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• W1979231487 abstract "The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum(20.8%) and the C-terminal domain of the 3′ nucleotidase ofLeishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP >> CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed. The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum(20.8%) and the C-terminal domain of the 3′ nucleotidase ofLeishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP >> CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed. fetal bovine serum binding protein base pair contour-clamped homogeneous electric field kilobase 3′-nucleotidase/nuclease polymerase chain reaction polyacrylamide gel electrophoresis endoplasmic reticulum single strand double strand phosphate-buffered saline 4,6-diamidino-2-phenylindole single-stranded circular Leishmania sp. are dimorphic intracellular parasites that cause a wide spectrum of human diseases, ranging from self-limited cutaneous to the more severe diffuse cutaneous and visceral forms. The parasite exists as a flagellated promastigote within the alimentary tract of its insect vector, the phlebotomine sand fly; within the mammalian host, the parasite transforms into the amastigote stage and resides in the phagolysosomal vacuole of the macrophage.Leishmania pifanoi, a member of the Leishmania mexicana complex, is associated with both simple and diffuse cutaneous leishmaniasis in the New World (1Grimaldi G. Tesh R.B. Clin. Microbiol. Rev. 1993; 6: 230-250Crossref PubMed Scopus (456) Google Scholar). The latter form of the disease is characterized by large histocytoma-like cutaneous nodules containing heavily parasitized macrophages and by a parasite-specific impairment of the cell-mediated immune response (2Peterson E. Neva A.F. Barral A. Correa-Coronas R. Bogaertz-Diaz H. Martinez D. Ward F.E. J. Immunol. 1984; 132: 2603-2606PubMed Google Scholar); patients with diffuse cutaneous leishmaniasis are generally resistant to current forms of chemotherapy (1Grimaldi G. Tesh R.B. Clin. Microbiol. Rev. 1993; 6: 230-250Crossref PubMed Scopus (456) Google Scholar). Over the past decade, leishmanial vaccine research has gained significant attention as clinical treatment failure is becoming increasingly common in many areas; furthermore, drugs used for therapy can be associated with significant adverse effects. However, problems exist with standard live vaccines employing virulent organisms (3Modabber F. Scand. J. Infect. Dis. 1990; 76 (suppl.): 72-78Google Scholar, 4Handman E. Parasitol. Today. 1997; 13: 236-238Abstract Full Text PDF PubMed Scopus (40) Google Scholar); consequently, a focus in leishmanial vaccine development is the identification of defined protective immunogens (5Russell D.G. Alexander J. J. Immunol. 1988; 140: 1273-1279Google Scholar, 6Yang D.M. Fairweather N. Button L.L. McMaster W.R. Kahl L.P. Liew F.Y. J. Immunol. 1990; 145: 2281-2285PubMed Google Scholar, 7McMahon-Pratt D. Rodriguez D. Rodriguez J.-R. Zhang Y. Manson K. Bergman C. Rivas L. Rodriguez J.F. Lohman K.L. Ruddle N.H. Esteban M. Infect. Immun. 1993; 61: 3351-3359Crossref PubMed Google Scholar, 8Mougneau E. Altare F. Wakil A.E. Zheng S. Coppola T. Wang S.-Z. Waldmann R. Locksley R.M. Glaichenhaus N. Science. 1995; 268: 563-566Crossref PubMed Scopus (326) Google Scholar, 9Russell D.G. Talamas-Rohana P. Immunol. Today. 1989; 10: 328-333Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Antigens specific for the amastigote (intracellular-mammalian host) stage of the parasite have been of interest in the construction of a leishmanial vaccine, as such developmentally regulated molecules may be biologically important for the intracellular survival of the parasite. Furthermore, the amastigote is the parasite stage responsible for the pathology associated with disease. Relatively little is known about the mechanisms of amastigote adaptation and survival within the degradative milieu of the macrophage phagolysosome (10Mosser D.M. Rosenthal L.A. Semin. Cell Biol. 1993; 4: 315-322Crossref PubMed Scopus (59) Google Scholar). Metabolic differences are known to exist between the promastigote and amastigote stages (11Zilberstein D. Gepstein A. Mol. Biochem. Parasitol. 