The Giardia duodenalis 14-3-3 Protein Is Post-translationally Modified by Phosphorylation and Polyglycylation of the C-terminal Tail
2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês
10.1074/jbc.m509673200
ISSN1083-351X
AutoresMarco Lalle, Anna Maria Salzano, Marco Crescenzi, Edoardo Pozio,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe flagellated protozoan Giardia duodenalis (syn. lamblia or intestinalis) has been chosen as a model parasite to further investigate the multifunctional 14-3-3s, a family of highly conserved eukaryotic proteins involved in many cellular processes, such as cell cycle, differentiation, apoptosis, and signal transduction pathways. We confirmed the presence of a single 14-3-3 homolog gene (g14-3-3) by an in silico screening of the complete genome of Giardia, and we demonstrated its constitutive transcription throughout the life stages of the parasite. We cloned and expressed the g14-3-3 in bacteria, and by protein-protein interaction assays we demonstrated that it is a functional 14-3-3. Using an anti-peptide antibody raised against a unique 18-amino acid sequence at the N terminus, we observed variations both in the intracellular localization and in the molecular size of the native g14-3-3 during the conversion of Giardia from trophozoites to the cyst stage. An affinity chromatography, based on the 14-3-3 binding to the polypeptide difopein, was set to purify the native g14-3-3. By matrix-assisted laser desorption ionization mass spectroscopy analysis, we showed that polyglycylation, an unusual post-translational modification described only for tubulin, occurred at the extreme C terminus of the native g14-3-3 on Glu246, Glu247, or both and that the Thr214, located in the loop between helices 8 and 9, is phosphorylated. We propose that the addition of the polyglycine chain can promote the binding of g14-3-3 to alternative ligands and that the differential rate of polyglycylation/deglycylation during the encystation process can act as a novel mechanism to regulate the intracellular localization of g14-3-3. The flagellated protozoan Giardia duodenalis (syn. lamblia or intestinalis) has been chosen as a model parasite to further investigate the multifunctional 14-3-3s, a family of highly conserved eukaryotic proteins involved in many cellular processes, such as cell cycle, differentiation, apoptosis, and signal transduction pathways. We confirmed the presence of a single 14-3-3 homolog gene (g14-3-3) by an in silico screening of the complete genome of Giardia, and we demonstrated its constitutive transcription throughout the life stages of the parasite. We cloned and expressed the g14-3-3 in bacteria, and by protein-protein interaction assays we demonstrated that it is a functional 14-3-3. Using an anti-peptide antibody raised against a unique 18-amino acid sequence at the N terminus, we observed variations both in the intracellular localization and in the molecular size of the native g14-3-3 during the conversion of Giardia from trophozoites to the cyst stage. An affinity chromatography, based on the 14-3-3 binding to the polypeptide difopein, was set to purify the native g14-3-3. By matrix-assisted laser desorption ionization mass spectroscopy analysis, we showed that polyglycylation, an unusual post-translational modification described only for tubulin, occurred at the extreme C terminus of the native g14-3-3 on Glu246, Glu247, or both and that the Thr214, located in the loop between helices 8 and 9, is phosphorylated. We propose that the addition of the polyglycine chain can promote the binding of g14-3-3 to alternative ligands and that the differential rate of polyglycylation/deglycylation during the encystation process can act as a novel mechanism to regulate the intracellular localization of g14-3-3. 