Aminopeptidase N1 (EtAPN1), an M1 Metalloprotease of the Apicomplexan Parasite Eimeria tenella, Participates in Parasite Development

ABSTRACT Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs.

[1]  Ben M. Webb,et al.  Comparative Protein Structure Modeling Using MODELLER , 2016, Current protocols in bioinformatics.

[2]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[3]  S. G. Monteiro,et al.  Meta-analysis of the performance variation in broilers experimentally challenged by Eimeria spp. , 2013, Veterinary parasitology.

[4]  M. Olivier,et al.  Leishmania evades host immunity by inhibiting antigen cross-presentation through direct cleavage of the SNARE VAMP8. , 2013, Cell host & microbe.

[5]  M. Katrib,et al.  Stage-specific expression of protease genes in the apicomplexan parasite, Eimeria tenella , 2012, BMC Genomics.

[6]  M. Katrib,et al.  Eimeripain, a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella , 2012, PloS one.

[7]  J. Whisstock,et al.  Fingerprinting the Substrate Specificity of M1 and M17 Aminopeptidases of Human Malaria, Plasmodium falciparum , 2012, PloS one.

[8]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[9]  Ludovic C. Gillet,et al.  Cysteine Cathepsins S and L Modulate Anti-angiogenic Activities of Human Endostatin* , 2011, The Journal of Biological Chemistry.

[10]  J. Whisstock,et al.  Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases , 2011, Proceedings of the National Academy of Sciences.

[11]  S. Dalal,et al.  Distribution and Biochemical Properties of an M1-family Aminopeptidase in Plasmodium falciparum Indicate a Role in Vacuolar Hemoglobin Catabolism* , 2011, The Journal of Biological Chemistry.

[12]  Jung-Mi Kang,et al.  Plasmodium vivax: collaborative roles for plasmepsin 4 and vivapains in hemoglobin hydrolysis. , 2011, Experimental parasitology.

[13]  A. Tewari,et al.  Control of poultry coccidiosis: changing trends , 2011, Journal of parasitic diseases : official organ of the Indian Society for Parasitology.

[14]  J. Whisstock,et al.  Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase. , 2011, Journal of medicinal chemistry.

[15]  M. Katrib,et al.  Chasing the golden egg: vaccination against poultry coccidiosis , 2010, Parasite immunology.

[16]  J. Nyalwidhe,et al.  Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole , 2010, Malaria Journal.

[17]  M. Bogyo,et al.  Aminopeptidase Fingerprints, an Integrated Approach for Identification of Good Substrates and Optimal Inhibitors* , 2009, The Journal of Biological Chemistry.

[18]  J. Yates,et al.  Proteomic comparison of four Eimeria tenella life‐cycle stages: Unsporulated oocyst, sporulated oocyst, sporozoite and second‐generation merozoite , 2009, Proteomics.

[19]  S. Dalal,et al.  Evidence for Catalytic Roles for Plasmodium falciparum Aminopeptidase P in the Food Vacuole and Cytosol* , 2009, The Journal of Biological Chemistry.

[20]  M. Tomita,et al.  Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs , 2009, Proceedings of the National Academy of Sciences.

[21]  L. Knoll,et al.  The ins and outs of nuclear trafficking: unusual aspects in apicomplexan parasites. , 2009, DNA and cell biology.

[22]  Jun O. Liu,et al.  Fumagillin and fumarranol interact with P. falciparum methionine aminopeptidase 2 and inhibit malaria parasite growth in vitro and in vivo. , 2009, Chemistry & biology.

[23]  J. Whisstock,et al.  Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase , 2009, Proceedings of the National Academy of Sciences.

[24]  B. Matthews,et al.  Structural basis for the unusual specificity of Escherichia coli aminopeptidase N. , 2008, Biochemistry.

[25]  S. Dalal,et al.  Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum* , 2007, Journal of Biological Chemistry.

[26]  C. Bourdieu,et al.  Eimeria tenella enolase and pyruvate kinase: a likely role in glycolysis and in others functions. , 2006, International journal for parasitology.

[27]  Ben M. Webb,et al.  Comparative Protein Structure Modeling Using Modeller , 2006, Current protocols in bioinformatics.

[28]  K. Miska,et al.  PARTIAL PURIFICATION AND CHARACTERIZATION OF AN AMINOPEPTIDASE FROM EIMERIA TENELLA , 2005, The Journal of parasitology.

[29]  L. Callado,et al.  Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain , 2005, Neuroscience Letters.

[30]  L. Holm,et al.  The Pfam protein families database , 2005, Nucleic Acids Res..

[31]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[32]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[33]  G. Mulcahy,et al.  Comparative development of Eimeria tenella (Apicomplexa) in host cells in vitro , 2003, Parasitology Research.

[34]  I. Florent,et al.  Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase , 2002, Parasitology.

[35]  P. Okhuysen,et al.  Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene. , 2002, International journal for parasitology.

[36]  M. Löhn,et al.  Cell Cycle Retardation in Monocytoid Cells Induced by Aminopeptidase N (CD13) , 2002, Leukemia & lymphoma.

[37]  M. Rodier,et al.  Toxoplasma gondii: purification and characterization of an immunogenic metallopeptidase. , 2000, Experimental parasitology.

[38]  P. Brown,et al.  A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein. , 2000, Molecular and biochemical parasitology.

[39]  R. Williams.  A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry. , 1999, International journal for parasitology.

[40]  M. Monsigny,et al.  A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages. , 1998, Molecular and biochemical parasitology.

[41]  U. Lendeckel,et al.  Inhibition of alanyl aminopeptidase induces MAP-kinase p42/ERK2 in the human T cell line KARPAS-299. , 1998, Biochemical and biophysical research communications.

[42]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[43]  M. F. Nankya-Kitaka,et al.  Plasmodium chabaudi chabaudi and P. falciparum : inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin , 1998, Parasitology Research.

[44]  B. Roques,et al.  Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights into its mechanism of action. , 1998, Biochemistry.

[45]  N. Rawlings,et al.  Evolutionary families of peptidases. , 1993, The Biochemical journal.

[46]  R. Ashmun,et al.  Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules results in loss of epitopes that mediate binding of inhibitory antibodies. , 1992, Blood.

[47]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[48]  C. C. Norton,et al.  The oocyst sporulation time of Eimeria species from the fowl , 1983, Parasitology.

[49]  L. Mcdougald,et al.  Comparative in vitro development of precocious and normal strains of Eimeria tenella (Coccidia). , 1976, The Journal of protozoology.

[50]  T. K. Jeffers.  Attenuation of Eimeria tenella through selection for precociousness. , 1975, The Journal of parasitology.

[51]  W. Raether,et al.  Improved techniques for the in vitro cultivation ofEimeria tenella in primary chick kidney cells , 2004, Parasitology Research.

[52]  N. Rawlings,et al.  Evolutionary families of metallopeptidases. , 1995, Methods in enzymology.