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• W2147334621 abstract "A novel Plasmodium falciparum gene,MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M r of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2protein is distributed as a ∼120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a ∼66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of theMB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target. A novel Plasmodium falciparum gene,MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M r of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2protein is distributed as a ∼120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a ∼66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of theMB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target. circumsporozoite cell-surface retention signal immunoelectron microscopy glutathione S-transferase nuclear localization signal open reading frame parasitophosporous vacuole amino acid(s) untranslated region base pair(s) merozoite surface protein thrombospondin related anonymous protein Plasmodium falciparum is the most virulent etiological agent of human malaria, responsible for over 90% of mortality due to the disease. Each year 300–500 million people are infected by malaria parasites, and this results in 1.5–3 million deaths (1World Health Organization Fact Sheet N94. World Health Organization, Geneva, Switzerland1998Google Scholar). Efforts to eradicate malaria generally have failed, and currently the disease is endemic in more than 90 countries throughout the tropics. Widespread and increasing drug and insecticide resistance have exacerbated the situation, undermining the effectiveness of existing malaria control methods that depend on chemotherapy and vector control, respectively. Novel means to fight the disease are needed urgently, and a vaccine is predicted to have the greatest impact in addition to being the most cost-effective control measure (2Miller L.H. Hoffman S.L. Nat. Med. 1998; 4: 520-524Crossref PubMed Scopus (103) Google Scholar). Experimental support for the development of a vaccine for human malaria was provided first by the use of radiation-attenuated sporozoites as immunogens (3Clyde D.F. Most H. McCarthy V.C. Vanderberg J.P. Am. J. Med. Sci. 1973; 266: 169-177Crossref PubMed Scopus (407) Google Scholar). The success of this experimental vaccination provided the impetus for the search for mechanisms of protective immune responses and the target antigens involved. The circumsporozoite (CS) protein was identified as the major surface antigen ofPlasmodium sporozoites (4Yoshida N. Nussenzweig R.S. Potocnjak P. Nussenzweig V. Aikawa M. Science. 1980; 207: 71-73Crossref PubMed Scopus (341) Google Scholar, 5Zavala F. Cochrane A.H. Nardin E.H. Nussenzweig R.S. Nussenzweig V. J. Exp. Med. 1983; 157: 1947-1957Crossref PubMed Scopus (212) Google Scholar, 6Dame J.B. Williams J.L. McCutchan T.F. Weber J.L. Wirtz R.A. Hockmeyer W.T. Maloy W.L. Haynes J.D. Schneider I. Roberts D. Science. 1984; 225: 593-599Crossref PubMed Scopus (547) Google Scholar). The CS protein has been a leading vaccine candidate antigen because irradiated sporozoite-induced, protected human volunteers have high titers of anti-CS1 antibodies (7Herrington D. Davis J. Nardin E.H. Beier M. Cortese J. Eddy H. Losonsky G. Hollingdale M.R. Sztein M. Levine M. Nussenzweig R.S. Clyde D.F. Edelman R. Am. J. Trop. Med. Hyg. 1991; 5: 539-547Crossref Scopus (145) Google Scholar), and CS-specific monoclonal antibodies and cytotoxic T-lymphocytes could adoptively transfer protection in a rodent malaria model system (8Weiss W.R. Berzovsky J.A. Houghten R.A. Sedegah M. Hollingdale M.R. Hoffman S.L. J. Immunol. 1992; 149: 2103-2109PubMed Google Scholar). However, attempts to induce protection in humans using P. falciparum CS-based vaccines, despite recent improvement in their immunogenicity, have repeatedly yielded only partial success (9Ballou W.R. Hoffman S.L. Sherwood J.A. Hollingdale M.R. Neva F.A. Hockmeyer W.T. Gordon D.M. Schneider I. Wirtz R.A. Young J.F. Wasserman G.F. Reeve P. Diggs C.L. Chulay J.D. Lancet. 