14-3-3s are a family of highly conserved dimeric proteins with an approximate molecular mass of 30 kDa. With the exception of prokaryotes, they have been found in all eukaryotes studied to date, including protozoa, yeasts, plants, and animals (1Rosenquist M. Sehnke P. Ferl R.J. Sommarin M. Larsson C. J. Mol. Evol. 2000; 51: 446-458Crossref PubMed Scopus (171) Google Scholar). Generally, unicellular organisms have either one or two 14-3-3s (e.g. the slime mold Dictyostelium discoideum has one, and the yeast Saccharomyces cerevisiae has two), whereas multicellular organisms contain several. Mammals, including Homo sapiens, contain seven 14-3-3 genes (i.e. β, γ, ∈, σ, ζ, τ, η), and 15 genes coding for 14-3-3s have been identified in the plant Arabidopsis thaliana. 14-3-3 homologs have also been found in several protozoan and metazoan parasites (1Rosenquist M. Sehnke P. Ferl R.J. Sommarin M. Larsson C. J. Mol. Evol. 2000; 51: 446-458Crossref PubMed Scopus (171) Google Scholar, 2Siles Lucas del Mar M. Gottstein B. Trends Parasitol. 2003; 19: 575-581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The crystal structure of both animal and plant 14-3-3s has been shown to be conserved. The monomers, consisting of nine α-helices organized in a cup-like shape, are able to interact by their N-terminal portions to form U-shaped dimers (3Liu D. Bienkowska J. Petosa C. Collier R.J. Fu H. Liddington R. Nature. 1995; 376: 191-194Crossref PubMed Scopus (437) Google Scholar, 4Xiao B. Smerdon S.J. Jones D.H. Dodson G.G. Soneji Y. Aitken A. Gamblin S.J. Nature. 1995; 376: 188-191Crossref PubMed Scopus (400) Google Scholar, 5Wurtele M. Jelich-Ottmann C. Wittinghofer A. Oecking C. EMBO J. 2003; 22: 987-994Crossref PubMed Scopus (269) Google Scholar).The main property of 14-3-3s is their ability to bind other proteins containing a consensus Ser/Thr-phosphorylated binding sequence by the interaction with conserved residues located in the amphipathic groove of each monomer (6Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 7Petosa C. Masters S.C. Bankston L.A. Pohl J. Wang B. Fu H. Liddington R.C. J. Biol. Chem. 1998; 273: 16305-16310Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). 14-3-3s were first described in mammals as activators of the brain enzymes tyrosine and tryptophan hydroxylases (8Ichimura T. Isobe T. Okuyama T. Yamauchi T. Fujisawa H. FEBS Lett. 1987; 219: 79-82Crossref PubMed Scopus (234) Google Scholar). To date, 14-3-3 interactors, identified in both animal and plant cells, have been shown to include enzymes, transcriptional factors, and structural proteins (9Moorhead G. Douglas P. Cotelle V. Harthill J. Morrice N. Meek S. Deiting U. Stitt M. Scarabel M. Aitken A. MacKintosh C. Plant J. 1999; 18: 1-12Crossref PubMed Scopus (229) Google Scholar, 10Pozuelo-Rubio M. Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. Biochem. J. 2004; 379: 395-408Crossref PubMed Google Scholar, 11Jin J. Smith F.D. Stark C. Wells C.D. Fawcett J.P. Kulkarni S. Metalnikov P. O'Donnell P. Taylor P. Taylor L. Zougman A. Woodgett J.R. Langeberg L.K. Scott J.D. Pawson T. Curr. Biol. 2004; 14: 1436-1450Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). The phosphorylation (or dephosphorylation) of the 14-3-3 ligands determines their intracellular localization, complex formation, enzyme activation, and conformational changes through the association or dissociation with 14-3-3s.With regard to protozoan parasites, only limited information is available on 14-3-3s. Preliminary studies, which have mainly focused on expression and localization, have been conducted on Plasmodium falciparum, Plasmodium knowlesi, Toxoplasma gondii, and Eimeria tenella, and the results have suggested that 14-3-3s play a role in the transmission of the regulatory signals involved in a wide array of biological processes, including the proliferation and migration of the parasite during infection (2Siles Lucas del Mar M. Gottstein B. Trends Parasitol. 