1993; 61: 197-205Crossref PubMed Scopus (40) Google Scholar, 12Glaser T.A. Baatz J.E. Kreishman G.P. Mukkada A.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7602-7606Crossref PubMed Scopus (74) Google Scholar, 13Hart D.T. Coombs G.H. Exp. Parasitol. 1982; 54: 397-409Crossref PubMed Scopus (133) Google Scholar, 14Rainey P.M. MacKenzenie N.E. Mol. Biochem. Parasitol. 1991; 45: 307-316Crossref PubMed Scopus (42) Google Scholar); in addition, several leishmanial molecules have been demonstrated to be up-regulated or specifically associated with the amastigote stage. These include specific glycosphingolipids, parasite lysosomal enzymes (cysteine proteinase(s); arylsulfatase), the Leishmania donovani A2 gene, superoxide dismutase, and the proteophosphoglycan molecule(s) (15Peters C. Sierhof Y.D. Ilg T. Infect. Immun. 1997; 65: 783-786Crossref PubMed Google Scholar, 16Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (131) Google Scholar, 17Paramchuk W.J. Ismail S.O. Bhatia A. Gedamu L. Mol. Biochem. Parasitol. 1997; 90: 203-221Crossref PubMed Scopus (90) Google Scholar, 18Straus A.H. Valero V.B. Takizawa C.M. Levery S.B. Toledo M.S. Suzuki E. Salyan M.E. Hakomori S. Barnieri C.L. Takahashi H.K. Braz. J. Med. Biol. Res. 1997; 30: 395-399Crossref PubMed Scopus (10) Google Scholar, 19Straus A.H. Levery S.B. Jasiulionis M.G. Salyan M.E. Steele S.J. Travassos L.R. Hakomori S. Takahashi H.K. J. Biol. Chem. 1993; 268: 13723-13730Abstract Full Text PDF PubMed Google Scholar). The biological functions of these stage-specific molecules are of interest in terms of their potential role(s) in parasite virulence, pathogenicity, and intracellular survival. The leishmanial superoxide dismutase is considered to be involved in the detoxification of host cell radical oxygen intermediates known to be deleterious to the intracellular amastigote. The proteophosphoglycan molecule appears to have a role in parasite vacuole formation within the infected macrophage. Although not essential for survival, experimental studies of Leishmania genetically deficient in either the A2 or cysteine proteinase genes indicate that these molecules are important in parasite virulence. We have previously reported that three purified antigens (P-2, P-4, and P-8), up-regulated or selectively expressed in the amastigote stage, provide partial to complete protection in BALB/c mice against infection with L. pifanoi and Leishmania amazonensis (20Soong L. Duboise S.M. Kima P. McMahon-Pratt D. Infect. Immun. 1995; 63: 3559-3566Crossref PubMed Google Scholar). The enhanced resistance to infection in mice immunized with the P-4 antigen correlates with an increased interferon-γ (Th1/Tc1) response. More recently, we have found that the P-4 antigen also can elicit a preferential Th1-like response in patients with American cutaneous leishmaniasis (21Coutinho S.G. Oliveira M.P. DaCruz A.M. De Luca P.M. Mendonca S.C.F. Bertho A.L. Soong L. McMahon-Pratt D. Exp. Parasitol. 1996; 84: 144-155Crossref PubMed Scopus (71) Google Scholar). For future vaccine studies of leishmaniasis and to understand better the potential biological function of the P-4 amastigote protein, we have cloned and sequenced the gene encoding the P-4 antigen from L. pifanoi. DNA-derived protein sequence data indicate that P-4 is a single strand-specific nuclease. Biochemical analyses have demonstrated that P-4 has both endo- and exonuclease activities and cleaves both RNA and single strand DNA substrates. The specific nuclease/ribonuclease activities, as well as developmental regulation of this molecule, suggest a potential role for P-4 in intracellular survival of these protozoan parasites. L. pifanoi (MHOM/VE/60/Ltrod) amastigotes were maintained at 31 °C in F-29 medium containing 20% heat-inactivated fetal bovine serum (FBS,1 Life Technologies, Inc.), as previously reported (22Landford C.K. Ullman B. Landfear S.M. Exp. Parasitol. 