1987; 1: 1277-1281Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 10Herrington D. Clyde D.F. Losonsky G. Cortesia M. Murphy F.R. Davis J. Baqar A. Felix A.M. Heimer E.P. Gillessen D. Nardin E.H. Nussenzweig R.S. Nussenzweig V. Hollingdale M.R. Levine M.M. Nature. 1987; 328: 257-259Crossref PubMed Scopus (408) Google Scholar, 11Hoffman S.L. Franke E.D. Rogers W.O. Mellouk S. Good M.F. Saul A.J. Molecular Immunologic Considerations in Malaria Vaccine Development. CRC Press, London1993: 149-167Google Scholar, 12Stoute J.A. Slaoui M. Heppner D.G. Momin P. Kester K.E. Desmons P. Wellde B.T. Garcon N. Krzych U. Marchand M. N. Engl. J. Med. 1997; 336: 86-91Crossref PubMed Scopus (757) Google Scholar, 13Stoute J.A. Kester K.E. Krzych U. Wellde B.T. Hall T. White K. Glenn G. Ockenhouse C.F. Garcon N. Schwenk R. Lanar D.E. Sun P. Momin P. Wirtz R.A. Golenda C. Slaoui M. Wortmann G. Holland C. Dowler M. Cohen J. Ballou W.R. J. Infect. Dis. 1998; 178: 1139-1144Crossref PubMed Scopus (221) Google Scholar). The inability to develop a vaccine based on the CS protein was interpreted to indicate that additional antigens play a role in irradiated sporozoite-mediated protection against infection (14Galey B. Druilhe P. Ploton I. Desgranges C. Asavanich A. Harinasuta T. Marchand C. Brahimi K. Charoenvit Y. Paul C. Young J. Gross M. Beaudoin R.L. Infect. Immun. 1990; 9: 2995-3001Crossref Google Scholar). It then becomes important to identify antigens that may act independently, additively or synergistically with the CS protein in the development of a multicomponent vaccine. Here we report the characterization ofMB2, a novel gene encoding a P. falciparumsporozoite surface antigen identified by screening a CS-depleted sporozoite expression cDNA library with serum from a human volunteer protected by the bites of P. falciparum-infected and irradiated mosquitoes. The MB2 gene is expressed in sporozoites, the exoerythrocytic stages, the asexual blood stages, and gametocytes, but the gene product is localized differentially in these developmental stages. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine and drug target. A CS-depleted sporozoite cDNA library was constructed from a P. falciparumsalivary gland sporozoite cDNA library (strain NF54; Ref. 15Fidock D.A. Nguyen T.V. Ribeiro J.M. Valenzuela J.G. James A.A. Exp. Parasitol. 2000; 95: 220-225Crossref PubMed Scopus (2) Google Scholar) using a hydroxyapatite column-based subtractive hybridization technique (16Usui H. Falk J.D. Dopazo A. Lecea L.D. Erlander M.G. Sutcliffe J.G. J. Neurosci. 1994; 14: 4915-4926Crossref PubMed Google Scholar). Briefly, to prepare the target cDNA sense-strands, DNA from the unsubtracted library was linearized with NotI and used as a template to transcribe antisense cRNAs with T7 RNA polymerase (Megascript, Ambion). Template DNA was removed by DNase treatment and the antisense cRNA strands were used to generate cDNA sense strands in a reaction using SuperScript (Life Technologies, Inc.) reverse transcriptase. To prepare the driver cRNA, a CS clone, G89 (17McCutchan T.F. Hansen J.L. Dame J.B. Mullins J.A. Science. 1984; 225: 625-628Crossref PubMed Scopus (91) Google Scholar), was linearized with NotI. Digestion products were used to generate antisense CS cRNA with T7 RNA polymerase (Megascript, Ambion). The target cDNA sense strands were allowed to reassociate with a 50-fold excess of the driver cRNA antisense strands. The reassociation mix was loaded onto a hydroxyapatite column and non-duplex, single-stranded target cDNA was separated from duplex cDNA/cRNA by elution with a high molarity phosphate buffer. Primers specific for the UniZap λ phage vector (Stratagene) were used to amplify the subtracted cDNA, and the amplification products were subcloned into the phage arms of the UniZap vector and packaged. Phage were plated and lifted onto nitrocellulose membranes that were soaked in 10 mmisopropylthio-β-d-galactoside and air-dried prior to use. Membranes were incubated in