2003; 19: 575-581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Al-Khedery B. Barnwell J.W. Galinski M.R. Mol. Biochem. Parasitol. 1999; 102: 117-130Crossref PubMed Scopus (25) Google Scholar, 13Assossou O. Besson F. Rouault J.P. Persat F. Brisson C. Duret L. Ferrandiz J. Mayencon M. Peyron F. Picot S. FEMS Microbiol. Lett. 2003; 224: 161-168Crossref PubMed Scopus (32) Google Scholar).To date, no analyses of 14-3-3s have been performed for Giardia duodenalis. The flagellate protozoan G. duodenalis (syn. lamblia or intestinalis), which parasitizes the upper part of the small intestine of mammals, including humans, is one of the major causes of non-bacterial diarrhea worldwide (14Thompson R.C. Int. J. Parasitol. 2000; 12-13: 1259-1267Crossref Scopus (356) Google Scholar). This parasite belongs to the order Diplomonadidae, the earliest known branching eukaryotic lineage. Giardia is an "ancient" eukaryote, as inferred from the phylogenetic analysis of different genes and proteins (15Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (586) Google Scholar, 16Hedges S.B. Blair J.E. Venturi M.L. Shoe J.L. BMC Evol. Biol. 2004; 4: 2Crossref PubMed Scopus (433) Google Scholar) and from the finding that it retains many prokaryotic characteristics, such as the size of the small subunit rRNA and the presence of bacterial-like metabolic enzymes. Moreover, it lacks typical eukaryotic structures (i.e. peroxisomes) and has a reduced Golgi apparatus and mitochondrial remnant organelles (known as "mitosomes"), probably as an adaptation to parasitism (17Lloyd D. Harris J.C. Trends Microbiol. 2002; 10: 122-127Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The Giardia life cycle consists of two main stages, (i) the trophozoite, a teardrop-shaped binucleated cell that colonizes the host intestine and reproduces through binary fission, and (ii) the cyst, an infective stage that is able to survive in the external environment. Given that the life cycle can be reproduced in vitro and that the entire genome is available (18McArthur A.G. Morrison H.G. Nixon J.E. Passamaneck N.Q. Kim U. Hinkle G. Crocker M.K. Holder M.E. Farr R. Reich C.I. Olsen G.E. Aley S.B. Adam R.D. Gillin F.D. Sogin M.L. FEMS Microbiol. Lett. 2000; 189: 271-273Crossref PubMed Google Scholar), Giardia is a suitable model for analyzing the functions of 14-3-3s in eukaryotes and the peculiar role of this class of protein in parasites.In the present study we describe the cloning and expression of the 14-3-3 protein homolog of G. duodenalis. The recombinant 14-3-3 was biochemically characterized, and the expression and intracellular localization of the 14-3-3 protein were studied during the parasite life stages using specific antibodies. Moreover, mass spectroscopy analysis of the purified protein demonstrated that the Giardia 14-3-3 is phosphorylated and, for the first time, that it is a target for polyglycylation.MATERIALS AND METHODSChemicals—[γ-32P]ATP and [α-32P]dCTP (specific activities, 110 TBq/mmol) were from MP Biomedicals (Irvine, CA); PreScission protease was from Amersham Biosciences. The catalytic subunit of protein kinase A, dithiothreitol and iodoacetamide were from Sigma. The soluble Raf259p phosphopeptide, LSQRQRST(pS)TPNVHMV (pS, phosphoserine) was synthesized, and the anti-g14-3-3 polyclonal antibody was produced by NeoMPS (Strasbourg, France). Chemicals for gel electrophoresis were from Bio-Rad. All other reagents were of analytical grade.Cell Culture and Differentiation—Trophozoites of the G. duodenalis WB-C6 strain were axenically grown for 72 h at 37 °C in TYI-S-33 medium supplemented with 10% bovine serum and bovine bile at pH 7.0. Encystation was induced essentially as described (19Kane A.V. Ward H.D. Keusch G.T. Pereira M.E. J. Parasitol. 