Molecular and Biochemical Parasitology 91 (1998) 237–249
Identification and heterologous expression of a new densegranule protein (GRA7) from Toxoplasma gondii1
Dirk Jacobs a,*, Jean-Francois Dubremetz b, Anne Loyens b, Fons Bosman a,Eric Saman a
a Innogenetics N.V., Industriepark 7, B-9052 Gent, Belgiumb Institut National de la Sante et de la Recherche Medicale, Unite 42, Villeneu6e d’Ascq, France
Received 8 July 1997; received in revised form 28 October 1997; accepted 29 October 1997
Toxoplasma gondii is an intracellular protozoanparasite which infects humans and warm-bloodedanimals. Although toxoplasmosis is generally clin-ically asymptomatic in healthy individuals, it maycause severe complications in pregnant womenand immunocompromised patients [1,2].
In recent years, several genes and their corre-sponding proteins—suspected to be important in
D. Jacobs et al. / Molecular and Biochemical Parasitology 91 (1998) 237–249238
the process of cell invasion and establishment ofthe parasite inside the cell—have been cloned andcharacterized. Rhoptries and dense granules are,among others, organelles that secrete suchproteins. Rhoptry proteins (ROP) are released bythe tachyzoite very early in invasion [3], and theyare also detected in the emerging parasitophorousvacuole (PV) [4,5] in which the tachyzoite resides.
The nascent PV membrane (PVM), delimitingthe PV, matures by incorporation of several com-ponents released from the dense granules. Somecomponents of the dense granules are targeted tothe vacuolar network whereas others are directedto the vacuolar space [6]. The dense granule(GRA) proteins are believed to play a role inintracellular survival and the nutrient/waste ex-change mechanism with the host cell [6]. It hasbeen demonstrated that the PVM does not con-tain membrane-specific markers from the host cell[7]. In contrast to phagosomes, the PV does notacidify and lysosomes do not fuse with it [8,9].After differentiation of tachyzoites into brady-zoites, GRA proteins are associating with the cystwall [10].
Seven GRA proteins have been identified untilnow: GRA1 (23 kDa) contains two calcium bind-ing domains and is targeted to the PV network[11]; GRA2 (28 kDa) contains two amphipathica-helices suggesting linkage to lipids and it isassociated with the PV network [12–14]; GRA4(40 kDa) and GRA6 (32 kDa) both contain atransmembrane domain, and are also localizedpredominantly in the PV network [15,16]; GRA3(30 kDa) and GRA5 (21 kDa) are both localizedin the PVM as shown by immunofluorescence andby immunogold electron microscopy [17,18]; NT-Pase has also been demonstrated to be a GRAprotein [19,20].
We describe here the molecular characteristicsof a new toxoplasma antigen and provide evi-dence for its classification as a new GRA protein.Since this is the seventh protein in this series, wesuggest to designate it GRA72.
2. Materials and methods
2.1. Reagents
All reagents were of analytical grade and ob-tained from Merck (Darmstadt, Germany), Sigma(St Louis, MO) or Bio-Rad Laboratories (Rich-mond, CA). Restriction enzymes and DNA modi-fying enzymes were purchased from BoehringerMannheim (Brussels, Belgium) and were used ac-cording to the manufacturer’s instructions.Protein concentrations were determined by thebicinchoninic acid method (Pierce, Rockford, IL)
The patients’ sera were obtained from routinescreening for toxoplasmosis and were positive inimmunofluorescense as well as in ELISA assays.Discrepant results were confirmed by the Sabin-Feldman dye test.
2.2. Bacteria and 6ectors
E. coli Y1090, E. coli Y1089, E. coli DH5 a F’and pBluescript KS+ vector were from Strata-gene (La Jolla, CA).
2.3. Parasites and lysates
The RH and Wiktor strains of T. gondii weremaintained by serial passage in the peritonealcavity of Swiss mice. Tachyzoites were collectedeither from the peritoneal cavity of mice or grownin vitro in African green monkey kidney cells(AGMK) (Flow Laboratories, Brussels, Belgium)as described [21]. After washing, tachyzoites werestored at −80° C until use.