1992; 74: 360-361Crossref PubMed Scopus (34) Google Scholar). L. amazonensis(MHOM/BR/77/LTB0016), Leishmania major (MHOM/IS/79/LRCL251) strain WR309, Leishmania braziliensis(MHOM/BR/75/M2903), and L. donovani (MHOM/ET/67/L82) strain LV9 promastigotes were grown at 23 °C in Schneider'sDrosophila medium supplemented with 20% FBS. The P-4 antigen was purified from detergent-solubilized L. pifanoi amastigote membrane preparations by monoclonal antibody affinity chromatography as described previously (20Soong L. Duboise S.M. Kima P. McMahon-Pratt D. Infect. Immun. 1995; 63: 3559-3566Crossref PubMed Google Scholar). Isolated P-4 protein was then concentrated and further separated by SDS-PAGE. After staining of the proteins with Coomassie Blue, gel slices containing either the 33- or 35-kDa protein were excised and subjected to in-gel enzymatic digestion with either trypsin or chymotrypsin (Roche Molecular Biochemicals). Peptides were isolated by high pressure liquid chromatography on a Vydac C-18 column and subjected to amino acid sequence analysis at the Yale University School of Medicine Protein and Nucleic Acid Chemistry Facility. For N-terminal sequence analysis of the 33-kDa protein, proteins separated by SDS-PAGE were electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), stained with Coomassie Blue, and subjected to gas phase sequence analysis. L. pifanoi amastigote cDNA was generated from isolated total mRNA and oligo(dT) primers using a cDNA cycle kit (Invitrogen, San Diego, CA). The cDNA was immediately amplified via PCR using a GeneAmp kit (PerkinElmer Life Sciences) in the presence of pairs of primers specific for P-4, oligo(dT), or spliced leader. Based upon the protein/peptide amino acid sequences obtained (Table I), and the codon usage (G/C bias in the third codon position) reported for other Leishmania genes (22Landford C.K. Ullman B. Landfear S.M. Exp. Parasitol. 1992; 74: 360-361Crossref PubMed Scopus (34) Google Scholar), degenerate sense and antisense oligonucleotide primers were synthesized. Mixed nucleotides are indicated in parentheses; inosines (I) were used to minimize degeneracy. The sequences of primers used in reverse transcriptase-PCR to amplify the P-4 cDNA were as follows: A1 (sense), 5′-CAGCTIGA(T/C)CTIG A(A/G)AACGA(A/G)GA-3′; A4 (antisense), 5′-TAIGT(T/C)TCIACIAGCTT(A/G)TCIGC-3′; A5 (sense), 5′-GA(A/G)AACA AGGA(A/G)GTIAT(T/C/A)CAGAAGATGG-3′. Cycling conditions were 95 °C for 3 min, followed by 35 cycles at 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min. Amplification products were examined by electrophoresis in ethidium bromide-agarose gels. Isolated DNAs were ligated into a pCRTM II vector provided in a TA cloning kit (Invitrogen) and then transformed into Escherichia coli. Positive colonies were selected for plasmid isolation, restriction endonuclease analysis, and DNA sequencing.Table IAmino acid sequences of the N-terminal and internal peptides of the P4 33-kDa proteinPeptidesSequenceDesigned primers 1-aDegenerate oligonucleotide primers were synthesized based on underlined amino acids.N-terminalX 1-bX designates an undetermined residue. GXVGHMLLAEIAIAQLDLENEEKIQKIIXXRRQLDLENEEKIQKA1IIIMLENKEVIQKMAAVWA5IVHTISRYVXVXTSYPGVTPGGTLVIYTDLFVIIXLSATADKLVETYA4VIIIFSEELETLVDVMAIHEE(S)1-a Degenerate oligonucleotide primers were synthesized based on underlined amino acids.1-b X designates an undetermined residue. Open table in a new tab To obtain a complete P-4 gene copy, a genomic library constructed with partially Sau3A-digested L. pifanoi genomic DNA ligated into the BamHI site of EMBL3cos was screened employing a 32P-labeledEcoRI fragment from clone TA6.2. This 540-bp DNA fragment was