1991; 77: 974-981Crossref PubMed Scopus (83) Google Scholar). Briefly, the cells were grown until confluence, and the medium was replaced with TYI-S-33 supplemented with 10% bovine serum and 10 mg/ml bovine bile at pH 7.8 and then incubated at 37 °C for the indicated time as shown in the figures.Nucleic Acid Isolation—Genomic DNA was isolated from 109 cells of G. duodenalis using the phenol/chloroform extraction method. Total RNA was extracted from 107 trophozoites, or encysting parasites, using the RNAeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Plasmid DNA was isolated from bacteria using the QIAPrep kit (Qiagen).PCR Amplification—The sequence encoding for the g14-3-3 protein without any intron was amplified directly from the genomic DNA of the WB-C6 isolate using the primers Gd14forw (5′-ggatccatggccgaggcatttacgcgt-3′) and Gd14rev (5′-gaatcctcatcacttctcctcggcattatcgtc-3′), designed to anneal with, respectively, the ATG and the stop codons of the coding sequence (the BamHI and EcoRI restriction sites are underlined). PCR reactions were performed in a final volume of 50 μl using 5 μl of 10 × buffer containing 20 mm MgCl2 (Takara Holdings Inc., Kyoto, Japan), 50 μm dNTPs (Takara), 20 pmol of each primer, and 1.25 units of ExTaq (Takara). Reactions were performed on a GeneAmp 2400 thermocycler (Applera Corp., Norwalk, CT). Amplification conditions were 1 cycle at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 1 min, and 1 cycle at 72 °C for 7 min. For the Northern blot analysis, a 725-bp fragment of the G. duodenalis cyst wall protein 1 gene (cwp1) and a 1011-bp fragment of the glyceraldehyde-3-phosphate dehydrogenase gene (gap1) were amplified using, respectively, the designed primers CWP1forw (5′-ATGATGCTCGCTCTCCTTGC-3′) and CWP1rev (5′-TCAAGGCGGGGTGAGGC-3′) and primers GAP1forw (5′-ATGCCTATTCGCCTCGG-3′) and GAP1rev (5′-TTAGCAGCCCTTGGACC-3′). The PCR conditions were the same as those used to amplify the g14-3-3 fragment.Northern Blot Analysis—Total RNA (10 μg) was separated on 1.2% agarose gel containing 37% formaldehyde. RNA was capillary-transferred with 20× standard saline phosphate-EDTA onto Hybond-N nylon membranes (Amersham Biosciences), and Northern hybridization was performed at 50 °C, as described by Sambrook et al. (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.39-7.52Google Scholar), using specific probes previously labeled with [α-32P]dCTP using the RadPrime DNA labeling system kit (Invitrogen) following the manufacturer's instructions.Vector Construction—The g14-3-3 coding sequence was amplified by PCR as described above. For overlay experiments, a short sequence, coding for a protein kinase A phosphorylation site, was introduced by PCR at the 5′ end of the g14-3-3 coding sequence using the designed primers Gd14PKforw (5′-ggatcccgtcgtgcatctgttatggccgaggcatttacgcgt-3′) (the BamHI site is underlined, and the phosphorylation site coding sequence is in italics) and Gd14rev to produce the modified gPK14-3-3 protein. The PCR conditions were the same as those described for g14-3-3 amplification. Both fragments were cloned in the BamHI and EcoRI sites of the pGEX6-P1 vector (Amersham Biosciences) in-frame with the glutathione S-transferase (GST) 3The abbreviations used are: used: GST, glutathione S-transferase; LC, liquid