Lysates were obtained by resuspending 109
tachyzoites in 5 ml phosphate-buffered saline(PBS), followed by sonication (Soniprep 150,MSE, Abcoude, The Netherlands) on ice at 50 Wwith seven pulses of 20 s each.
2.4. Molecular biology methods
Digestions with restriction enzymes, ligations ofDNA, blunting of DNA fragments were all car-ried out as described [22]. Purification of DNAfragments after agarose gel electrophoresis wasdone with the Geneclean II kit (Bio 101, La Jolla,CA).
2 Note: After completion of this manuscript a gene with anidentical sequence has been found in GenBank under accessionnumber Y 13863.
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2.5. cDNA-library clones and screening
The cDNA clones used in this study were de-scribed by Saavedra et al. [23]. Briefly, a cDNAlibrary in lgt11 was made after total RNA extrac-tion (T. gondii Wiktor strain), poly(A)+ RNApreparation, and cDNA synthesis. After primaryimmunoscreening either with a pool of immunehuman IgG or mouse IgG, 196 positive cloneswere identified, 44 of which were purified as de-scribed before [23].
2.6. Crude lysate preparation from lgt11 lysogens
From the purified clones, lysogens were pre-pared by infection of E. coli Y1089(r-) with lgt11recombinants. Crude lysates of these lysogenswere obtained after induction with isopropyl-b-D-1-thiogalactopyranoside (IPTG) as described byHuynh et al. [24]. Cell pellets originating from 1ml of cell culture were dissolved in 200 m l ofsample buffer.
2.7. Gel electrophoresis and Western blotting
The total E. coli extracts or toxoplasma lysateswere analyzed by SDS-PAGE (12.5%) in the pres-ence of b-mercaptoethanol as described byLaemmli [25]. When necessary, proteins weretransferred to nitrocellulose membranes by thewet Western blotting technique [26] in carbonatebuffer (10 mM NaHCO3, 3 mM Na2CO3,methanol 20% (v/v)).
The membrane was saturated with 5% fat-freemilk in 10 mM Tris, 150 mM NaCl, and 0.05%Tween 20 (TNT) for 1 h, followed by two washesin TNT. The membranes were incubated withmonoclonals, appropriately diluted, or humansera, diluted 1/100 in TNT containing 1% BSAfor 90 min. For screening purposes, sera werepreabsorbed on ice for 30 min using 10% E. colilysate (protein concentration 16 mg ml–1) in thedilution buffer (TNT+1% BSA). After threewashes with TNT, the bands were revealed withrabbit anti-mouse IgG conjugate (Dako,Glostrup, Denmark) or rabbit anti-human IgGalkaline phosphatase-labelled conjugate (Dako).Conjugates were diluted 1/2000. AP activity was
detected by using the chromogenic substrate ni-troblue tetrazolium 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) in 50 mM Tris–HCl (pH9.5), 150 mM NaCl, 5 mM MgCl2 buffer [27].
Ponceau S (Serva, Heidelberg, Germany) stain-ing of Western blots was done to estimate theprotein load of each lysogen. The quantity of eachprotein was estimated by eye and assigned a scorefrom 1 to 10. The reciprocal of this score wasmultiplied by the intensity of the reaction in West-ern blot which was also estimated visually andgiven a value between 1 and 10. For each lysogenthe serum reactivity was expressed relative to theTg47 reactivity (Tg47=1). This numerical expres-sion of the results for the different lysogens makesthem mutually comparable. When the serum reac-tivity for a lysogen was greater than 1, the reac-tion was considered positive (Table 1).
2.8. DNA sequencing
Sequence analysis of the DNA fragmentscloned in plasmid pBKS+ was performed usingthe chain termination procedure [28], adapted toallow analysis on an automated DNA sequencer(Applied Biosystems, Foster City, CA). Sequenc-ing reactions were carried out using the dye-termi-nator technology, as described by themanufacturer, using the universal or reverse M13primers. In order to obtain the complete sequenceof the fragment, five internal primers were customsynthesized (Pharmacia, Uppsala, Sweden) to en-able internal priming. Sequence manipulationswere performed using the Intelligenetics softwarepackage (Mountain View, CA).