isolated and random prime-labeled with [α-32P]dCTP in 1% low melt agarose using a random primer DNA labeling system (Life Technologies, Inc.), and was used as a probe for colony hybridization and Northern and Southern blots. After four rounds of isolation and hybridization, four phage were chosen for further analysis. The DNAs from two of these phage clones, when digested with various restriction endonucleases (BamHI, PstI, EcoRV,SphI, HindIII, HincII; alone and in double digest combinations), revealed after Southern blot hybridization a similar fragment pattern to that observed for total genomic L. pifanoi DNA. The resulting 2.4-kb PstI fragments of each of these phage DNAs were cloned into pUC 19 and further restriction-mapped with EcoRV, HindIII, andSphI; the PstI subclones, based upon restriction mapping, appeared to be similar. Plasmid DNAs derived from genomic clones or cDNA from TA clones were isolated using a Qiagen Qiaquick plasmid miniprep kit (Qiagen, Chatsworth, CA). Both strands of each DNA clone were sequenced using the dideoxy chain termination method employing [35S]dATP and a Sequenase 2.0 DNA sequencing kit (U. S. Biochemical Corp.) or the Fidelity Sequencing Kit from Oncor (Gaithersburg, MD); alternatively, clones were sequenced using automated sequencing (Keck Sequencing Facility, Yale University) using an Applied Biosystems 377 Gel Sequencers and Capillary ABI 3700 DNA Analyzers. Multiple colonies for each clone were sequenced; each colony was sequenced in both directions at least three times. Analyses of the derived nucleotide and amino acid sequences were performed using the Swiss Institute of Bioinformatics ExPASy Proteomics Server. Northern blot analysis was performed using total mRNA isolated from cultured parasites using a micro RNA isolation kit (Stratagene). The mRNA was fractionated electrophoretically on 1.2% agarose gels containing 2.2 m formaldehyde (23Campos-Neto A. Soong L. Cordova J.L. Sant'Angelo D. Skeiky Y.A.W. Ruddle N.H. Reed S.G. Janeway C. McMahon-Pratt D. J. Exp. Med. 1995; 182: 1423-1433Crossref PubMed Scopus (51) Google Scholar) and transferred to Nytran filters (Schleicher & Schuell). Blots were hybridized with the TA6.2 probe at 42 °C in 2× SSC, 0.5% SDS, 50% formamide and washed at 42 °C with 2× SSC, 0.5% SDS. The filters were exposed to Kodak X-Omat AR film at −70 °C with a Cronex Lighting Plus intensifier screen. For Southern blot analysis, genomic DNA of L. pifanoiamastigotes was digested with endonuclease as indicated, subjected to electrophoresis in 0.8% agarose gels, and transferred onto Nytran filters. Filters were hybridized with the TA6.2 probe at 65 °C in 5× SSC, 0.5% SDS, washed at 65 °C, and processed for radioautography. For molecular karyotype analysis,Leishmania chromosomes were prepared in 1% agarose plugs as described (24Beverley S.M. Nucleic Acids Res. 1988; 16: 925-938Crossref PubMed Scopus (135) Google Scholar) and stored at 4 °C in lysis buffer (0.5 mm EDTA, 1% Sarkosyl, 0.25 mg/ml proteinase K, pH 9.5). Pulsed field gel electrophoresis (25Chu G., D. Vollrath D. Davis R.W. Science. 1986; 234: 1582-1585Crossref PubMed Scopus (1092) Google Scholar) was performed in a Bio-Rad CHEF-DR II apparatus, using 0.5× TBE buffer (45 mm Tris, 45 mm boric acid, 1 mm EDTA, pH 8.3) at 175 V employing a 60–150-s ramp for 30 h. The gels were transferred onto Nytran, hybridized with the TA6.2 probe at 60 °C in 5× SSC, 0.5% SDS, washed at 60 °C, and processed for autoradiography. The P-4 protein was isolated as indicated above and assessed for purity by SDS-PAGE analysis using Coomassie Blue staining as described previously (20Soong L. Duboise S.M. Kima P. McMahon-Pratt D. Infect. Immun. 