chromatography; ESI, electrospray; MS, mass spectrometry; MS/MS, tandem MS; MS3, triple stage MS; α-cyano, α-cyano-4-hydroxycinnamic acid; PBS, phosphate-buffered saline; Ab, antibody; MOPS, 4-morpholinepropanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; DHB, dihydroxybenzoic acid. and introduced in Escherichia coli JM109 competent cells. The cloned fragments were verified by DNA sequencing. The plasmids were named p14-X and pPK14-X, and the expressed proteins were named g14-3-3 and gPK14-3-3, respectively.The mutants PK-K53E and T214A were obtained by site-directed mutagenesis of the g14-3-3 coding sequence using the QuikChange site-directed mutagenesis methods (Stratagene, La Jolla, CA). The plasmid pPK14-X was used as template to produce the PK-K53E mutant, and the plasmid p14-X was used as a template for the T214A mutant. The primers used for mutagenesis were: K53Eforw (5′-ctgctttctgtagcctacgagaacgtcatcggc-3′) and K53Erev (5′-gcgggggccgatgacgttctcgtaggctacaga-3′) and T214Aforw (5′-acagatctggacaagctggccgaggagtcttac-3′) and T214Arev (5′-cgagtccttgtaagactcctcggccagcttgtc-3′) (the mutated triplets are underlined). After 18 cycles of PCR (95 °C for 30 s, 53 °C for 1 min, and 68 °C for 8 min), 10 units of DpnI were added to the mixture to digest the templates, and the reactions were carried out at 37 °C for 2 h. Twenty μl of each reaction mixture was used to transform E. coli JM109 competent cells. The presence of the mutations was determined by DNA sequencing.The plasmid pSCM138A, containing the difopein coding sequence fused to the enhanced green fluorescent protein (21Masters S.C. Fu H. J. Biol. Chem. 2001; 276: 45193-45200Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), was kindly provided by Dr. Haian Fu. A 169-bp EcoRI difopein fragment was excised from the plasmid and cloned into the EcoRI-digested and dephosphorylated pGEX-6P1 vector in-frame with the GST coding sequence to produce the plasmid pDIF-X.Expression and Purification of the Recombinant Proteins—E. coli- transformed cells were grown in SOB medium (2% bacto tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mm KCl, 10 mm MgCl2, pH 7.2) at A600 = 0.6, and the expression of recombinant proteins was induced in the presence of 1 mm isopropyl thio-β-d-galactoside at 37 °C for 4 h. GST-fused proteins were purified by affinity chromatography on glutathione-Sepharose 4B (Amersham Biosciences) and eluted with 10 mm reduced glutathione, pH 8.0, or released from GST by digestion with the appropriate amount of PreScission protease (Amersham Biosciences) in digestion buffer (50 mm Tris-HCl, 15 mm NaCl, 1 mm dithiothreitol, and 1 mm EDTA, pH 7.5) at 4 °C for 16 h following the manufacturer's instructions.Protein Preparation—Total proteins from soluble and membrane fractions were prepared according to the method of Moss et al. (22Moss D.M. Mathews H.M. Visvesvara G.S. Dickerson J.W. Walker E.M. J. Clin. Microbiol. 1990; 28: 254-257Crossref PubMed Google Scholar), with minor modifications. Briefly, 2 × 109 trophozoites or encysting trophozoites were collected by chilling on ice and washed 3 times with cold PBS (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.2), and the cell pellet was frozen at -70 °C overnight. Cells were resuspended in 2 volumes of extraction buffer (30 mm Tris-HCl,1mm dithiothreitol, and 1 mm EDTA, pH 7.4), supplemented with a protease-inhibitor mixture (P8340, Sigma) and a phosphatase-inhibitor mixture (P2850, Sigma), and then destroyed by sonication (5 times for 30 s at 60% power and 10% duty cycle) with a Sonoplus ultrasonic homogenizer (Bandelin electronic, Berlin, Germany). The lysate was centrifuged at 24,000 × g for 30 min at 4 °C, and the supernatant was collected and designated