2.9. Heterologous expression and purification ofrTg20
The vector pmTNFMPH [29] was used forheterologous expression in E. coli. This vectorenables expression of recombinant proteins as N-terminal fusions with a short (25 residues) mousetumour necrosis factor (mTNF) peptide followedby six consecutive histidine residues. The mTNFpeptide contains an antigenic epitope for which aspecific monoclonal antibody is available. Thepolyhistidine allows purification using immobi-
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Table 1Insert size and specific immunoreactivity for lysogens
a For each lysogen the serum reactivity is expressed relative to the Tg47 reactivity, which expresses only b-galactosidase.b Lysogen containing ROP2 gene.c ROP2 reactive reference serum.d Not determined.
lized metal ion affinity chromatography (IMAC)[30]. Transcription of the heterologous genecloned in this vector is initiated by the earlyleftward lambda promotor (Pl) which is con-trolled by the C1 repressor. The host cell used forexpression is E. coli strain MC1061 [pAC1], con-tained a compatible plasmid which carries theC1-857 mutant gene, encoding a temperature-sen-sitive variant of the C1 repressor [31]. This allowsthe initiation of expression of heterologous genesby shifting the temperature of the culture from 28to 42°C.
Bacteria from an induced culture (5 l) wereharvested by centrifugation. The cell pellet wasresuspended in lysis buffer (100 mM KCl, 10 mMTris–HCl [pH 6.8], 5 mM EDTA, 20 mM o-amino-caproic acid, 1 mM dithiotreitol, 1 mMphenyl-methyl-sulphonyl-fluoride) and passedthree times through a French press. The lysatewas centrifuged to obtain a pellet containing therecombinant protein; this pellet was extractedwith 6 M guanidine HCl 50 mM phosphate (pH7.2). A column containing 20 ml chelating Sep-harose fast flow (Pharmacia, Uppsala, Sweden)
was activated with NiCl2 and washed with 6 Mguanidine HCl 50 mM phosphate (pH 7.2) bufferas described by the manufacturer. The extract wasthen loaded onto the column and elution wascarried out with an imidazole step gradient (30, 60and 100 mM) in the same buffer. The fusionprotein was purified to at least 95% homogeneity,as determined by gel electrophoresis andCoomassie blue staining.
2.10. Immunofluorescence assay
Vero cells were grown on 12 mm diametercoverslips in 24 well plates; they were infectedwith tachyzoites, and fixed 24 h later with 3%formaldehyde in PBS for 30 min, washed withPBS, and permeabilized in acetone at –20°C.Primary antibodies were added and incubationswere continued at room temperature (20°C) usingascitic fluid diluted 1:100, followed by fluorescein-conjugated rabbit anti-mouse-IgG antibodies. Im-munofluorescence (IFA) was also performed onfree tachyzoites that had been dried on standardIFA slides, following the same procedure.
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2.11. Immunoelectronmicroscopy
Vero cells that had been infected by tachyzoites24 h earlier were fixed with 4% formaldehyde and0.05% glutaraldehyde in PBS for 15 min at roomtemperature, dehydrated in ethanol at −20°C,and embedded in LR white (London Resin, Read-ing, Berks, UK). Thin sections were collected onparlodion-carbon-coated nickel grids and floatedfor 30 min on 2.5% fat-free dry milk and 0.1%Tween 20 in PBS (PBS-milk-Tween). The gridswere transferred successively for 1 h each ondilutions of mouse ascitic fluid, anti-mouse IgG–IgM rabbit antibodies, and protein A-gold (8 nm)in PBS-milk-Tween. Sections were stained with4% uranyl acetate in water, then with lead citrate,and observed with a H600 Hitachi electron micro-scope.