1995; 63: 3559-3566Crossref PubMed Google Scholar). Phosphomonoesterase activity of purified P-4 protein was assayed by measuring the inorganic phosphate liberated following the hydrolysis of the indicated substrates. As described previously (26Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1983; 7: 303-317Crossref PubMed Scopus (79) Google Scholar), the enzymatic activity was assessed using reaction mixtures (0.1 ml) that were composed of 50 mm Tris maleate, pH 8.5, 100 mm KCl, 1 mm CoCl2, 2.5 mm substrate (e.g. 3′-AMP), and varying amounts of the protein fraction being tested. Nuclease P1 from Penicillin citrinum (Sigma) was used as a positive control. After incubation at 42 °C for 30 min, liberated Pi was measured using a detection buffer containing 0.045% malachite green hydrochloride and 4.2% ammonium molybdate (27Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1810) Google Scholar), as described by Zlotnick and Gottlieb (28Zlotnick G.W. Gottlieb M. Anal. Biochem. 1986; 153: 121-125Crossref PubMed Scopus (22) Google Scholar). The reaction was terminated by the addition of 34% sodium citrate; the absorbances at 660 nm were immediately determined spectrophotometrically. Appropriate dilutions of KH2PO4 were used as standards. Results are expressed as μm of inorganic phosphate(Pi) released per 30 min. Single strand nuclease activity of purified P-4 protein was assayed by measuring the release of acid-soluble nucleotides at 260 nm, following the hydrolysis of either heat-denatured DNA or RNA. As described previously (29Vogt V.M. Eur. J. Biochem. 1973; 33: 192-200Crossref PubMed Scopus (624) Google Scholar, 30Shishido K. Habuka N. Biochim. Biophys. Acta. 1986; 884: 215-218Crossref PubMed Scopus (17) Google Scholar), the standard reaction consisted of 30 μg of single strand salmon sperm DNA (sonicated and then boiled for 15 min before use; Sigma) or yeast tRNA (Life Technologies, Inc.) in 0.2 ml of buffer containing 30 mm sodium acetate, pH 5.0, 100 mm NaCl, 2 mm ZnCl2, and varying amounts of the protein fraction being tested. Penicillium citrinum P1 or mung bean nuclease (New England Biolabs, Beverly, MA) was used as a positive control. Incubation was carried out at 37 °C for 30 min and terminated by chilling and addition of 0.4 ml of ice-cold 10% trichloroacetic acid. The sample was clarified by centrifugation, and the absorbance at 260 nm was determined. Result are expressed as nanomoles of nucleotide released per 30 min. The endonuclease activity of the P-4 protein was assessed using covalently closed single-stranded M13mp DNA, a “bubble” DNA substrate, and single strand oligonucleotides. Briefly, covalently closed single-stranded M13mp DNA (0.5 μg) was incubated with different concentrations of P-4 protein at 25 °C in 30 μl of buffer containing 50 mm sodium acetate, 30 mmNaCl, and 1 mm ZnSO4 for 2 h. The reaction was stopped, followed by electrophoresis in 1% agarose gel; DNA was stained with ethidium bromide and then photographed using Polaroid type 55 film. The P-4 protein was tested for its ability to cleave bubble-structured DNA consisting of a central unpaired region of 29 nucleotide in one strand and 30 nucleotides in the other strand flanked by 30 base pairs on both sides. Substrate was formed by annealing one 90-mer oligonucleotide and one 89-mer oligonucleotide (as indicated below); one of the strands (89- or 90-mer) was labeled with [γ-32P]ATP at the 5′-end. The bubble substrate was gel-purified after annealing the oligonucleotide 5′-CCAGTGATCACATACGCTTTGCTAGGACATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAGTGCCACGTTGTATGCCCACGTTGACCG-3′ to the oligonucleotide 5′-CGGTCAACGTGGGCATACAACGTGGCACTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATGTCCTAGCAAAGCGTATGTGATCACTGG-3′ (31O'Donovan A. Davis A.A. Moggs J.G. West S.C. Wood R.D. Nature. 