the serum of human volunteer 5 2W. O. Rogers, unpublished data. at a 1:100 dilution, and horseradish peroxidase-conjugated anti-human IgG+IgM were used to detect positive antibody reactions by the ECL system (Amersham Pharmacia Biotech). Positive phage were screened a second time to isolate single phage clones. Additional cDNA and genomic clones (strain ITO) were recovered using MB2-derived32P-labeled probes and standard library-screening techniques (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The primary nucleotide sequences of all clones were determined by the dideoxynucleotide chain termination method (19Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52605) Google Scholar) using a 33P nucleotide terminator kit (Amersham Pharmacia Biotech). Specific oligonucleotide primers for sequencing were made by Heligen Laboratories (Huntington Beach, CA). Contiguity of clones was verified by gene amplification of genomic DNA using the following primers: a, 5′-GGTGATGACATTGAAGATATGAATG-3′; b, 5′-CAATAGAATAGATATAATCACC; c, 5′-CTGGGTCATCATATGGAAAAGTG-3′; and d, 5′-CAATACACCCTGCAACCTTTCC-3′. P. falciparum genomic DNA was isolated using a phenol/chloroform-based procedure (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) from blood-stage parasites (strain FCR3) cultured in vitro. The DNA was digested with various restriction endonucleases, and Southern blots were prepared as described (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The probe for Southern blot analyses was prepared by labeling the sporozoite cDNA clone, spz-MB2, with radioactive [32P]ATP using the Megaprime DNA system (Amersham Pharmacia Biotech). Total RNA was isolated from blood-stage parasites of the same strain cultured in vitro using the Trizol® reagent (Life Technologies, Inc.). 15–20 µg of total RNA were electrophoresed and Northern blots were prepared as described (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Two 32P-labeled probes consisting of nucleotides 1–580 and 2393–2836 of the coding sequence ofMB2 were used separately on filters to which RNA from blood-stage parasites had been transferred. Segments of the MB2 open reading frame (ORF) were expressed in bacteria as GST-MB2–6xHis fusion proteins from the dual-affinity expression vector, pAK1–6H (20Stratmann T. Schmid S.R. Harper J. Kang A.S. Protein Expression Purif. 1997; 11: 72-78Crossref PubMed Scopus (7) Google Scholar). NcoI and SmaI cloning sites were created for each insert by amplifying NF54 strain genomic DNA. The primer pair, 5′-GATGCCATGGAATATAATAGAATATGCTCA-3′ and 5′-GATCCCGGGTTTTTATTATTAGAAGAATCA-3′, was used to amplify a sequence that encodes a peptide, designated MB2-B, that overlaps amino acids (aa) 95–206. The primer pair, 5′-GATGCCATGGATTCTTCTAATAATAAAAAT-3′ and 5′-ATGCATCCCCGGGTCATTTTTTATTTGAAGAATTCTC-3′, was used to amplify a sequence that encodes a second peptide, designated MB2-C, that overlaps aa 200–316. The primer pair, 5′-GTATGCCATGGTCCACGAAAATAAAGAATATAATTCAAG-3′ and 5′-GATCCCGGGTCATCGAGCGATTCATTTTGGTC-3′, was used to amplify a sequence encoding the peptide, MB2-FA, that overlaps aa 764–945. Finally, the primer pair, 5′-GATGCCATGGATGG TAATAGAACAAATAATGAC-3′ and 5′-GATCCCGGGTACGCTTCGATTATATCGTTTGGCTC-3′, was used to amplify a sequence that encodes the peptide, MB2-IF2, that overlaps aa 1337–1606. The amplification products were digested withNcoI and SmaI and ligated into pAK1–6H. The ligation mixture was used to transform Escherichia coliDH10B, and transformants were selected. Bacterial cells were grown at 37 °C in SuperBroth (Life Technologies, Inc.) to anA 600 = 0.6 and induced in 1 mm final concentration of isopropylthio-β-d-galactoside for 3–4 h to express recombinant proteins. Purification of recombinant proteins was done using the ProBond resin (Invitrogen) modified by the inclusion of imidazole at 85 mm final concentration in the washing buffer. Eluted fractions were analyzed for the presence of recombinant proteins by SDS-polyacrylamide gel electrophoresis and immunoblotting. 