3. Results
3.1. Isolation of a new gene
In a continuous effort to identify T. gondiiantigens useful for detection of humoral responseupon infection with this parasite, the performanceof the ROP2 C-terminal fragment (ROP2C), com-prising the terminal two-thirds of ROP2, wasrecently evaluated in serology [32]. Upon evalua-tion of a more extended set of sera, a sensitivity of79% was obtained with this fragment. In order toidentify other antigens complementary to thisROP2C fragment, a selection of sera that wereunreactive or borderline reactive with the ROP2Cfragment were analyzed for reactivity with recom-binant phage clones from a cDNA library de-scribed previously [23]. The clones were selectedbased on their reactivity with human and/ormouse T. gondii-positive sera. Selected phageswere used to produce E. coli lysogens and thelysogenized cells were induced at 42°C. Overallseven different lysogens were analyzed in additionto a positive control (Tg34) previously character-ized as containing the ROP2 gene [33] and anegative control (Tg47) producing only b-galac-tosidase (Table 1).
Total cell extracts of the induced lysogens wereseparated by SDS-PAGE in quadruplicate, blot-ted onto nitrocellulose, and immunoscreened withthree human sera that were non-reactive in aROP2C ELISA (sera numbers 4, 21 and 33). Onepanel was stained with Ponceau S in order toestimate the amount of recombinant b-gal fusionpresent on the membrane. Lysogens Tg13, 19 and27 showed very low reactivity and were not ana-lyzed further with other sera (Table 1). LysogensTg6, 18, 20, 46, 34 and b-gal control were probedwith nine different human sera (Nos. 17, 39, 45,53, 63, 68, 79, 83 and 87). A highly ROP2C-reac-tive serum (No. 18) was used as a positive control.
On Western blot, clone Tg20 reacted with hightiter serum number 18. Moreover, this clone re-acted strongly with sera numbers 21, 33 and 87and weakly with sera numbers 4, 39, 68, 79 and 83(Table 1). Clone Tg6 showed strong reaction withsera numbers 4, 21, 33, 53, 68, 79, 87 and 18.None of the lysogens showed reaction with seranumbers 17, 45 and 63.
Although all sera used failed to react withROP2C in ELISA, clearly some sera were reactivewith ROP2 on Western blot. This indicates thatROP2C lacks an important antigenic region. Onthe other hand, serum number 18 which reactedwell in the ROP2C ELISA was only weakly reac-tive on Western blot. This illustrates the limitedcorrelation between ELISA and Western blot re-sults.
Based on the results shown in Table 1, clonesTg20 and Tg6 were selected for more detailedstudy.
3.2. Isolation and characterization of cDNA
Lambda gt11 phage DNA of both recombinantphages was isolated and digested with EcoRI torecover the inserts. The inserts were then ligatedinto the EcoRI site of the pBluescript KS+plasmid to give rise to pBKS+Tg20 andpBKS+Tg6. One deletion clone containing a 404bp EcoRI-HindIII fragment was constructed fromTg20.
Sequence analysis showed that Tg6 containedthe GRA1 gene described before [11]. The se-quence of the full Tg20 insert is shown in Fig. 1.
D. Jacobs et al. / Molecular and Biochemical Parasitology 91 (1998) 237–249242
Fig. 1. Nucleotide sequence and predicted amino acid sequence of GRA7. The in-frame stop codons upstream and downstream aredouble underlined; signal peptidase cleavage sites are indicated by an arrow head. The putative membrane-spanning domain isunderlined. The gene fragment derived from EST sequences (GenBank) is shown in lower case letters. Single letter amino acid codeis used, except for the GenBank derived fragment.
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An open reading frame of 696 nt was identified,coding for a protein with a theoretically calcu-lated molecular mass of 25 362 Da. A putativeinitiation codon was found at position 91.
By comparison of this sequence with the Gen-Bank™ EST database, more than 100 matchingsequences were found and 65 EST sequencesmatched with the first 200 nt of the Tg20 gene. Inmost of these EST sequences, differences with theTg20 sequence are found, resulting often in-frameshift and premature termination. SeveralEST sequences extend further into the 5’ end ofthe gene and upon comparison of four EST se-quences [accession numbers T62422, W63473,N61771 and N61541], a consensus sequence wasderived that extended the Tg20 sequence at its 5’end. The Tg20 protein was thereby enlarged at theN-terminal end by four extra amino acids includ-ing a methionine [Met-Ala-Arg-His] (Fig. 1). Fur-ther upstream, the gene was extended with apresumably 5’ untranslated region containing anin-frame stop codon [TAA] at position –33, indi-cating that translation initiation of Tg20 can notoccur further upstream from this position (Fig. 1).