as soluble cytosolic fraction. The sediment containing the membranous material was washed twice with cold PBS and centrifuged both times for 30 min at 24,000 × g at 4 °C. The pellet was then resuspended in 3 ml of 8 m urea, 0.05 m Tris-HCl, 0.3 m KCl, and 0.002 m EDTA, pH 8.0, and constantly stirred at 4 °C overnight. Further solubilization was achieved by sonication (5 times for 30 s at 50% power and 20% duty cycle), and centrifugation was performed at 24,000 × g for 30 min at 4 °C. The supernatant was collected, and the urea was removed by diafiltration against TBE (40 mm Tris, 54 mm boric acid, 1 mm EDTA, pH 8.3) using a PM-5 membrane and then concentrated using Centricon 10 (Millipore Corp., Bedford, MA). The protein concentration was measured with the method of Bradford (52Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar) (Bio-Rad), and the material was stored at -70 °C.Western Blot Analysis—Proteins were separated on SDS-PAGE (23Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (206048) Google Scholar) and transferred onto polyvinylidene difluoride membranes with 39 mm glycine, 48 mm Tris, 0.1% SDS, and 10% methanol membrane using a semidry apparatus (Bio-Rad). Membranes were blocked with 5% non-fatty dried milk in T-TBS (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.05% Tween 20) for 1 h and then incubated with the primary antibody (Ab) in 3% non-fatty dried milk/T-TBS. After incubation with an appropriate horseradish peroxidase-conjugated secondary Ab (1:2000-1:4000), the antibody/antigen interaction was revealed with the ECL system (Amersham Biosciences). The anti-g14-3-3 polyclonal Ab was produced in rabbit against the g14-3-3 N-terminal peptide (EAFTREDYVFMAQLNENA), and the serum (N14) was used at a 1:4000 dilution, whereas the AXO49 mouse monoclonal Ab, kindly provided by Dr. M.H. Brè, was used at a 1:2000 dilution.Overlay Assay—The overlay assay was carried out according to Lalle et al. (24Lalle M. Visconti S. Marra M. Camoni L. Velasco R. Aducci P. Plant Mol. Biol. 2005; 59: 713-722Crossref PubMed Scopus (33) Google Scholar). GST-gPK14-3-3 and the GST-PK-K53E mutant were labeled with [32γ P]ATP on a cAMP-dependent protein kinase phosphorylation site using the protein kinase A catalytic subunit and then released from GST by cleavage with the PreScission protease. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane with 25 mm Tris, 192 mm glycine, and 0.01% SDS, pH 8.3, and blocked for 1 h with 5% nonfat dried milk in HT buffer (20 mm Hepes-KOH pH 7.6, 75 mm KCl, 5 mm MgCl2,1mm dithiothreitol, 0.1 mm EDTA, 0.04% Tween 20). The membrane was incubated at 4 °C overnight in HT buffer with 3% nonfat dried milk and with 1 μg/ml 32P-labeled gPK14-3-3 or PK-K53E (9 kBq/ml). Where indicated, 100 μm concentrations of the soluble phosphopeptide Raf259p (LSQRQRST(pS)TPNVHMV), reproducing one of the 14-3-3 binding motifs of the human Raf-1 protein, was added during the incubation with 32P-gPK14-3-3. After thorough washing with HT buffer, the interaction was revealed by autoradiography. For Pep-spot binding assay, kindly provided by Drs. G. Cesareni and S. Panni, 100 nmol/cm2 of phosphorylated and unphosphorylated forms of the Raf259 and Raf621 peptides were directly synthesized on cellulose-(3-amino-2-hydroxy-propyl) ether membranes, and after prewashing the filter with 100% ethanol and HT buffer, the overlay assay was performed as described above.Immunofluorescence Microscopy—Trophozoites or encysting cells were collected and fixed at 37 °C with 4% formaldehyde in PBS for 30 min. Fixed cells were stored in PBS at 4 °C until permeabilization, or they were immediately permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min and