400 µg of purified recombinant protein were injected subcutaneously into a rabbit four times at 2-week intervals. Ten days following the last injection, high titer sera were obtained from the rabbit. The sera were depleted of anti-GST antibodies by chromatography on GST-bound nickel columns. P. falciparumparasites and parasite-infected cells or tissues were fixed for 30 min at 4 °C with 1% formaldehyde, 0.1% glutaraldehyde in a 0.1m phosphate buffer, pH = 7.4. Fixed samples were washed, dehydrated and embedded in LR White resin (Polysciences, Inc.). Thin sections (70–80 nm) were blocked in a phosphate buffer containing 5% w/v nonfat dry milk and 0.01% v/v Tween 20 (21Aikawa M. Atkinson C.T. Adv. Parasitol. 1990; 29: 151-214Crossref PubMed Scopus (67) Google Scholar). Grids were incubated at 4 °C overnight in solutions containing variable concentrations of rabbit antiserum reactive to domain-specific recombinant proteins diluted in the blocking buffer. Pre-immune sera were used as negative controls. After washing, grids were incubated for 1 h in 15 nm gold-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech) diluted 1:40 in phosphate buffer containing 1% bovine serum albumin and 0.01% Tween 20. Following the 1-h incubation, grids were rinsed with phosphate buffer containing 1% bovine serum albumin and 0.01% Tween 20 and fixed with glutaraldehyde to stabilize the gold particles. Samples were stained with uranyl acetate and lead citrate and examined by electron microscopy. Protein extracts from parasites were prepared by boiling them in sample buffer for 10 min (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206999) Google Scholar). For the sporozoite stage, parasites were isolated from dissected salivary glands of infected mosquitoes. For the asexual blood stages, parasites were obtained from a saponin lysis of infected red blood cells grown in culture (23Hyde J.E. Read M. Methods Mol. Biol. 1993; 21: 133-143PubMed Google Scholar). Protein extracts were fractionated on 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were incubated in rabbit antiserum diluted 1:500 for 1 h. Horseradish peroxidase-conjugated anti-rabbit IgG was used to detect positive signals using the ECL kit (Amersham Pharmacia Biotech). Preimmune sera and lysates from uninfected human red blood cells were used as negative controls. To evaluate the efficiency of CS depletion in the subtracted cDNA library, 100 ng of the recovered single-stranded cDNA were amplified with oligonucleotide primers complementary to the cloning vector to generate a heterogeneous mixture of fragments of 200–1000 bp in size. The products were amplified again with gene-specific primers. Although two marker genes, TRAP(24Robson K.J. Hall J.R. Jennings M.W. Harris T.J. Marsh K. Newbold C.I. Tate V.E. Weatherall D.J. Nature. 1988; 335: 79-82Crossref PubMed Scopus (324) Google Scholar, 25Rogers W.O. Malik A. Mellouk S. Nakamura K. Rogers M.D. Szarfman A. Gordon D.M. Nussler A. Aikawa M. Hoffman S.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9176-9180Crossref PubMed Scopus (195) Google Scholar) and P2 (26Fidock D.A. Nguyen T.V. Dodemont H.J. Eling W.M. James A.A. Exp. Parasitol. 1998; 89: 125-128Crossref PubMed Scopus (8) Google Scholar), were detected, no signal corresponding to the CS gene was amplified, indicating that the subtraction technique was highly efficient (data not shown). The amplification products were subcloned into the UniZap vector and packaged in λ phage, producing a library of 1.45 × 106 primary phage. A screening of 1 × 104 primary phage with the human volunteer serum led to the selection of 18 candidate phage clones. These were rescreened, and 12 phage again were reactive for antibodies. Southern analyses showed that 1 of the 12 secondary clones hybridized specifically to P. falciparum genomic DNA and showed patterns of hybridization consistent with a single-copy gene (Fig.1 A). This 496-bp sporozoite cDNA clone was designated spz-MB2 and was selected for further characterization. Northern analyses of RNA isolated from blood-stage parasites cultured in vitro and