The completed sequence contains two methion-ine codons in the N-terminal part, the first ofwhich complies almost perfectly with the transla-tion initiation rules for Toxoplasma as describedby Seeber [34]: the ATG codon is followed by a Gresidue and is preceded from position –4 to −1by the sequence CAAA, as found in the majorityof the known T. gondii genes. The calculatedmolecular mass of the presumably completeprotein is 25 857 Da.
Still another matching sequence was found un-der accession number EMBL A19554 covering226 nt of the 3’ end of Tg20 (Fig. 1, nt 659–885).
3.3. Characteristics of the new protein
Following the putative initiation codon, a se-quence with the characteristics of a signal peptidewas present, as defined by PC GENE PSIGNALanalysis (Intelligenetics, Mountain View, CA) onthe complete protein. Three possible signal pepti-dase cleavage sites were found (positions 18, 21and 26, respectively) (Fig. 1).
Two more regions with high hydrophobicitywere present at the C-terminus of this protein,with only the last one being identified as a puta-tive membrane-spanning domain (Fig. 1) (residues177–193) (PC GENE SOAP). These features sug-gested this protein to be secreted and membrane-associated, which are properties expected for adense granule or rhoptry protein [5,15–18].
The signal sequence contained the only cysteineof the protein. There were no tryptophan residuespresent and the proline content was 5.5% which isin the same range as that for GRA3 (5.9%), and islow compared to GRA4 (12%).
One putative N-glycosylation site was predictedat position 209 (Asn) (PC GENE PROSITE).
3.4. Recombinant expression
The insert of pBKS+Tg20 was recovered afterEcoRI digestion. The insert was blunted with T4DNA polymerase. Expression vector pmT-NFMPH was digested with ApaI, blunted, anddephosphorylated. Ligation of both fragmentsgenerated an in-frame fusion between the ORF ofTg20 and the mTNF-his6 leader peptide (pmT-NFMPHTg20). This fusion protein contained 272amino acids, 37 residues of which were providedby the leader peptide (calculated molecular mass29 846 Da).
Plasmid pmTNFMPHTg20 was transformedinto E. coli strain MC1061 [pAC1]. Induction wasdone as described in Section 2. Crude lysate wasdirectly loaded on SDS-PAGE gels or recombi-nant Tg20 was purified using Ni-IMAC chro-matography (Fig. 2A). The apparent molecularmass of the recombinant protein was 33 kDa, inreasonable agreement with the calculated molecu-lar mass.
3.5. Reacti6ity with monoclonals and with T.gondii-positi6e sera
Monoclonals, reacting with T. gondii antigenssmaller than 40 kDa, described by Saavedra et al.[21], were incubated with nitrocellulose membranestrips prepared by Western blotting of crudelysates from recombinant E. coli producing theTg20 fusion protein. Of these monoclonals, onlyBATO 214 reacted (data not shown).
D. Jacobs et al. / Molecular and Biochemical Parasitology 91 (1998) 237–249244
Fig. 2. SDS-PAGE and Western blot showing the size of rTg20 and natural GRA7. (A) Coomassie blue stained gel with purifiedrTg20 (500 ng) (lane2). Size of marker proteins (lane 1) is indicated in kDa. (B) Western blot showing reaction with the Tg20-specificmonoclonal BATO 214. Lane 1, marker proteins kDa; lane 2, rTg20 [50 ng]; lane 3, lysate of T. gondii Wiktor strain [5 mg]; lane4, T. gondii RH strain [5 mg]; lane 5, AGMK cells lysate [5 mg].