1994; 371: 432-435Crossref PubMed Scopus (392) Google Scholar). The bubble-structured DNA (2 ng) was incubated with different concentrations of P-4 at 25 °C for different times. Reactions were stopped by adding an equal volume of denaturing solution (95% (v/v) formamide, 10 mm EDTA, pH 8.0, 0.1% bromphenol blue, and 0.1% xylene cyanol), and samples were heated to 95 °C for 3 min. Products were separated on a denaturing 12% polyacrylamide gel and visualized by autoradiography and photographed using a DC 220 ZOOM camera. Size markers were made by labeling of the 10-bp DNA ladder with [γ-32P]ATP. To determine the nucleotide preference of the P-4 nuclease, 5′-end-labeled oligonucleotide 5′-CGGTCAACGTGGGCATACAACGTGGCACTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATGTCCTAGCAAAGCGTATGTGATCACTGG-3′ or 5′-CCAGTGATCACATACGCTTTGCTAGGACATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAGTGCCACGTTGTATGCCCACGTTGACCG-3′ was used as substrate. P-4 digestion was conducted as indicated above, and the products were visualized by autoradiography after resolution in denaturing 12% polyacrylamide gel. All incubations were carried out on ice. After washing three times in PBS, L. pifanoi amastigotes were incubated in PBS containing 4% paraformaldehyde for 15 min and then washed once in PBS. Fixed cells were then permeabilized by incubation for 5 min, in PBS containing 0.05% Triton X-100, washed once in PBS, and then incubated in PBS containing 5% FBS and 5% normal goat serum. After washing twice with PBS, amastigotes were then either incubated for 45 min with either normal rabbit serum or anti-P-4 monoclonal antibody and rabbit anti-Trypanosoma brucei binding protein (BiP; generously provided by Dr. J. Bangs, University of Wisconsin) diluted in PBS, 5% FBS. The parasites were then washed three times with PBS containing 5% FBS and 0.05% Tween 20. Washed amastigotes were then incubated for 45 min with fluorescein-conjugated goat anti-rabbit IgG (1:100; Molecular Probes), rhodamine-conjugated goat anti-mouse IgG (1:100; Jackson Laboratories, Inc.), and DAPI (1:1000; Sigma). Organisms were then washed, air-dried onto poly-l-lysine-coated slides, and mounted in aqueous mounting medium (Biomeda Corp., Foster City, CA). Fluorescence was visualized using either a Nikon Microphot-FXA microscope; images were digitalized with a film scanner equipped with the microscope. Alternately, the localization of P-4 and/or BiP proteins were examined using confocal microscopy employing a Zeiss axiovert 100 and LSM 510 software. We have previously shown that on SDS-PAGE, affinity purified P-4 appears as a doublet of proteins with estimated molecular masses of 33 and 35 kDa (20Soong L. Duboise S.M. Kima P. McMahon-Pratt D. Infect. Immun. 1995; 63: 3559-3566Crossref PubMed Google Scholar). Pulse-chase immunoprecipitation studies, examining the in vivobiochemical processing of these proteins, indicated that the 35-kDa protein is the precursor of the 33-kDa protein (65Duboise S.M. Developmentally Regulated Antigens of Leishmania pifanoi Amastigotes: Characterization, Patterns of Expression, and Immunoprophylactic Potential. Ph.D. Thesis. Yale University, New Haven, CT1994Google Scholar). In agreement with this, the alignment of high pressure liquid chromatography elution profiles of trypsin-digested 33- and 35-kDa proteins indicated that these two proteins were closely related, if not identical (data not shown). The amino acid sequences of the N terminus and eight internal tryptic and/or chymotryptic peptides (Table I) from the 33-kDa P-4 protein were obtained (see “Experimental Procedures”). Based on amino acid sequences of peptides II, III, and VII, degenerate oligonucleotides A1, A4, and A5, respectively, were synthesized (see “Experimental Procedures”) using a mixed primer strategy (32Sirakova T.D. Markaryan A. Kolattukudy P.E. Infect. Immun. 