hybridized with probes derived from both the 5′ and 3′ ends of the complete ORF (described below) produced a single positive signal at ∼7.5 kilobases (Fig. 1 B). A comparison of the size of the spz-MB2 cDNA with the mRNA detected in the Northern analyses indicates that it is not a full-length cDNA. Furthermore, primary sequencing ofspz-MB2 showed that it lacked a translation termination codon and represented an incomplete ORF. Sequence complementary toMB2 was detected in an asexual blood-stage cDNA library using specific gene amplification primers, and therefore the library was screened with the spz-MB2 cDNA. Two overlapping blood-stage cDNAs, c3–1-18 and c18–4-23, were identified (Fig. 2 A). Nucleotide sequence analysis revealed that the reading frame ofspz-MB2 was contained entirely within a contig formed by these two cDNAs. The c3–1-18 clone contained a putative translation initiation codon and a 435-bp 5′ end untranslated region (UTR). The 3′ end termini of the c3–1-18 andc18–4-23 cDNAs each have what appear to be polyadenylation (poly(A)) sequences characteristic of the 3′ end termini of processed mRNAs. However, there were no translation termination codons located to the 5′ end of the poly (A) tracks in either of the cDNAs, and the overlap of c3–1-18 withc18–4-23 revealed that the 17 terminal A nucleotides inc3–1-18 comprise an internal A-rich nucleotide stretch inc18–4-23. Therefore, we concluded that the oligo(dT) primed the mRNA for cDNA synthesis from within the coding region. To obtain additional 3′ end sequence of MB2, aSau3AI genomic library (strain ITO) was screened using as a probe the 400 nucleotides at the 3′ end of c18–4-23. A genomic clone, g2–4-4#5, was identified having overlapping and contiguous sequence with c18–4-23. The sequence ofg2–4-4#5 confirmed that the 17-A region at the 3′ end ofc18–4-23 is an internal A-rich nucleotide track, supporting the conclusion that these A-rich internal nucleotide tracks were primed by oligo(dT). To obtain additional 3′ end cDNA sequence, the 600 nucleotides at the 3′ end of g2–4-4#5 were used to screen the blood-stage cDNA library, resulting in the identification of the cDNA clone, c3–4-29. Sequencing ofc3–4-29 revealed that it was contiguous withc18–4-23. In addition, there are three stop codons at the 3′ end of c-3–4-29, commencing at nucleotides 5266, 5272, and 5293, and there is a putative poly(A) region near the 3′ end of the last stop codon. The positions of the stop codons and the authenticity of the poly(A) of the MB2 cDNA were supported by the genomic clone, g6–2-2, identified by screening the genomic library with a probe derived from the 3′ end of c3–4-29. Similarly, the 5′ end UTR and the start codon of MB2 also were verified by the clone g2–6-8, isolated from the genomic library using a probe derived from the 5′ end ofc3–1-18. The overlapping primary sequences of the three blood-stage cDNA clones and the contiguity of their reading frames allowed us to assemble a complete ORF of MB2 that is 4830 nucleotides in length, of which 77% of the bases are A-T pairs (data not shown). No nucleotide polymorphisms were observed among the cDNA and genomic sequences, indicating that there is a single allele of the MB2 gene encoded and expressed in the parasite strains used in our analyses. Because the nucleotide sequences of the three genomic clones did not overlap, we designed gene amplification primers a, b, c, and d (Fig.2 A) to assess the contiguity of the MB2 gene in the parasite genome. Amplification products produced by the primer pairs a+b and c+d with parasite genomic DNA as the template gave the predicted product sizes, ∼700 and ∼1000 bp, respectively (data not shown), indicating that MB2 is organized as a contiguous, single-exon gene in the parasite genome. MB2 encodes a putative translation product that is 1610 aa in length with an approximate molecular mass of 187 kDa (Fig. 2, B and C). The predicted protein is rich in asparagine (15%) and lysine (13%) and is strongly basic with a calculated net charge of +20 at pH 7 and a pI of 