then spotted on slides. Slides were blocked with 1% gelatin in PBS for 30 min and then incubated at room temperature with polyclonal anti-g14-3-3 antiserum (1:100) for 1 h. After extensive washes with PBS, the parasites were incubated with a fluorescein isothiocyanate-conjugated anti-rabbit secondary Ab (Kirkegard and Perry Laboratories, Gaithersburg, MD) and the Texas Red-conjugated anti-CWP monoclonal Ab A300-TR at 1:30 dilution (Waterborne Inc., New Orleans, LA), and the nuclei were stained with 300 nm 4′,6-diamidino-2-phenylindole. After washes with PBS, the slides were mounted with an anti-fading agent (Vectashield, Vector Laboratories, Burlingame, CA), and microscopy was performed using a Leica (Bensheim, Germany) confocal laser-scanning microscope with appropriate filter sets. A series of 20 optical sections with an approximate z distance of 0.3 μm was performed for each cell. The image processing was performed using the Huygen program (Scientific Volume Imaging BV, Hilversum, The Netherlands) and the Imaris program (Bitplane, Zurich, Switzerland).Pull-down Assay—For pull-down assays, 15 μg of glutathione-Sepharose immobilized GST or GST-difopein were incubated with 3 mg of Giardia cytosolic fractions from trophozoites or from trophozoites grown for 12 h in encystation medium (12 h encysting cells) in HT buffer at 4 °C for 2 h. After extensive washes with HT buffer, the 14-3-3 proteins bound to the beads were eluted with 100 μl of 5 mm soluble Raf259p phosphopeptide in HT buffer. An aliquot was run in SDS-PAGE and subjected to immunoblot with the N14 serum as described above.Mass Spectrometry Analysis—Aliquots of the PreScission-cleaved recombinant g14-3-3, the T214A mutant or the purified native g14-3-3 were separated on a one-dimensional gel NuPAGE 4-12% (Novex, Invitrogen) run in MOPS buffer and stained with the Colloidal Blue staining kit (Invitrogen). Slices were excised, treated essentially as previously described (25Shevchenko S. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar), and digested with modified trypsin, sequencing grade (Promega Corp.). Peptide mixtures were desalted on a POROS-R2 (Applied Biosystems) handmade microcolumn or directly analyzed by MALDI-time of flight on a Voyager-DE STR mass spectrometer (Applera Corp.) in linear and/or reflector positive mode. As a matrix, we used re-crystallized α-cyano-4-hydroxycinnamic acid (α-cyano, Sigma) dissolved in 50% CH3CN, 0.1% trifluoroacetic acid (10 mg/ml) or 2,5-dihydroxybenzoic acid (DHB) (Sigma) in 50% CH3CN, and 1% orthophosphoric acid (30 mg/ml), which has been reported to enhance the efficiency of phosphopeptide ionization in MALDI by diminishing the loss of phosphoric acid (26Kjellstrom S. Jensen O. Anal. Chem. 2004; 76: 5109-5117Crossref PubMed Scopus (180) Google Scholar). The presence of phosphopeptides in MALDI-MS spectra was confirmed by direct comparison between spectra acquired using 1% orthophosphoric DHB and α-cyano; the metastable decomposition product of the phosphopeptide is prominent with α-cyano-4-hydroxycinnamic acid, whereas it is present in lower quantities or not detectable in a 1% orthophosphoric DHB spectrum. Samples were loaded onto the instrument target using the dried droplet technique. Spectra were externally calibrated using a standard peptide mixture.For the liquid chromatography-mass spectrometry analysis, LC/ESI-MS/MS and the LC/ESI-MS3 experiments were performed on an LCQ-DECA XP instrument equipped with a Surveyor MS pump (Thermo Electron Corp.). Peptide mixtures were analyzed on a capillary column, BioBasic C18, 100 × 0.180 mm, 5-μm particle size (Thermo Electron Corp.); the operating flow (2 μl/min) was obtained by the Surveyor MS pump through