1994; 62: 4208-4218Crossref PubMed Google Scholar, 33Frohman M.A. Snincky J.J. White T.J. PCR Protocols. Academic Press, San Diego, CA1990: 28-38Google Scholar). Reverse transcriptase-PCR amplification of L. pifanoi amastigote RNA employing either A1/A4 or A5/A4 primer sets each yielded a single fragment of approximately 540 or 520 bp, respectively. These PCR products were cloned into pCRTM II vectors; the DNA sequences obtained encoded a polypeptide that included five peptides of trypsin/chymotrypsin-digested P-4 (peptides I–IV, VI, and VII), clearly indicating that these PCR products represented a segment of a cDNA encoding the P-4 protein. To obtain a complete copy of the P-4 gene, a L. pifanoi EMBL3cosgenomic library was screened using a radiolabeled TA6.2 cDNA clone as probe. Two separate phage clones containing genomic P-4gene copies were isolated; subfragments containing the P-4genes were subcloned and sequenced from each phage clone. The two cloned copies of the P-4 gene were found to have identical sequences. The sequence of the cDNA clone TA6.2 was contained within the genomic clones; some differences in the derived protein sequence appeared to exist between the TA6.2 cDNA clone and genomic sequences. These difference did not involve residues involved in either zinc-binding sites nor hypothetically involved in the active site of the enzyme. Furthermore, the completely derived P-4 protein sequence included the chymotryptic peptides V and VIII (Table I), not found within derived protein sequence of the TA6.2 cDNA clone. The final DNA sequence encoding the P-4 protein is shown in Fig.1 A. The gene encoding P-4 is 948 nucleotides in length (Fig. 1 A); the open reading frame encodes a polypeptide of 316 amino acids with a predicted mass of 35.1 kDa and predicted pI of 8.9. Based upon the structural features signal peptide, a putative signal peptidase recognition site (34Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (734) Google Scholar) is predicted between Gly-30 (amino acid residue 30) and Trp-31 (Fig.1 A, marked with ▾). This is consistent with the known N-terminal sequence of the 33-kDa protein (Table I). The deduced protein sequence contains two putative N-linked glycosylation sites ( NIT and NTS , Fig. 1 B) at amino acid residues 108–110 and 251–253, as well as potential casein kinase II phosphorylation sites and protein kinase C phosphorylation sites. The existence of such post-translational modifications, however, needs to be confirmed experimentally. The P-4 proteins have been demonstrated to be membrane-associated (20Soong L. Duboise S.M. Kima P. McMahon-Pratt D. Infect. Immun. 1995; 63: 3559-3566Crossref PubMed Google Scholar). Based on the prediction of Gerberet al. (35Gerber L.D. Kodukula K. Udenfriend S. J. Biol. Chem. 1992; 267: 12168-12173Abstract Full Text PDF PubMed Google Scholar), it is not likely that P-4 is a glycosylphosphatidylinositol-anchored protein. Sequence analyses, however, predicted three putative transmembrane domains from residues 1–38, 134–151, and 286–299. However, based upon the enzymatic activity (see below) of the protein, which is dependent upon zinc-binding site included in residues 138–151, it is unlikely that these residues represent a membrane-spanning region of the protein. To define the biological function of the P-4 protein(s), the nucleotide and amino acid sequences of P-4 were compared with those in the data banks. These analyses indicated similarities in amino acid sequences among the mature P-4 protein (amino acid residues 31–316), and the C-terminal region of L. donovani 3′-nucleotidase/nuclease (3′-NT/Nu; GenBankTM accession number L35078, with an open reading frame of a 477 amino acids), and the zinc-dependentP. citrinum nuclease P1 (GenBankTM accession number P24289, with an open reading frame of a 270 amino acids). Within these regions, P-4 shared" @default.
• W1979231487 created "2016-06-24" @default.
• W1979231487 creator A5016390393 @default.
• W1979231487 creator A5020114169 @default.
• W1979231487 creator A5020985367 @default.
• W1979231487 creator A5070884217 @default.
• W1979231487 creator A5089914375 @default.
• W1979231487 date "2000-12-01" @default.
• W1979231487 modified "2023-10-02" @default.
• W1979231487 title "The Immunologically Protective P-4 Antigen ofLeishmania Amastigotes" @default.
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