluated. The enzyme showed a preference for substrates containing N-terminal Ala residues, followed by Tyr and Cys. The rTgAPN3 activity was significantly inhibited by bestatin and phebestatin. In general, TgAPN3 was a structurally conserved member of the M1 family, although it also displayed unique biochemical characteristics. These results lay the foundation for a functional study of TgAPN3 and constitute its putative identification as a drug target.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

    Article  Google Scholar 

  • Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res

  • Carruthers VB, Sibley LD (1999) Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol. 31(2):421–428

    Article  CAS  Google Scholar 

  • Dalal S, Klemba M (2007) Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum. J Biol Chem 282:35978–35987

    Article  CAS  Google Scholar 

  • Drag M, Bogyo M, Ellman JA, Salvesen GS (2010) Aminopeptidase fingerprints, an integrated approach for identification of good substrates and optimal inhibitors. Journal of Biological Chemistry 285:3310–3318

    Article  CAS  Google Scholar 

  • Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL, Bateman A (2008) The Pfam protein families database. Nucleic Acids Res 36:D281–D288

    Article  CAS  Google Scholar 

  • González-Bacerio J, Maluf SEC, Méndez Y, Pascual I, Florent I, Melo PMS, Budu A, Ferreira JC, Moreno E, Carmona AK, Rivera DG, Alonso D, Rivero M, Gazarini ML (2017) KBE009: an antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library. Bioorg Med Chem 25(17):4628–4636

    Article  Google Scholar 

  • Gras S, Byzia A, Gilbert FB, McGowan S, Drag M, Silvestre A, Niepceron A, Lecaille F, Lalmanach G, Brossier F (2014) Aminopeptidase N1 (EtAPN1), an M1 metalloprotease of the apicomplexan parasite Eimeria tenella, participates in parasite development. Eukaryot Cell 13(7):884–895

    Article  Google Scholar 

  • Gumpena R, Kishor C, Ganji RJ, Addlagatta A (2011) Discovery of α, β- and α, γ-diamino acid scaffolds for the inhibition of M1 family aminopeptidases. ChemMedChem 6(11):1971–1976

    Article  CAS  Google Scholar 

  • Harbut MB, Velmourougane G, Dalal S, Reiss G, Whisstock JC, Onder O, Brisson D, McGowan S, Klemba M, Greenbaum DC (2011) Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases. Proc Natl Acad Sci U S A 108:E526–E534

    Article  CAS  Google Scholar 

  • Jia H, Nishikawa Y, Luo Y, Yamagishi J, Sugimoto C, Xuan X (2010) Characterization of a leucine aminopeptidase from Toxoplasma gondii. Mol Biochem Parasitol 170:1–6

    Article  CAS  Google Scholar 

  • Kim KI, Baek SH, Hong YM, Kang MS, Ha DB, Goldberg AL, Chung CH (1995) Purification and characterization of protease Ci, a cytoplasmic metalloendoprotease in Escherichia coli. J Biol Chem 270:29799–29805

    Article  CAS  Google Scholar 

  • Li Q, Jia H, Cao S, Zhang Z, Zheng J, Zhang Y (2017) Biochemical characterization of aminopeptidase N2 from Toxoplasma gondii. J Vet Med Sci 79(8):1404–1411

    Article  CAS  Google Scholar 

  • Page MJ, Di Cera E (2008) Evolution of peptidase diversity. J Biol Chem 283(44):30010–30014

    Article  CAS  Google Scholar 

  • Poreba M, McGowan S, Skinner-Adams TS, Trenholme KR, Gardiner DL, Whisstock JC, To J, Salvesen GS, Dalton JP, Drag M (2012) Fingerprinting the substrate specificity of M1 and M17 aminopeptidases of human malaria, Plasmodium falciparum. PLoS One 7:e31938

    Article  CAS  Google Scholar 

  • Rawlings ND, Barrett AJ (1993) Evolutionary families of peptidases. Biochem J 290:205–218

    Article  CAS  Google Scholar 

  • Rawlings ND, Barrett AJ (1995) Evolutionary families of metallopeptidases. Methods Enzymol 248:183–228

    Article  CAS  Google Scholar 

  • Rieux A, Gras S, Lecaille F, Niepceron A, Katrib M, Smith NC, Lalmanach G, Brossier F (2012) Eimeripain, a cathepsin B-like cysteine protease, expressed throughout sporulation of the apicomplexan parasite Eimeria tenella. PLoS One 7:e31914

    Article  CAS  Google Scholar 

  • Skinner-Adams TS, Stack CM, Trenholme KR, Brown CL, Grembecka J, Lowther J, Mucha A, Drag M, Kafarski P, McGowan S (2010) Plasmodium falciparum neutral aminopeptidases: new targets for anti-malarials. Trends Biochem Sci 35:53–61

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  CAS  Google Scholar 

  • Zheng J, Cheng Z, Jia H, Zheng Y (2016) Characterization of aspartyl aminopeptidase from Toxoplasma gondii. Sci Rep 6:34448

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. K.A. Joiner (Yale University) for providing the pHXNTPHA and pDMG plasmids generated by Dr. X. Xuan (Obihiro University of Agriculture and Veterinary Medicine).

Funding

This work is supported by a grant awarded to J.Z. by the National Natural Science Foundation of China (Grant 31402184) and by the Natural Science Foundation of Heilongjiang Province of China (Grants C2015063 and C2015065).

Author information

Authors and Affiliations

  1. Department of Animal Medicine, Yanbian University, Yanji, 133000, China

    Wenhua Lu & Cheng Lu

  2. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, China

    Shinuo Cao, Zhaoxia Zhang, Honglin Jia & Jun Zheng

  3. State Key Laboratory of Veterinary Biotechnology, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China

    Qian Zhang

Authors
  1. Wenhua Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cheng Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shinuo Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhaoxia Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Honglin Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jun Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Cheng Lu or Jun Zheng.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Section Editor: David S. Lindsay

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lu, W., Lu, C., Zhang, Q. et al. Localization and enzyme kinetics of aminopeptidase N3 from Toxoplasma gondii. Parasitol Res 119, 357–364 (2020). https://doi.org/10.1007/s00436-019-06512-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-019-06512-6

Keywords

Search

Navigation