a homemade splitting device. The LCQ-ESI probe was equipped with a 34-gauge inner diameter metal needle (Thermo Electron Corp.). Peptides were eluted from the column in 50 min using a linear 5-60% acetonitrile gradient in 0.1% formic acid. The acquisition method for LC/ESI-MS/MS experiments was set to perform MS/MS data-dependent scanning on the three most abundant ions, enabling the dynamic exclusion function (repeat count 2). For LC-MS3 experiments, parent ions at m/z 1054.6 (for MS/MS) and 1006.4 (for MS3) were isolated (3.0 m/z window) and fragmented using 35% collision energy.In Vitro Kinase Assays—Approximately 20 μg of the glutathione-Sepharose immobilized recombinant GST-g14-3-3 or GST-T214A mutant protein was mixed with 50 μl of the reaction buffer containing 100 mm Tris-HCl, pH 7.5, 20 mm MgCl2, 200 μm ATP, 2.5 μCi of [32γ-P]-ATP, protease and phosphatase inhibitors, and 0.4 mg of the trophozoite cytosolic fraction. After incubation at 30 °C for 1 h, the reactions were stopped by adding 5 mm EDTA, and the beads were extensively washed with T-TBS. The recombinant wild type g14-3-3 and the T214A mutant protein were recovered by digestion with the PreScission protease, as described above, and 2 μg of each protein was run in 12% SDS-PAGE and stained with Coomassie, and the phosphorylated polypeptides were detected by autoradiography of dried gels.Densitometric Analysis—The image was acquired using the GS-690 Imaging Densitometer (Bio-Rad) and analyzed with the Multi-Analyst 1.1 software (Bio-Rad).Sequence Analysis—General homology searches with DNA and protein sequences were conducted on non-redundant GenBank™ databases using the BLAST algorithm, available at www.ncbi.nlm.nih.gov/BLAST. Giardia genome databases were analyzed on-line (www.mbl.edu/Giardia) by submitting the protein sequence on translated databases (TBLASTN). Sequence data were provided by the National Centre for Biotechnology Information. Multiple alignments were performed using the ClustalW program at www.ebi.ac.uk/clustalw.RESULTSIdentification and Cloning of the 14-3-3 Coding Sequence from G. duodenalis—A single putative 14-3-3 homolog gene has been annotated in the genome of the G. duodenalis WB-C6 clone (www.mbl.edu/Giardia). To verify the presence of other 14-3-3 homologs, we carried out an in silico screening of the G. duodenalis genome using the BLAST algorithm. As probes, we selected two amino acid sequences, RNLLS-VAYKN(V/I) and SYKDSTLIMQLL(R/H)DNLTLWTD(S/A), previously defined as 14-3-3 signatory motifs (27Wang W. Shakes D.C. J. Mol. Evol. 1996; 43: 384-398Crossref PubMed Scopus (190) Google Scholar). The search confirmed the presence of the single open reading frame of 744 bp (GenBank™/EBI accession number AACB01000009), previously identified by the Giardia genome project (18McArthur A.G. Morrison H.G. Nixon J.E. Passamaneck N.Q. Kim U. Hinkle G. Crocker M.K. Holder M.E. Farr R. Reich C.I. Olsen G.E. Aley S.B. Adam R.D. Gillin F.D. Sogin M.L. FEMS Microbiol. Lett. 2000; 189: 271-273Crossref PubMed Google Scholar). The gene lacks introns and codes for a polypeptide of 248 amino acids with a predicted molecular mass of 28.5 kDa and a theoretical isoelectric point of 5.1 (data not shown). The sequence was confirmed by PCR amplification and sequencing of a 2.5-kilobase region spanning the ATG and the stop codon of the open reading frame (data not shown). This gene was named g14-3-3 (GenBank™/EBI accession number DQ146480).Using the ClustalW program, we performed a multiple alignment between the g14-3-3 protein and 117 sequences of different 14-3-3 isoforms from protozoa, fungi, plants, and animals, obtained from the GenBank™ d
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