GRA 14, a novel dense granule protein from Neospora caninum.

Dense granule protein (GRA) is a secreted protein in Neospora caninum and Toxoplasma gondii, which plays an important role in forming parasitophorous vacuole (PV) [1–4]. GRAs are thought to remodel and maintain the environment of the PV for intracellular survival and replication in T. gondii [5]. The precise function of GRAs is still unknown [6–9]. TgGRA14 protein is a new GRA protein that it is highly expressed and targeted to PV membrane and intravacuolar [5]. However, the GRA14 of N. caninum (NcGRA14) has not been identified. The NcGRA14 gene was predicted in ToxoDB (http ://toxodb.org/toxo/). NcGRA14 has 60.5% nucleotide similarity with the TgGRA14. DNA sequencing analysis indicated that NcGRA14 gene contained a long open reading frame (1215 bp) without introns. NcGRA14 appeared to contain one methionine translation start codon in the N-terminal portion of the encoded protein (Fig. 1). The whole amino acid (aa) sequences is 404 aa. The predicted potential site of signal peptidase is in aa 1–36, while the transmembrane domain is in aa 285–304. The predicted size of the mature peptide after removal of the signal sequence is about 44.6 kDa. The bioinformatics prediction of NcGRA14 protein is a transport protein involved in telomerase ribonucleoprotein complex–RNA binding. To determine whether the putative NcGRA14 gene encoded a functional protein, the cDNA of NcGRA14 was amplified by PCR. Primers used for amplification of the full length of GRA14 gene were P1F (50-ATGCAGGG CGCAACGGGG-30) and the P1R (50-TTAGTAGACCG AGTTACCTGAGG-30). In addition, the primers P2F (50cgGGATCCATGGGCTTGGGCGAGATTTCG-30) and P2R (50-ccgCTCGAGCCGAGACTTGCCTCCGGAT-30) were designed to amplify the expression sequence of NcGRA14 (747 bp). Then the product was cloned into the prokaryotic expression vector pET-28a (Novagen, Gibbstown, USA) and transformed into Escherichia coli. The recombinant NcGRA14 (rNcGRA14) fused to a His6-tag was expressed and purified using HisTrap FF purification columns (Novagen). The purified protein was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot analysis using sera from mice immunized with Nc1 tachyzoites as previously described for Toxoplasma tachyzoites [10]. A polyclonal antibody against the rNcGRA14 recognized an approximate 45-kDa GRA14 protein (Fig. 2). To confirm GRA localization, intracellular and extracellular immunoflourescence assay (IFA) was carried out as described previously [5]. For intracellular IFA, N. caninum tachyzoites were used to infect the human foreskin fibroblasts on coverslips for 16–24 h. The coverslips were fixed in 3.5% formaldehyde/phosphate buffered saline (PBS) for 15 min. The coverslips were then washed in PBS and blocked in PBS/3% bovine serum albumin (BSA). For complete permeabilization of formaldehyde-fixed samples, cells were permeabilized in a solution containing PBS/3% BSA/0.25% Triton X-100 for 30 min. Samples were then incubated with primary antibody (1:100) in PBS/3% BSA or PBS/3% BSA/0.25%Triton X-100 for 1 h. The samples were then washed five times in PBS and incubated with the fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (Protein Tech Group, Chicago, USA) diluted 1:100 in PBS/3% BSA. For extracellular parasites IFA, N. caninum tachyzoites were harvested and purified. Then tachyzoites were resuspended in 200 ml of cold PBS, spotted on glass slides, air-dried, fixed, permeabilized and then processed for immunoflourescence as intracellular IFAs described above. All observations were performed on a confocal microscope (Leica TCS SP5 II; Leica, Solms, Germany). Immunoflourescence analysis showed that NcGRA14 protein existed in tachyzoites and secreted to the PV in 24 h (Fig. 3). In conclusion, we present a novel secreted GRA protein in N. caninum that has not been reported, further work needs to be done in the future including (i) whether NcGRA14 protein is a diagnosis antigen or cross-reacting antigen in T. gondii? (ii) whether the NcGRA14 protein is a vaccine candidate? (iii) can overexpression or knockdown of GRA14 strains affect the invasion in vitro or in vivo? Acta Biochim Biophys Sin 2013, 45: 607–609 | a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt036. Advance Access Publication 30 May 2013