title: The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.
abstract: NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided.
  
  
  
  Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points.
  
  
  
  We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress.
  
  
  
  The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
authors.forename: Iván
authors.initials: I
authors.lastname: Pastor-Fernández
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Regidor-Cerrillo
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
authors.forename: Gema
authors.initials: G
authors.lastname: Álvarez-García
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Marugán-Hernández
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
authors.forename: Paula
authors.initials: P
authors.lastname: García-Lunar
authors.affiliation: Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, CH-3012, Berne, Switzerland.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hemphill
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. luis.ortega@vet.ucm.es.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Ortega-Mora
mesh_terms: D002118:Calcium; D015971:Gene Expression Regulation, Enzymologic; D018688:Neospora / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D020080:Tandem Repeat Sequences
chemical_list: D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D043583:Nucleoside-Triphosphatase; D002118:Calcium
keywords: Dense granule protein; NTPase; Neospora caninum; Tachyzoite lytic cycle