After purification of the recombinant Tg20 onNi-IMAC, the rTg20 was probed again andshowed strong reaction with anti-mouse-TNFmonoclonal and with BATO 214. A strong reac-tion was observed with Toxoplasma-positive hu-man sera (Fig. 3, lanes 4–9), while no reactionwas seen with negative human sera (Fig. 3, lanes14 and 15). Positive human sera not reactive withROP2 [32], but positive in the screening experi-ment where Tg20 was identified, also tested posi-tive for reaction with rTg20 (Fig. 3, lanes 10–13).
3.6. Characterization of the natural T. gondiiequi6alent
A polyacrylamide gel was run with a lysate of
the T. gondii Wiktor and RH strains, as well aspurified rTg20. After transferring the proteins tonitrocellulose, the membrane was probed withmonoclonal BATO 214.
For both Toxoplasma strains, a band migratingat 26 kDa was revealed (Fig. 2B). The main signalof rTg20 was observed at 33 kDa. Cleaving at oneof the putative signal peptidase sites would reducethe calculated size of the protein to 24, 23.7 or23.3 kDa, respectively, depending on which site isused. This is in good agreement with the 26 kDafound in both T. gondii strains. However, the sizeof the natural protein as determined by SDS-PAGE agreed perfectly with the calculated size ofthe complete uncleaved protein. The larger size ofthe recombinant protein was due to the presence
D. Jacobs et al. / Molecular and Biochemical Parasitology 91 (1998) 237–249 245
of the mTNF tag peptide and the natural T.gondii signal sequence which was apparently notcleaved by the E. coli proteases.
IFA of dried tachyzoites with monoclonalBATO 214 showed several fluorescent dots bothon the apical and basal sides of the nucleus (notshown). IFA of infected cells with the same mon-oclonal produced a typical vacuole membranefluorescence that corresponded to the outline ofthe vacuole (as verified by phase-contrast mi-croscopy). In addition, fine strands of fluorescencewere found in many infected cells extending fromthe vacuolar membrane into the cell cytoplasm(Fig. 4).
On thin sections, the immunogold label wasfound specifically on dense granules in tachy-zoites, both intra- and extra-cellularly. In intra-cellular stages, gold particles were also found onthe tubulo-membranous network and on the PVM(Fig. 5 A/B). In contrast to IFA, no significant
Fig. 4. IFA detection with monoclonal BATO 214 on T.gondii-infected cell (upper) and phase contrast image of thesame field (lower). Note the reactivity of the entire vacuole andfine strands in the host cell cytoplasm. (bar=1 mm).
Fig. 3. Western blot of purified rTg20. Lane 1, markerproteins (kDa); lane 2, probed with anti-TNF monoclonal;lane 3, monoclonal BATO 214; lanes 4–9, positive sera (Nos.18, 22, 25, 28, 29 and 30, respectively); lanes 10–13, positivesera used for screening (Nos. 21, 33, 79 and 87, respectively);lanes 14 and 15, negative sera (Nos. 210 and 213, respectively).
reactivity was found in the host cell cytoplasm,which may be explained by the lower sensitivity ofthis method as compared to IFA.
4. Discussion
Screening of a lcDNA expression library withT. gondii-positive human sera selected for theirfailure to react with the previously describedROP2 antigen [32] led to the discovery of a newT. gondii antigen which belongs to the family ofdense granule (GRA) proteins. We therefore sug-gest that it be designated GRA7.
The complete cDNA sequence encoding thisnew antigen contained two possible translationinitiation codons, similar to what is also found for
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Fig. 5. Immunoelectronmicroscopic detection with monoclonal BATO 214. (A) On extracellular tachyzoite, the dense granules (D)are specifically labeled. (B) On a parasitized cell, the parasitophorous vacuole membrane and the tubulo membranous network arealso labeled (arrows). (Magnification: A, 45 000× ; B, 50 000× ; bar=1 mm in both cases) (R, roptry, G, golgi; NU, nucleus; HC,host cell; N, network).
other GRA proteins [14–18]. However, the initia-tion codon located closest to the 5’ end probablyis the genuine initiation codon since it complieswith the T. gondii consensus sequence for transla-tion initiation [34] and precedes a peptide whichcould function as a secretion signal. The presenceof a signal sequence is in keeping with the local-ization of the protein in the dense granules [14–18]. At the 3’ end of the sequence, a consensuspoly A addition signal is found, indicating that atthe 3’ end site, the cDNA contains nearly thecomplete transcript.