8.3. The primary amino acid sequence can be separated into three distinct, linear domains, the first of which is an amino-terminal basic domain of 490 residues (aa 1–490) with a calculated net charge of +30 and a pI of 9.4. We have designated this the “B” domain. This domain contains a region of six 9-aa imperfect repeats (aa 211–264) with the consensus sequence L, N, S, K, K, N, D/N, N, T/S. The central acidic domain, designated “A,” encompasses 496 residues (aa 491–986) with a calculated net charge of −6.2 and a pI of 6.1. The boundary between the B and A domains was selected to maximize the basic and acidic properties of the respective domains. The A domain contains two regions of imperfect repeats of 5 amino acids. The first region (aa 493–542) contains 10 repeats with a consensus sequence of D, N, Q/P, N, Y. The second region (aa 870–914) contains nine repeats with a consensus of I/M, N/D, V, Q, D. No similarities to any sequences of known function deposited in the data bases were detected for either the B or A domains. Finally, a 624-residue carboxyl-terminal domain (aa 987–1610) with sequence similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2, IF2, as revealed by the BLAST search program (27Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59748) Google Scholar) has been designated “G.” The boundary between the A and G domains was selected based on the start of the regions of similarity of theMB2 protein with known IF2 molecules. In contrast to its overall hydrophilic nature, the MB2 polypeptide contains at the amino terminus a strongly hydrophobic region (aa 1–25) mapped by a Kyte-Doolittle hydrophobicity plot. The PSORT computer program (28Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1365) Google Scholar) predicted an uncleavable signal peptide in the hydrophobic amino-terminal region of MB2. However, the SignalP program (29Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4927) Google Scholar) predicted that the signal peptide could be cleaved between a pair of S-S residues at aa 27–28 (Fig. 2 C). Currently, we have no experimental data that support one alternative over the other. The PSORT program also predicted a number of nuclear localization signals (NLS), PKKK (aa 120–123), RRKK (aa 173–176), KKKKK (aa 652–656), and a bipartite NLS, KKNKELPFNNKFKKIIK (aa 718–734), within the B and A domains. Multiple putative sites for N-glycosylation,N-myristoylation, and phosphorylation were detected by the ScanProsite program (Ref. 30Appel R.D. Bairoch A. Hochstrasser D.F. Trends Biochem. Sci. 1994; 19: 258-260Abstract Full Text PDF PubMed Scopus (512) Google Scholar; data not shown). There is a polybasic motif, KKKKKGKSRKK (aa 956–966), just before the start of the G domain, that could function as a plasma membrane localization signal as well as a cell-surface retention sequence (CRS) (31Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (844) Google Scholar). This sequence also could be a putative NLS, although PSORT failed to identify it as such. The similarity of the G domain to the GTP-binding domains of the prokaryotic IF2 proteins includes the conservation of sequence and spacing of three motifs, GX 4GK (aa 999–1005), DX 2K (aa 1046–1049), and NKXD (aa 1100–1104), common to this family of proteins (32Dever T.E. Glynias M.V. Merrick W.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1814-1818Crossref PubMed Scopus (466) Google Scholar). There is a small variation in the third motif, TKXD, in MB2 as compared with the consensus seen in other G proteins (Fig. 2 C). Immunoelectron microscopy (IEM) was used to study the subcellular localization of theMB2 antigen. All rabbit antisera prepared against recombinant peptides derived from the B and A domains (Fig.2 A), and reacted with sectioned material containing sporozoites, showed that MB2 protein was localized predominantly to the surface (Fig. 3,A–D). This was true of sporozoites in salivary glands (Fig.3, A and B), as well as those that invadedin vitro cells of the human liver cell line, HepG2-A16 (Fig.3, C and D). No