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Identification and characterization of serine proteinase inhibitors from Neospora caninum
Susana Bruno1, Vilma G Duschak, Bibiana Ledesma
1Departamento de Parasitología, ANLIS Dr. Carlos G. Malbran, Ciudad de Buenos Aires, Buenos Aires, Argentina.
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.
Insights
This study identifies and characterizes NcPI-S, a novel serine proteinase inhibitor from Neospora caninum. The recombinant NcPI-S protein effectively inhibits bacterial subtilisin, indicating its potential role in parasite biology.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Neospora caninum is an apicomplexan parasite causing significant economic losses in the livestock industry.
- Serine proteinase inhibitors (serpins) are crucial regulators of proteolytic activity in various organisms, including parasites.
- Understanding N. caninum serpins can provide insights into parasite survival and host-pathogen interactions.
Purpose of the Study:
- To identify and characterize novel serine proteinase inhibitors from Neospora caninum.
- To investigate the functional properties and potential inhibitory activity of a specific N. caninum serpin, NcPI-S.
- To explore the cross-reactivity of N. caninum serpins with Toxoplasma gondii serpins.
Main Methods:
- Expressed Sequence Tag (EST) analysis to identify putative serpin genes in N. caninum.
- Recombinant expression of NcPI-S in Escherichia coli using a 6 histidine tag.
- Generation of specific rabbit antiserum against recombinant NcPI-S.
- Immunoblot assays using SDS-PAGE of parasite homogenates.
- Enzyme inhibition assays using bacterial subtilisin, human neutrophil elastase, trypsin, and chymotrypsin.
Main Results:
- Two cDNA clones with homology to Toxoplasma gondii serpins were identified in N. caninum, named NcPI-H and NcPI-S.
- NcPI-S encodes a protein with a putative signal peptide and a single non-classical Kazal domain.
- Recombinant NcPI-S demonstrated complete inhibition of bacterial subtilisin and partial inhibition of human neutrophil elastase, trypsin, and chymotrypsin.
- Immunoblot assays detected NcPI-S in N. caninum homogenates and revealed no cross-reactivity with anti-TgPI-1 serum, suggesting the presence of other serpins.
Conclusions:
- NcPI-S is a novel single-domain serpin identified in Neospora caninum.
- The functional characterization of NcPI-S suggests its potential role in inhibiting bacterial proteases, possibly contributing to parasite survival.
- Further investigation is warranted to elucidate the precise biological function and host interactions of NcPI-S and other N. caninum serpins.