Three putative signal peptidase cleavage sitesare present. To date, on the basis of the Westernblot, it is impossible to conclude whether theprotein is cleaved at one of these sites. The molec-ular mass of the natural GRA7 on Western blot isin agreement with the calculated size of the un-cleaved protein. However, if we assume that themigration of the natural GRA7 in SDS-PAGE isslowed down to the same extent as the recombi-nant protein (3 kDa), the natural GRA7 couldwell be cleaved at one of the three putative cleav-age sites and hence give rise to an apparent molec-
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ular mass of 26 kDa. Definitive conclusions con-cerning the signal cleavage can only be drawnafter purification and N-terminal sequencing ofthe natural GRA7 protein.
Upon expression of the GRA7 protein in E. colias a fusion with the short mTNF tag peptide, aprotein of about 33 kDa was obtained, while 30kDa was expected for the uncleaved protein, and24 kDa was expected when cleavage at the pre-dicted site would occur. The observed size indi-cates that the signal peptide cleavage site ofGRA7 is very inefficiently (or not at all) recog-nized in E. coli.
The proline content of the protein is rather high(5.5%) and this property could contribute to itsslightly aberrant migration in SDS-PAGE gels.For other T. gondii proteins, aberrant elec-trophoretic migration and high proline contenthave been causally related [GRA4 (12%), GRA3(5.9%), ROP1 (10.3%), ROP2 (7.7%)] [17].
GRA7 possesses one potential N-glycosylationsite. In other GRA proteins, showing the presenceof a potential N-glycosylation site, glycosylationhas not been observed [14,17]. To further clarifythis aspect, labelling experiments using [3H]-la-belled sugars or deglycosylation by endoglycosi-dase treatment of the natural GRA7 should beperformed.
In the C-terminal part of the protein, two re-gions with high hydrophobicity have been iden-tified, one of which has the characteristics of amembrane-spanning domain [35] (Fig. 1). This isa feature also described for other GRA proteins[15,18] and is in agreement with the extracellularlocalization of these proteins after cell invasion(see below).
The initial purpose of the screening methodwhich resulted in the identification of GRA7, wasto obtain an immunodiagnostic antigen comple-menting ROP2C. Preliminary results obtained ina Western blot experiment indicate that GRA7 isindeed reactive with sera that are unreactive in aROP2C ELISA. Investigations on the perfor-mance of GRA7 in ELISA serology are in pro-gress.
From a battery of monoclonals raised againstthe parasite tachyzoites [21], one monoclonal rec-ognized the recombinant GRA7 fusion protein as
well as the native protein and thus allowed us tostudy the ultrastructural localization of the newprotein in some detail. The pattern of distributionof the immunolabelling was exactly as describedfor the other dense granule protein GRA3 [36,37]by immunofluorescence and immunoelectronmi-croscopy. The fine strands of fluorescence foundin the host cells are typical of vacuolar membraneproteins derived from dense granules; they havebeen found also for GRA3 and GRA5. However,GRA5 seems to differ from GRA3 and the newGRA7 as it is exclusively targeted to the vacuolarmembrane, whereas both GRA3 and GRA7 arealso found in the vacuolar network. The molecu-lar basis for these differences in targeting remainsto be investigated.
Screening of the GenBank database for se-quences related to GRA7 has revealed a sequence[accession U72991] of the apicomplexan parasiteNeospora caninum, which is closely related to T.gondii. Although the overall similarities at thenucleotide (40%) and protein levels (34%) arerather low, the region of the putative transmem-brane domain seems to be rather well conserved(11/17 residues, 65%). A few other regions alsoshow higher than average similarity, and since thecorresponding N. caninum protein is also consid-ered to be a GRA protein, the question remainswhether these two proteins have a similar functionin their respective host cells, despite the low over-all similarity.
Acknowledgements
The authors wish to thank T. Scarcez for theDNA sequence analysis and F. Shapiro for edito-rial comments.
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