antibody reaction was detected with sporozoites using the anti-G domain antibody (data not shown). Preimmune control sera for all reagents were negative (data not shown). In contrast, the majority of the MB2 protein detected in blood-stage parasites using both antisera against the B domain was localized in the nucleus, with some antibody reactivity detected in the cytoplasm (Fig. 3, E and F) and data not shown). Rabbit antisera against the A and G domains detected protein only in the cytoplasm of these parasites (Fig. 3 G and data not shown). Furthermore, the numbers of gold particles observed in sections of parasites exposed to antibodies against the A and G domains were low when compared with the signal produced by the anti-B domain antisera, indicating probably that the majority of MB2 protein present at the blood stages does not contain the A and G domains. MB2 protein was detected in the cytoplasm, nucleus, and parasitophorous vacuole (PV) space of gametocyte-stage parasites using the anti-B domain antiserum (Fig. 3 H). MB2protein detected by the anti-A domain antiserum was localized only in the PV space (Fig. 3 I), indicating that the protein detected in the nucleus and cytoplasm with the anti-B domain antiserum does not contain the A domain. The anti-G domain antiserum produced a high background signal, making it difficult to interpret any specific localization pattern. Finally, we attempted to use IEM to look at the localization ofMB2 protein in the exoerythrocytic stages of the parasite. A section of the liver of an infected Aotus monkey was reacted with the anti-B domain antiserum. It is hard to locate the parasites in these sections, but we were able to find some that revealed that the protein is localized mostly in the cytoplasm with some in the PV space (Fig. 3 J). Sections reacted with the anti-A domain antiserum had high backgrounds obscuring any evidence of a specific localization pattern. A series of immunoblotting experiments were performed with parasite protein extracts prepared from the sporozoite and blood stages to determine the relative size of theMB2 protein (Fig. 4). Both anti-B and anti-A domain antisera detected a single polypeptide of ∼120 kDa at the sporozoite stage (Fig. 4, A andB). No immunoblot analyses were done on sporozoite preparations with anti-G domain antiserum because of the negative results obtained in the IEM analyses. Immunoblotting using anti-B domain antibody detected a single polypeptide of ∼66 kDa at the blood stages (Fig. 4 C). Furthermore, the 66-kDa polypeptide was not detected with either the anti-A or anti-G domain antibodies (Fig.4 D and data not shown). These data are consistent with the IEM study and indicate that the MB2 polypeptide located at the surface of sporozoites consists of only the B and A domains, and the polypeptide translocated into the nucleus of parasites at the blood stages consists primarily of the B domain. A major difficulty in immunoscreening of expression libraries to identify P. falciparum sporozoite antigens is the abundant and immunodominant characteristics of the CS protein. The abundant expression of the CS gene results in its representation in high frequency in cDNA libraries (15Fidock D.A. Nguyen T.V. Ribeiro J.M. Valenzuela J.G. James A.A. Exp. Parasitol. 2000; 95: 220-225Crossref PubMed Scopus (2) Google Scholar), and its immunodominant repeat domain induces a high antibody titer in the host providing the screening antiserum (7H" @default.
• W2147334621 created "2016-06-24" @default.
• W2147334621 creator A5014158535 @default.
• W2147334621 creator A5039756363 @default.
• W2147334621 creator A5055537743 @default.
• W2147334621 creator A5062473075 @default.
• W2147334621 creator A5071314941 @default.
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• W2147334621 date "2001-07-01" @default.
• W2147334621 modified "2023-09-30" @default.
• W2147334621 title "Stage-dependent Localization of a Novel Gene Product of the Malaria Parasite, Plasmodium falciparum" @default.
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