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Neospora caninum is an obligatory intracellular Apicomplexan protozoan parasite that must invade cells for its development. Thrombospondin-related proteins are Acomponents of the parasite micronemes, organelles that have an important role in Apicomplexa adhesion and invasion and are currently promising targets for malaria vaccine research. In Neospora caninum, two thrombospondin-related protein homologues have been described: NcMIC2 and NcMIC2-like1. This work extends the information about microneme proteins in N. caninum by employing mass spectrometry data on tachyzoite proteins. The proteins were isolated by two-dimensional SDS-PAGE, uncovering some details not described in previous works that were based on one-dimensional SDS-PAGE. The compilation of NcMIC2-like1 and NcMIC2 data in a unique approach will guide future studies related to the blockage and manipulation of these proteins, improving the alternatives for neosporosis prevention.
Neosporosis is a disease related to economic losses in livestock industry due to fetal abortion, stillbirth, clinical and subclinical diseases, impaired milk production, neonatal deaths, and reduced fertility. Neospora caninum is an obligate intracellular parasite that invades host cells by a conserved mechanism, which is typical to the Apicomplexa phylum. The invasion process is active, parasite-coordinated and multifactorial, involving the release of proteins from organelles such as micronemes, rhoptries, and dense granules. Apicomplexans use a substrate locomotion process called gliding motility that involves both the parasite cytoskeleton and the host cell. In this context, the thrombospondin-related anonymous proteins (TRAP or MIC2 proteins) are considered key elements, since they interact simultaneously with host cell receptors and the actin-myosin motor of the parasite. TRAPs or MIC2 proteins in Apicomplexan parasites have two types of extracellular adhesive domains, one von Willebrand factor type A domain (or integrin IA) and at least one Thrombospondin type 1 domain (TSP-1). There are also a signal peptide, a transmembrane region with a rhomboid cleavage site and an acidic cytoplasmic tail with a conserved tryptophan residue close to the C-terminal end. TRAP family members include Plasmodium falciparum (PfTRAP), Toxoplasma gondii (TgMIC2), and N. caninum (NcMIC 2 and NcMIC2-like1) homologues.
The total disruption of Tgmic2 confirmed the role of this protein in parasite replication and growth. Based on the relevance of Apicomplexan TRAP proteins and their potential in the life cycle of N. caninum, our research group is interested in the molecular characterization of NcMIC2-like1. The present work includes a proteomic study of MIC2 proteins of N. caninum (NcMIC2-like1 and NcMIC2), improving the data about this protein family, closely related to the parasite invasion mechanism.
The aim of this work was to detect and identify TRAP proteins from N. caninum (NcMIC2-like1 and NcMIC2) using 2D SDS-PAGE and mass spectrometry.
A shotgun (LC-MS/MS) proteomic approach on N. caninum, both from N. caninum tachyzoite lysate and secreted fraction (ESA), enabled the identification of a massive number of proteins, including NcMIC2-like1 and NcMIC2. In total, 490 peptide spectrum matches (PSMs) from NcMIC2-like1 and 168 from NcMIC2 were obtained, with a considerably higher presence of NcMIC2-like1 than NcMIC2 in the ESA (448 and 126 PSMs, respectively) and the same number of PSMs (42) in the tachyzoite extract (Fig. 1A). Among all the 615 proteins identified in ESA, NcMIC2-like1 was the 21st most frequently identified and NcMIC2 ranked 93rd.
Since NcMIC2-like1 and NcMIC2 are predicted to be acidic (pI 4.5 for both), 2D gels were run carrying tachyzoite extracts using narrow acidic strips of 3–5.6 NL (nonlinear). Mass spectrometry revealed spots 1 and 2 (Fig. 1B) as NcMIC2-like1, whereas spots 3 and 4 presented tryptic fragments of both NcMIC2 (accession no. AAF01565.1) and NcMIC2-like1, with a clear predominance of the number of spectral counts from NcMIC2 (Fig. 1D). The 2D Western blot employing the anti-NcMIC2-like1 serum on the same sample matched with spots 1 and 2, observed between 100 and 120 kDa (Fig. 1C). These results are in contrast to the 77 kDa predicted for NcMIC2-like1 and 80 kDa for NcMIC2 and also in accordance with our previous work, when NcMIC2-like1 was localized with 78 kDa. However, in that work the strip employed was 3–11 NL, and MS identification was not obtained. On the other hand, the results are consistent with another study that described NcMIC2 between 100 and 120 kDa (bands), very similar to the two spots identified in this work (116–123 kDa, Fig. 1B). TgMIC2 (T. gondii MIC2) also has a higher molecular weight than predicted (113 versus 79.9 kDa). This is the first time that the NcMIC2 protein family is shown in multiple spots within the 100–120 kDa range. TgMIC2 has been identified in 2D gels from multiple and single spots.
In PfTRAP, the VWA and TSP-1 domains together are responsible for the binding to host cell heparin. PfTRAP is currently being tested in association with RTS,S/AS02 as a vaccine in phase 2a, corroborating with the idea that NcMIC2-like1 and NcMIC2 might represent valid antigens for further investigation, including immunization tests against neosporosis.
Several fundamental characteristics (molecular weight, isoelectric point, and abundance in tachyzoites) of Neospora caninum TRAPs (NcMIC2 and NcMIC2-like1) were achieved using proteomic approaches.
TRAP proteins usually are laborious to detect in SDS-PAGE due to the huge differences between predicted and observed molecular weight.
The detailed molecular features of N. caninum TRAPs are the initial steps to understand the function of these proteins in tachyzoite invasion. Several assays related to protein function (for example, genetic manipulation and preventive tools) depend on the data related to the exact molecular weight, pI, and abundance.
Tissue culture and parasite purification
Vero cell cultures were maintained in RPMI-1640 medium (Sigma) supplemented with 5% fetal calf serum (Cultilab), 2.05 mM glutamine, 50 U penicillin/streptomycin in T-25 or 75 tissue culture flasks at 37°C and 5% CO2. The cultures were trypsinized at least once a week. N. caninum tachyzoites from the Nc-1 isolate were maintained in Vero cell monolayers and purified by exclusion chromatography in Sephadex G-25 (PD-10 columns; GE).
The N. caninum data from high-resolution mass spectrometry-based proteomics was obtained as previously described. Proteins present in the ESA and in the discharged tachyzoites (parasites submitted to an ethanol secretion stimulus) were identified using shotgun LC-MS/MS. The protein IDs were generated after separation of the N. caninum proteins by strong cation exchange chromatography. From this work, 615 proteins were identified in ESA and 2,011 proteins quantified in the discharged tachyzoites; however, few properties about MIC2 proteins family were deeply analysed. Therefore, in this article, all the PSMs were analyzed in detail for NcMIC2-like 1 and NcMIC2 identification/quantification.
The N. caninum tachyzoite protein extract, prepared as described previously, was dissolved (50 μg) in a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% carrier ampholyte mixture (pH 3–5.6 NL in Immobiline Drystrip gels; GE), 20 mM dithiothreitol and supplemented with protease inhibitors (1/100, Protease Inhibitor cocktail; Sigma-Aldrich). The samples were loaded on 7 cm IPG strips (pH 3–5.6 NL), rehydrated and focused in an automated overnight run (IPGPhor) using 10–14 h of rehydration followed by a step voltage focusing procedure (20 min 100 V, 30 min 300 V, 1.2 h 1,000 V, 15 min 5,000 V, followed by 5,000 V until a total of 5 kVh was reached). After focusing, the strips were incubated with 30 mM dithiothreitol and 135 mM iodoacetamide in the equilibration buffer (50 mM Tris, 6 M urea, 2% SDS, 30% glycerol, pH 8.8) for 15 min. The proteins were separated by 10% SDS-PAGE and the gels stained with Coomassie Brilliant Blue G 250. Gel images were acquired with LabScan v5.0 software on a flatbed scanner (Image Scanner; GE).
The 2D gels of N. caninum tachyzoite protein extracts were transferred to a PVDF membrane (Immobilon-P; Millipore) with a semi-dry system (TE 77 PWR; Amersham Biosciences) using transfer buffer (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20% methanol) at 0.81 mA/cm2 for 1.15 h. The blot was blocked with 0.8% porcine gelatin (Sigma-Aldrich) diluted in phosphate-buffered saline containing 0.05% Tween (PBST) for 1 h at 37°C. The antiserum anti-NcMIC2-like1 was diluted (1/2,000) in 0.2% porcine gelatin PBST and incubated overnight at 4°C. After washing (three times with PBST), the blot was incubated with anti-rabbit-immunoglobulin conjugated to HRP (1/10,000; ZyMed-Invitrogen) for 1 h at 37°C and then washed three times with PBST. The signal was obtained with an enhanced chemiluminescent substrate for HRP detection (SuperSignal West Pico Chemiluminescent Substrate; Pierce). The blots were scanned using the Image Scanner.
The spots were washed once with milliQ water and twice with 50 mM ammonium bicarbonate (pH 8.0) followed by shrinkage with 100% acetonitrile (ACN). For protein digestion, the spot was incubated overnight with trypsin (Promega) at 37°C. The peptides were extracted with 100% ACN, dried in a SpeedVac vacuum concentrator, and analyzed by RP-nano-LC-MS/MS. After resuspension in 10% formic acid, the peptides were loaded into a LTQ Orbitrap XL ETD mass spectrometer (Thermo Electron, Bremen, Germany) coupled to an Agilent 1200 HPLC system (Agilent Technologies), using a 20×100 mm trap column (Aquat C18, 5 mm; Phenomenex, Torrance, CA) and a 40 cm × 50 mm analytical column (ReproSil-Pur C18-AQ, 3 mm; Dr Maisch GmbH, Ammerbuch, Germany). A 60 min run was carried out using elution gradients with solvent A (0.6% acetic acid) and with solvent B (0.6% acetic acid in 80% ACN) with a flow rate of 100 nL/min. The five most intense peptide ions were selected for fragmentation by collision-induced dissociation. The outputs were compared with data in the N. caninum predicted protein database (ToxoDB 28) using the Mascot software version 2.4.01 (Matrix Science).
Carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modifications, respectively. The database search was performed with a peptide tolerance of 50 ppm and product ion tolerance of 0.6 Da, allowing two missed cleavages.
We would like to acknowledge FAPESP for the PhD fellowship awarded to Pereira (2009/07713-7) and FAPESP (2005-3/53785-9) and CNPQ for the research grant 478767/2007-2.
The antiserum anti-NcMIC2-like1 was obtained from rabbits immunized with recombinant NcMIC2-like1. The animals were caged and handled according to local animal ethics committee (CEUA-FCFRP/USP, process 0611475334).