title: Identification and characterization of serine proteinase inhibitors from Neospora caninum.
abstract: Two cDNA clones obtained from the Neospora caninum Expressed Sequence Tag project were selected by their homology with the Toxoplasma gondii serine proteinase inhibitor (serpin) gene, TgPI-1 and TgPI-2. One of them, named NcPI-H, showed several premature stop codons. The other cDNA, named NcPI-S, encoded a 79 amino acid protein containing a putative signal peptide and only one non-classical Kazal domain. Two other N. caninum EST sequences (NcEST1 and NcEST2) and one from Eimeria tenella (EtPI-S) were retrieved from the database. Amino acid sequence analysis suggested that NcEST1 and NcEST2 might be the N. caninum counterparts of TgPI-1 and TgPI-2, respectively. EtEST-S, as NcPI-S, is a single domain serpin. The open reading frame encoding the mature version of NcPI-S was expressed as recombinant protein, fused to a 6 histidine tag in Escherichia coli. Specific rabbit antiserum generated against the recombinant NcPI-S was used in immunoblot assays. Bands of 20, 30, 40, and 66-kDa were detected by SDS-PAGE of whole parasite homogenate. In addition, when an anti-TgPI-1 serum was used, bands of 25 and 35-kDa were detected indicating that there is no cross-reactivity between both serpins, and showing as well, the presence of another putative serpin in N. caninum. The recombinant protein NcPI-S, inhibited bacterial subtilisin completely, and showed lower inhibitory capacity on human neutrophil elastase, animal trypsin, and chymotrypsin, suggesting differences in effectiveness.
authors.affiliation: Departamento de Parasitología, ANLIS Dr. Carlos G. Malbran, Ciudad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Susana
authors.initials: S
authors.lastname: Bruno
authors.forename: Vilma G
authors.initials: VG
authors.lastname: Duschak
authors.forename: Bibiana
authors.initials: B
authors.lastname: Ledesma
authors.forename: Marcela
authors.initials: M
authors.lastname: Ferella
authors.forename: Björn
authors.initials: B
authors.lastname: Andersson
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Guarnera
authors.forename: Sergio O
authors.initials: SO
authors.lastname: Angel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D018688:Neospora / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors