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Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus
Gene 332 (2004) 149 – 157 www.elsevier.com/locate/gene
Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus Silvana Sacchetti a, Simonetta Bartolucci a, Mose` Rossi b, Raffaele Cannio b,* a
Dipartimento di Chimica Biologica, Universita` degli Studi di Napoli ‘‘Federico II’’, Naples, Italy b Istituto di Biochimica delle Proteine, CNR, Via Pietro Castellino 111, 80131 Naples, Italy
Received 29 September 2003; received in revised form 2 February 2004; accepted 13 February 2004 Received by A.M. Campbell Available online 9 April 2004
Abstract An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 jC revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose. D 2004 Elsevier B.V. All rights reserved. Keywords: GDP-mannose pyrophosphorylase; Genome; Mannose metabolism; Cloning; Overexpression
1. Introduction Guanidine diphosphomannose (GDP-Man), the mannosyl donor for most Man-containing polymers, is formed by the transfer of mannose-1-phosphate (Man-1-P) to GTP to form GDP-Man and inorganic pyrophosphate (PPi). This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP) or GTP-a-D-
Man-1-P guanylyltransferase (EC 2.7.7.13), a key enzyme for the formation of mannose-containing glycoconjugates, such as protein N- and C-glycans, some O-glycans, glycosylphosphatidylinositol protein, as well as some glycolipid membrane anchors, known to have a variety of important functions (Varki, 1999). The enzyme GMPP catalyzes the synthesis of GDP-Man from Man-1-P and GTP by the following reversible reaction (Ning and Elbein, 2000): GTP þ Man -1 - P X GDP -Man þ Ppi:
Abbreviations: GDP-Man, guanidine diphosphomannose; GDP-Glc, guanidine diphosphoglucose; GMPP, guanidine diphosphomannose pyrophosphorylase; Man-1-P, mannose-1-phosphate; Glc-1-P, glucose-1-phosphate; PPi, inorganic pyrophosphate; LM, lipomannan; LAM, lipoarabidomannan; GST, glutathione S-tranferase; ORF, open reading frame; GSH, reduced glutathione; IPTG, isopropyl-1-thio-h-D-galactopyranoside; BSA, bovine serum albumin; PBS, phosphate buffered saline. * Corresponding author. Tel.: +39-816132285; fax: +39-816132248. E-mail address: [email protected] (R. Cannio). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.02.033
In eukaryotes, this activated form of mannose is the major mannosyl donor for the glycosylation of proteins in a biosynthetic pathway that produces N-linked oligosaccharides of many membrane and secretory glycoproteins. For this reason, mannose-containing glycans cover also an essential range of indirect functions that include the promotion of correct folding, solubility, stability and intracellular
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sorting of proteins. Particularly in lower eukaryotes, such as the Saccharomyces (Hashimoto et al., 1997) and Candida (Warit et al., 1998) genera of yeast, GMPP is involved in the multistep pathway of covalent linkages between the cell wall proteins and glucans (Warit et al., 2000). Mutations in the gene vig9 have been demonstrated to affect even sugar uptake and/or removal by increased sensitivity to antibiotic aminoglycosides (Hashimoto et al., 1997; Warit et al., 2000). In eukaryotic human pathogens, such as Leishmania, the GMPP gene is a virulence factor since its deletion causes loss of the capacity to infect macrophages and mice (Garami and Ilg, 2001). As another example of its broad biological role, GMPP provides also a key intermediate in the biosynthetic pathway of L-ascorbic acid (Vitamin C) in plants (Conklin et al., 1999). GMPPs have been reported to be the general enzymes catalyzing the formation of the activated sugar nucleotide precursors for both extracellular and capsular polysaccharide biosynthesis of bacteria. The enzyme participates in the elongation of the oligosaccharide moiety of mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), that are potent immunomodulators in tuberculosis and leprosy and hence it is important in light of the role of LAM in phagocytosis and generalized immunosuppression (Besra et al., 1997). Moreover, the GMPP gene is a component of a cluster responsible for the biosynthesis of the Yersinia enterocolitica O:8(YeO8) lipopolysaccharide O-side, the major virulence factor of this pathogen (Zhang et al., 1997). Only a few data are available in the literature about the distribution of this gene in the third domain of life, the Archaea. Recently, Empadinhas et al. (2001) have suggested its involvement in the defence against osmotic stress in the hyperthermophilic euryarchaeon Pyrococcus horikoshii; the compatible solute a-mannosylglycerate has been shown to be one of the most effective stabilizers of biomolecules like proteins (Ramos et al., 1997) and its biosynthetic pathway of mannosylglycerate requires GDP-Man as a substrate for the specific synthase in a fashion similar to the well-characterized mechanism in the thermophilic bacterium Rodothermus marinus (Martins et al., 1999). No data are available for the other archaeal subdomain of Crenarchaeota. Sulfolobus solfataricus, originally isolated from a solfataric field in the area of Naples, Italy, is a hyperthermophilic crenarchaeon able to grow chemoheterotrophically at acidic pH (pH 3 –5) and at high temperatures (80 – 87 jC). The inspection of the S. solfataricus genome, recently fully sequenced (She et al., 2001) and available at the web site http://www-archbac.u-psud.fr/projects/sulfolobus/, revealed an open reading frame (ORF) encoding a putative GDPMan pyrophosphorylase (SsoGMPP) with the predicted gene product showing significant similarity to corresponding proteins from all three domains of life. We describe here the cloning and heterologous expression of the Ssogmpp gene and the enzymatic characterization of its gene product in comparison with the yeast enzyme Vig9 as well as the
detection of the specific wild type protein in S. solfataricus cells. Moreover, we show that SsoGMPP is a thermophilic functional enzyme and that it is able to catalyze both the direct and reverse reaction for the synthesis and pyrophosphorolysis of GDP-Man in a highly specific manner. This is the first GDP-Man pyrophosphorylase identified in Archaea: its molecular characterization has implications for the function in vivo and the evolution of these enzymes.
2. Materials and methods 2.1. Strains, plasmids, enzymes and chemicals Escherichia coli TOP10F’ (purchased from Invitrogen) and Rb791 (Brent and Ptashne, 1981) strains were used as hosts for plasmid propagation and protein expression, respectively, and routinely cultured in Luria –Bertani medium, with ampicillin (100 Ag/ml). S. solfataricus strain P2 (DSM 1617) was supplied by Deutsche Sammlung von Mikroorganismen (DSM) (Braunschweig, Germany) and grown aerobically at 80 jC in Brock’s basal salt medium (Brock et al., 1972) containing 0.1% (w/v) yeast extract, 0.1% (w/v) casamino acids, and buffered at pH 3.7. For mRNA analysis simpler media containing 0.1% yeast extract and alternatively 0.2% (w/v) of sucrose, or mannose, or arabinose or lactose were tested. Nutrients and agar were purchased from Difco. Plasmid vectors pUC18 SmaI/BAP and pGEX-2TK were supplied by Pharmacia Biotech. Restriction/modification enzymes and Taq DNA polymerase were obtained from Roche and Promega, respectively. IPTG, X-Gal, all buffers, organic solvents and reagents were high grade pure and supplied by Sigma. Radioactive materials were from NEN-Dupont. 2.2. Isolation of the Ssogmpp gene from S. solfataricus P2 strain The gene encoding GDP-Man pyrophosphorylase (SsoGMPP) was amplified via PCR; the 5V primer used in the amplification contained the ATG start codon and a NcoI site (bold-faced letters and underlined in the sequence, respectively): manUP (5V-CATTTTTCTTACCATGG TATCCGCAAT-3V). The 3V primer had the following sequence: manDW (5V-CTAAGGATCATAATTGTCGAC TTATAG-3V), and was designed to insert a recognition site for the SalI endonuclease (underlined) immediately upstream of the termination codon (bold faced letters). The reaction was performed in a Perkin Elmer apparatus using as enzyme the Taq polymerase under the following conditions: denaturation at 95 jC for 10 min, followed by 35 cycles of denaturation at 95 jC for 1 min, annealing at 45 jC for 1 min and extension at 72 jC for 1 min. The resulting DNA fragment was ligated into the SmaI cloning site of the plasmid pUC18 and DNA fragments from four independent
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clones were sequenced by MWG-BIOTECH (Ebersberg, Germany). The complete coding sequence obtained was analyzed using Vector NTIk Suite program purchased by InforMax (North Bethesda, MD). Homology comparison and multiple alignment of the SsoGMPP protein with other nucleotidylsugar pyrophosphorylases were performed using Blast and ClustalW programs, respectively, available on the Internet. 2.3. RNA analyses 2.3.1. Total RNA extraction and Northern analysis S. solfataricus P2 cultures were grown in the media described above; cells were harvested when the cultures reached middle exponential phase, namely an absorbance at 600 nm of about 0.5 OD on the average. Total RNA was extracted by the guanidine thiocyanate method (Sambrook and Russel, 2001). For Northern blot analysis, the different RNAs extracted (14 Ag) were electrophoretically separated together with molecular weight RNA standards (MBI fermentas) in 1.2% agarose gel containing 10% formaldehyde and blotted onto a Hybond-XP nylon membrane (Amersham). Hybridization with [a-32P]-labelled Ssogmpp probe (1086-bp coding sequence) was carried out at 65 jC for 16 h in 5 Denhardt’s reagent, 6 SSC, 0.5% SDS, 100 Ag/ ml sonicated and denaturated salmon sperm DNA. After hybridization, blots were washed twice in 1 SSC, 0.1% SDS for 10 min at RT and three times in 0.5 SSC, 0.1% SDS for 15 min at 65 jC and signals visualized by autoradiography and quantified with a densitometric analysis. A replica filter was hybridized under the same conditions with the coding sequence of the Sso10b gene encoding the DNA binding protein ALBA (Wardlerworth et al., 2002) for the normalization of the specific signal quantification. 2.3.2. Cloning of the 5V Ssogmpp gene sequence from S. solfataricus P2 A 468-bp DNA fragment comprising 361-bp upstream of the S. solfataricus P2 Ssogmpp coding sequence was PCR amplified using oligonucleotides designed on the basis of the Sso0317 sequence on the P2 genome. The sequences of the 5V (GMPPup) and 3V (GMPPex) oligonucleotides were: 5 V- G G T G C A C TA G C A G A C T TA ATA A A C A A G - 3 V (corresponding to the region-361 to-335 from the A nucleotide in the translation start codon of the SsoGMPP gene) and 5V-CCCAGTATAGGTTTGTTTAAAATGGGG-3V (complementary to the region from + 81 to + 107 of the SsoGMPP coding sequence). The resulting amplicon was cloned into a pUC18 SmaI/BAP vector, producing the plasmid pUC-GMPPr.
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mmol) and purified on Sep-Pak cartridges (Waters) following the procedure described by Sambrook and Russel (2001). 105 cpm (106 cpm/pmol) of the oligonucleotide were co-precipitated with 50 Ag of total RNA isolated from S. Solfataricus cells grown in the simplified media described and were denatured, before coprecipitating with 1 pmol of the labelled oligonucleotide. The mixture was resuspended in 6 Al of reverse transcriptase buffer supplied by the manufacturer (Roche) and incubated for 3 min at 65 jC, placed on dry ice for 1 min and on ice for 5 min and then incubated for 40 min at 37 jC for the annealing step. Two microliters of deoxynucleotide triphosphates (2 mM each) suspended in reverse transcriptase buffer, 20 units reverse transcriptase (Roche) and 25 units ribonuclease inhibitor from human placenta were added and sample incubated at 48 jC for 30 min to drive cDNA synthesis. The synthesized cDNA was denatured at 100 jC and analyzed by electrophoresis on 6% acrylamide gel. The same primer was used for the DNA sequence performed on the 5V flanking region cloned in pUC18 to produce size standards. Both primer extension cDNA and the sequencing ladder were run on the same gel and the products visualized by autoradiography. 2.4. Gene expression and protein purification The Ssogmpp coding sequence obtained by PCR and cloned in pUC18 was digested with NcoI and HindIII, filled-in by Klenow treatment and inserted into the expression vector pGEX-2TK at the BamHI site made blunt by the same treatment. The cloning was designed to construct a plasmid, pGEX-SsR, in which an uninterrupted hybrid coding sequence was expressed as an unique inducible gene encoding GST-SsoGMPP fusion protein. E. coli RB791 competent cells were transformed with pGEX-SsR and grown at 22 jC in LB and 0.5% NaCl) until the A600 nm reached 2.0. IPTG (0.1 mM) was added, and the incubation was continued for 16 h. The procedure described by Hashimoto et al. (1997) was followed for the gene expression induction and production in E. coli BL21/pTgroE of the yeast Vig9 protein. Lysis for protein extract preparations was performed for both recombinant SsoGMPP and Vig9 as already described for recombinant thioredoxin from Bacillus acidocaldarius by Pedone et al. (1999), using PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) as the lysis buffer, and the cytosol was isolated by centrifugation 13,000 g. GST fusion proteins were loaded onto a Glutathione Sepharose 4B RediPack (Pharmacia) at 0.2 ml/min flow rate; after washing with 10 ml of PBS, the elution was obtained with 50 mM Tris – HCl pH 8.0 containing 10 mM GSH. 2.5. Protein determination and enzyme assays
2.3.3. Primer extension analysis The extension primer, the GMPPex oligonucleotide used for the 5V flanking region amplification, was radiolabelled using T4 polynucleotide kinase and [g-32P]dATP (3000 Ci/
Protein concentration was determined using the BioRad protein staining assay and BSA as the standard, following the instructions of the manufacturer.
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2.5.1. Assay in the forward direction The formation of [32P]GDP-Man was determined using [a-32P]GTP and Man-1-P as starting material. The incubation mixture (50 Al) contained: 40 mM HEPES pH 7.6, 8 mM MgCl2, 0.1 mM Man-1-P, 0.2 mM GTP, 0.1 mCi/ ml [a- 32 P]GTP (3000 Ci/mmol) and an appropriate amount of enzyme (affinity-purified GST-SsoGMPP, or crude extracts from S. solfataricus, as well as Vig9 and BSA, as positive and negative control, respectively). The assay was performed at 35 jC for Vig9 and in the range 35 – 60 jC for SsoGMPP and BSA; the reaction was stopped by mixing the samples with equal amounts of stop solution containing 100 mM EDTA and frozen in dry ice-ethanol. As a control of enzyme specificity, the assay was also performed on glucose-1P under the same conditions and using the same substrate concentrations and detection method. The formation of [32P]GDP-Man was detected and quantified by photostimulated luminescence, using a Phosphor Imager apparatus (BioRad) after separating the GDP-Man by thin layer chromatography on polyethyleneimine cellulose (Merck) performed with 1 M LiCl, 1 M HCOOH, as the solvent. Alternatively, for identification of the GDP-Man product, the reaction was performed in 50 Al of 20 mM Tris – HCl, 8.0 mM MgCl2, 4.0 mM GTP and 4.0 mM Man-1-P, pH 7.6 and 1.0 –2.0 Ag of the purified enzyme. Reaction reagents and products were separated by thin layer chromatography on silica gel with a solvent system composed of n-propanol and 25% ammonia (1:1, vol/vol) and visualized by spraying with a-naphthol-sulphuric acid solution followed by charring at 120 jC (Sampaio et al., 2003). Authentic standards of mannose-1-P and GDP-mannose were used for substrate and product band assignment, respectively. 2.5.2. Assay in the reverse direction The formation of [14C]Man-1-P was detected using GDP-[14C]Man and sodium pyrophosphate as starting material. The reaction mixture (50 Al) contained: 100 mM Tris – HCl, pH 7.5, 5 mM sodium pyrophosphate, 10 AM GDP-[14C]Man (330 mCi/mmol), 4 mM MgCl2 and appropriate amounts of GST-SsoGMPP or BSA. The samples were incubated for 30 min at 60 jC, and the reactions were stopped by mixing the samples with equal amounts of a stop solution consisting of 100 mM EDTA and frozen in dry ice-ethanol. The formation of [14C]Man1-P was analyzed by autoradiography after separating the GDP-Man by chromatography on a polyethyleneimine cellulose thin layer performed as described for the assay in the forward direction. 2.5.3. Electrophoretic analysis Electrophoretic runs were performed in a Bio-Rad Mini Protean II cell unit, at room temperature on 12% SDS polyacrylamide gels. Proteins were revealed by staining with Coomassie brilliant blue R250 (Bio-Rad). After the
staining reaction, gels were rinsed with water and fixed by storage in 10% acetic acid. For N-terminal sequences, protein samples after SDS/ PAGE electrophoresis were blotted onto a polyvinylidene fluoride membrane. Edman degradation was carried out on a pulsed liquid-phase sequencer model 477A (Applied Biosystems) equipped with a 120A analyzer for the on-line detection of phenylthiohydantoin amino acids.
3. Results 3.1. Identification and sequence analysis of SsoGMPP The sequence analysis of the S. solfataricus strain P2 genome (She et al., 2001) allowed the identification of an open reading frame (Sso0317), which was named Ssogmpp, potentially coding for a putative GDP-Man pyrophosphorylase. The derived amino acid sequence predicted a polypeptide of 361 residues with a calculated molecular mass of 40,519 Da. All non-redundant data bases were screened for entries showing similarity to this ORF with the BLASTP program, available at the site www.ncbi.nlm.nih.gov/ BLAST/. The predicted gene product deduced from the 1086-bp DNA sequence exhibits high similarity to several (deoxy)ribonucleotide diphosphate-sugar pyrophosphorylases, indicating that the protein is putatively involved in sugar activation-transfer mechanisms. In fact, the search for structural motifs involved in known biochemical functions, performed with the Protein Families database of Alignments and HMMs (PFAM) Program available at the web site www.sanger.ac.uk, pointed out the presence of a generally conserved N-terminal moiety (positions 3– 230) present also in bifunctional bacterial phosphomannose isomerase/guanosine diphospho-D-mannose pyrophosphorylase and, particularly, a block sequence (positions 4 to 28) presumably responsible for the sugar-1-P binding (Fig. 1, panel A). Moreover, the identification of a hexapeptide present in three repeats (located at 270 – 287, 310 –327 and 334– 351, Fig. 1, panels B, C and D), typical for a variety of bacterial transferases and shown to form a left-handed parallel h helix, is indicative of the nucleotide binding domain of the enzyme (Raetz and Roderick, 1995). Therefore, the new protein sequence was compared to orthologs that have been characterized biochemically (Fig. 1) and significant similarity was found with eukaryal and other archaeal representatives. In particular, the alignment with the GMPP from the yeast S. cerevisiae (Vig9) and from the plant Arabidopsis thaliana, produced the highest identity score (32%). Interestingly, this value is higher than that obtained in the comparison with the other archaeal GMPP from the hyperthermophile P. horikoshii (31%), confirming the evolutionary distance between the crenarchaeal and euryarchaeal sub-domains, when widespread and conserved sequences are considered as phylogenetic markers.
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Fig. 1. Multiple alignment of the Sulfolobus solfataricus GDP-Man pyrophosphorylase amino acid sequence with GMPPs from different sources. The sequences are ordered from the top to the bottom with decreasing degree of identity: Sulfolobus solfataricus (S.so, strain P2), S.ce (Vig9 from Saccharomyces cerevisiae), Arabidopsis thaliana (A.th), Pyrococcus horikoshii (P.ho), Leishmania mexicana (L.me). Conserved residues are boxed and the putative amino acid stretches involved in the sugar and nucleotide binding are separately indicated. Conserved amino acid stretches in the SsoGMPP sequence: Man-1-P binding Nterminal sequence in Panel A; GTP binding motif consisting of a three-fold hexapeptide repeat in Panels B, C and D.
3.2. RNA analysis The regulation of the expression of Ssogmpp was analyzed in S. solfataricus P2 cells grown in yeast extract media supplemented with different mono- and di-saccharides, evaluating RNA synthesis levels in exponential grown cultures; the Northern blot analysis using the Ssogmpp coding sequence as the probe revealed a single hybridization band, showing a signal intensity independent from the growth conditions (Fig. 2, panel A), namely from the specific sugar source in the single cultures. The molecular size, when compared to molecular weight RNA standards,
was calculated to be about 700 nt larger than the value of about 1100 expected for the length (1086 nt) of the gene coding sequence. The location of the transcription initiation site was determined by primer extension analysis using the same RNA samples extracted from S. solfataricus P2 cells in the mannose and arabinose cultures (Fig. 2, panel B). A single initiation site was identified for Ssogmpp corresponding to an A residue, belonging to the ATG translation start codon. Therefore, potential promoter sequences could be assigned: the most confident putative TATA box
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Fig. 2. Northern analysis and 5V mapping of the Ssogmpp transcript. (A) RNA extracted from cells harvested at middle exponential phase of growth and cultured in yeast media supplemented with different carbon sources (M: mannose; A: arabinose; L: Lactose; S: Sucrose) was analysed by electrophoresis on denaturing agarose gel stained with ethidium bromide (panel 1) and Northern blot using the Ssogmpp coding sequence as the probe (panel 2). A replica filter was hybridised with Sso10b gene encoding the DNA binding protein ALBA for signal normalization (panel 3). (B) Total RNAs from mannose and arabinose cultured cells were analysed by primer extension (lanes M1, M2, A1 and A2). The cDNA products were electrophoresed in increasing amounts with the sequence ladder generated by the same primer on the non coding strand of the Ssogmpp gene. The mapped transcription start site (the adenine nucleotide in the ATG translation start codon) and the identified TATA box consensus are indicated on the sequence by a circle and a box, respectively.
(TTTATC), not perfectly matching the archaeal consensus (T/CTTAT/AA), could be centered at 25 nt from the transcription/translation start site and an A/T-rich sequence located in between the TATA box and the ATG start codon, very frequent in archaeal promoters and indicated as ‘‘proximal promoter’’. No typical terminator T-rich sequence downstream of the stop codon, neither a sequence immediately upstream of the TATA box matching significantly the TFB-responsive element (BRE), which is involved in the orientation of the transcription preinitiation complex in archaea (Bell et al., 1999), could be identified. 3.3. Heterologous gene expression and purification of recombinant GST-SsoGMPP In order to demonstrate that the putative GDP-Man pyrophosphorylase encoded by the isolated Ssogmpp se-
quence catalyzes the formation of GDP-Man, a plasmid that produces a GST-SsoGMPP fusion protein was constructed as described in Materials and methods. E. coli Rb791 cells transformed with the hybrid gene were grown in liquid culture under different temperature, inducer concentrations and induction times in order to test their ability as a good source of the recombinant enzyme. Standard conditions, namely growth at 37 jC, sorted the recombinant protein almost exclusively to inclusion bodies (Fig. 3) at any IPTG concentration (0.1, 0.5 and 1.0 mM) tested. Therefore, lower temperature cultures were performed to increase the soluble cytosolic fraction of the enzyme and growth at 22 jC up to 2.0 OD, 0.1 mM IPTG and incubation over night resulted the best conditions for this purpose. Purification of the fusion protein, with free GST as the only contaminant, could be obtained by Glutathione Sepharose affinity chromatography as judged by Coomas-
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lane 9) and electrophoretic separation of the tag GST. The molecular mass of the protein (42.00 F 1.0 kDa) as determined by SDS-PAGE analysis was significantly close to the value calculated for the polypeptide translated from the isolated Ssogmpp gene, taking into account the additional amino acids left at the N-terminus after the thrombin cleavage. 3.4. Specificity of the enzyme activity
Fig. 3. SDS-PAGE analysis of insoluble fractions of E. coli transformed with the hybrid gene expressing the GST-SsoGMPP fusion. Growth of transformed cells and induction of the gene expression were performed at 22 and 37 jC. Solubilized pellets after lysis were loaded onto 12% gel. Lane M: molecular weight standards; lane 1: solubilized proteins from cells grown and induced at 22 jC; lane 2: cytosolic fraction from cells transformed with only the GST gene and grown at 37 jC; lane 3: cytosolic proteins from cells grown at 22 jC; lane 4: insoluble fraction from cells grown at 37 jC; lane 5: protein soluble fraction from cells grown at 37 jC. The expected molecular size of chimeric GST-SsoGMPP protein is indicated by the arrow.
sie staining analysis on SDS-PAGE gel. Fig. 4 shows the results of growth-induction and purification of the enzymes under the different conditions tested. Definitive identification of the GMPP expression product (indicated by the white arrow) was obtained by specific cutting in vitro performed with thrombin on the fusion protein eluted from the affinity column (Fig. 4,
The specific Man-1P guanylyl transferase activity direct assay allowed us to ascertain the biochemical function of the SsoGMPP and hence to confirm sequence analysis prediction by the identification of the specific GDP-Man product, performed with reference standards both on polyethyleneimine cellulose and silica plates thin layer chromatography. In fact, the affinitypurified GST-SsoGMPP protein had activity to synthesize [32P]GDP-Man from [a-32P]GTP and Man-1-P with values increasing in the range 35 –60 jC, whereas the fusion partner GST did not. More interestingly, the enzyme exhibited higher catalytic efficiency at 60 jC, when compared to the corresponding mesophilic Vig9 protein from yeast, assayed at its own optimum temperature of 35 jC. The enzyme specificity was definitively demonstrated in the assay of the catalytic efficiency in the reverse reaction: [14C]Man-1-P was specifically formed in the presence of the enzyme starting from GDP-[14C]Man and sodium pyrophosphate. The enzyme was also highly specific for GDP-Man synthesis as demonstrated by the barely detectable activity
Fig. 4. GST-SsoGMPP purification by affinity chromatography on glutathione sepharose. E. coli Rb791 cells transformed with the plasmid pGEX-SsR were grown and subjected to induction of the gene expression at 17 and 22 jC. The fusion protein (indicated by the black and white arrow) was detectable after affinity chromatography of the cleared protein extracts (lanes 1 and 5) and recovered in two fractions (lanes 3, 4, 7, and 8). Definitive identification of the GMPP expression product (indicated by the white arrow) was obtained by specific cut in vitro with thrombin (lane 9) and electrophoretic separation of the tag GST (indicated by a black arrow). Lane 2 and 6: flow-through fractions of the affinity chromatography; lane M: molecular weight standards.
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Fig. 5. Sugar-1-P specificity assay of SsoGMPP. Sugar-GDP synthesis activity was assayed using [a-32P]GTP and Man-1-P or Glc-1-P as starting material. The labelled products, formed in the reaction performed at 75 jC as described under Material and methods, were detected by chromatography on polyethyleneimine cellulose and imager analyzer. The arrow indicates the nucleotidyl-sugar products of the reactions. (A) Reaction performed using bovine serum albumin on [a-32P]GTP and Man-1-P as a negative control reaction. (B and C) GDP-Man and GDP-Glc formation. (D) Reaction performed at 35 jC using yeast Vig9 on [a-32P]GTP and Man-1-P as a positive control reaction.
when Glc-1-P instead of Man-1-P was used as the substrate (Fig. 5).
4. Discussion In the framework of a more general project aimed at the study of glycosylation mechanisms of biomolecules in S. solfataricus, a gene (Ssogmpp) encoding a Man-1-P guanylyl transferase/GDP-Man pyrophosphorylase was identified on the available analysis data from the recently sequenced genome of the P2 strain. In fact, the amino acid sequence assigned SsoGMPP to the general family of sugar-1-P (deoxy)ribonucleotidyl transferase, a group of enzymes that includes representatives from all three domains of life, namely from Archaea, Eukarya and Bacteria. Interestingly, the highest identity score (32%) was found in the comparison with the sequence of two eukaryal GMPPs from A. thaliana and S. cerevisiae. For this reason, the protein Vig9 from yeast was used along all this work as a positive control for both the heterologous gene expression in E. coli under lower temperature growth and induction conditions and for the identification of the enzyme specificity. The expression as a recombinant GST-fusion protein in E. coli was at fairly high levels and allowed the successful purification in only one affinity chromatography step and the production of enough material for further study. Therefore, the biochemical function of the enzyme could be confirmed by specific assays performed at high temperature, the activity showing thermophilicity as expected for a protein from S. solfataricus (Ladenstein and Antranikian, 1998). Differently from other well-characterized GMPPs
(Ning and Elbein, 2000), the enzyme showed substrate specificity towards both Man-1-P and GTP: barely detectable activity could be revealed when Glc-1-P was used or other nucleotides (ATP, CTP and UTP) were tested under the same conditions. The present study for the first time describes an enzyme from Archaea potentially involved in glycosylation of cell components such as proteins, lipids, and oligosaccharides, namely in those processes requiring GDP-Man as an activated form of mannosyl precursors and there are some evidences strongly supporting this hypothesis. For example, the annotation of the SsoGMPP sequence on the S. solfataricus P2 genome project indicates, on the basis of homology comparison performed by the authors themselves, that this gene could participate in the cell envelope biogenesis, as an outer membrane nucleoside-diphosphate-sugar pyrophosphorylase involved in lipopolysaccharide biosynthesis. In addition, three similar genes, encoding putative polysaccharide glycosyl transferases (Sso0837, Sso1624 and Sso1780), present on the genome of S. solfataricus P2, show significant similarity with genes of the rfb family, namely genes involved in the biosynthesis of lipid-anchored polysaccharides in Bacteria. Members of this family are integral membrane proteins and includes RfbX, part of the O antigen biosynthesis operon in pathogenic bacteria (Marolda et al., 1999) and SpoVB from Bacillus subtilis, which is involved in spore cortex biosynthesis (Popham and Stragier, 1991). These biosynthetic pathways require, among others, GDPMan as a sugar elongation precursor and hence its synthesizing enzyme GMPP. In plant pathogens such as Xanthomonas campestris, exopolysaccharides (xanthans) mediate cell– cell interaction by connecting cell wall components of the microbe and colonized cells (Ielpi et al., 1993). In Sulfolobus, the function of these cell-associated polysaccharides is still unknown but they likely serve as similar mediators for specific interactions of the cell envelope with environmental macromolecules or complex structures, such as plant components. The results of Northern analysis of the SsoGMPP transcript seem to provide a possible role for SsoGMPP in vivo; the specific messenger RNA detected was found to exceed the expected size of about 700 nt and this extra sequence can be explained with the existence of a polycistronic messenger RNA. In fact, a 711-bp ORF, indicated as Sso0318, is located at only 2-bp downstream of the Ssogmpp gene on the S. solfataricus genome sequence. Intriguingly, the translated protein showed significant identity with a chinase specific for the elongation of oligo-/poly-saccharide components of the cell surface. Therefore, this study reasonably strengthens the hypothesis of a specific involvement of SsoGMPP in the biogenesis of cell envelope. Similarly, only a few data are available on protein glycosylation in Sulfolobus but the study performed by Za¨hringer et al. (2000) on post-translational modifications of cytochrome b558/556 from the species acidocaldarius, indicates a specific pattern of Asn-glycosylation, with a
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highly branched hexasaccharide containing two mannose residues, again suggesting the essential contribution of GMPP in the oligosaccharide formation. Differently from the euryarchaeon P. horikoshii (Empadinhas et al., 2001) and as a confirmation of the relative evolutionary distance between the two archaeal subdomains, the involvement of GMPP in mannosylglycerate biosynthesis and more generally in the osmotic stress response can be definitively excluded since the specific compatible solute cannot be found in S. solfataricus cells both under normal (da Costa et al., 1998) or salt stress conditions (data not shown). This work highlights the power of using post-genomics techniques coupled with the S. solfataricus genome initiative to rapidly clone physiologically important genes. In this respect, the assignment of the specific functional role in vivo of the Ssogmpp and its involvement, if any, in the specific transglycosylation processes mentioned, is at present under investigation.
Acknowledgements We thank Prof. Koji Yoda (University of Tokyo) for providing us with the Vig9 expressing E. coli strain and Dr. Giuseppe Ruggiero for technical assistance in the S. solfataricus cell cultures. This work was supported by European Union project ‘‘Hypersolutes’’ (QLRT-2000-00640).
References Bell, S.D., Kosa, P.L., Sigler, P.B., Jackson, S.P., 1999. Orientation of the transcription preinitiation complex in Archaea. Proc. Natl. Acad. Sci. U. S. A. 96, 13662 – 13667. Besra, G.S., Morehouse, C.B., Rittner, C.M., Waechter, C.J., Brennan, P.J., 1997. Biosynthesis of mycobacterial lipoarabinomannan. J. Biol. Chem. 272, 18460 – 18466. Brent, R., Ptashne, M., 1981. Mechanism of action of the lexA gene product. Proc. Natl. Acad. Sci. U. S. A. 78, 4204 – 4208. Brock, T.D., Brock, K.M., Belly, R.T., Weiss, R.L., 1972. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Microbiol. 84, 54 – 68. Conklin, P.L., Norris, S.R., Wheeler, G.L., Williams, E.H., Smirnoff, N., Last, R.L., 1999. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 96, 4198 – 4203. da Costa, M.S., Santos, H., Galinski, E.A., 1998. An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv. Biochem. Eng. Biotechnol. 61, 117 – 153. Empadinhas, N., Marugg, J.D., Borges, N., Santos, H., da Costa, M.S., 2001. Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii. Biochemical and genetic characterization of key enzymes. J. Biol. Chem. 276, 43580 – 43588. Garami, A., Ilg, T., 2001. Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability. EMBO J. 20, 3657 – 3666. Hashimoto, H., Sakakibara, A., Yamasaki, M., Yoda, K., 1997. Saccharo-
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myces cerevisiae VIG9 encodes GDP-mannose pyrophosphorylase, which is essential for protein glycosylation. J. Biol. Chem. 272, 16308 – 16314. Ielpi, L., Couso, R.O., Dankert, M.A., 1993. Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris. J. Bacteriol. 175, 2490 – 2500. Ladenstein, R., Antranikian, G., 1998. Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water. Adv. Biochem. Eng. Biotechnol. 61, 37 – 85. Marolda, C.L., Feldman, M.F., Valvano, M.A., 1999. Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1). Microbiology 145, 2485 – 2495. Martins, L.O., Empadinhas, N., Marugg, J.D., Miguel, C., Ferreira, C., da Costa, M.S., Santos, H., 1999. Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase. J. Biol. Chem. 274, 35407 – 35414. Ning, B., Elbein, A.D., 2000. Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDPmannose and GDP-Glc pyrophosphorylases are different proteins. Eur. J. Biochem. 267, 6866 – 6874. Pedone, E., Cannio, R., Saviano, M., Rossi, M., Bartolucci, S., 1999. Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability. Biochem. J. 339, 309 – 317. Popham, D.L., Stragier, P., 1991. Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis. J. Bacteriol. 173, 7942 – 7949. Raetz, C.R., Roderick, S.L., 1995. A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase. Science 270, 997 – 1000. Ramos, A., Raven, N.D.H., Sharp, R.J., Bartolucci, S., Rossi, M., Cannio, R., Lebbink, J., Van der Oost, J., de Wos, W.M., Santos, H., 1997. Stabilization of enzymes against thermal stress and freeze-drying by mannosylglycerate. Appl. Environ. Microbiol. 63, 4020 – 4025. Sambrook, J., Russel, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sampaio, M.M., Santos, H., Boos, W., 2003. Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment. Appl. Environ. Microbiol. 69, 233 – 240. She, Q., et al., 2001. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. U. S. A. 98, 7835 – 7840. Varki, A., 1999. Exploring the biological role of glycans. In: Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G., Marth, J. (Eds.), Essentials of Glicobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 57 – 68. Wardlerworth, B.N., Russell, R.J., Bell, S.D., Taylor, G.L., White, M.F., 2002. Structure of Alba: an archaeal chromatin protein modulated by acetylation. EMBO J. 21, 4654 – 4662. Warit, S., Walmsley, R.M., Stateva, L.I., 1998. Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. Microbiology 144, 2417 – 2426. Warit, S., Zhang, N., Short, A., Walmsley, R.M., Oliver, S.G., Stateva, L.I., 2000. Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species. Mol. Microbiol. 36, 1156 – 1166. Za¨hringer, U., Moll, H., Hettmann, T., Knirel, Y.A., Schafer, G., 2000. Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose. Eur. J. Biochem. 267, 4144 – 4149. Zhang, L., Radziejewska-Lebrecht, J., Krajewska-Pietrasik, D., Toivanen, P., Skurnik, M., 1997. Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8. Mol. Microbiol. 23, 63 – 76.
Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus Silvana Sacchetti a, Simonetta Bartolucci a, Mose` Rossi b, Raffaele Cannio b,* a
Dipartimento di Chimica Biologica, Universita` degli Studi di Napoli ‘‘Federico II’’, Naples, Italy b Istituto di Biochimica delle Proteine, CNR, Via Pietro Castellino 111, 80131 Naples, Italy
Received 29 September 2003; received in revised form 2 February 2004; accepted 13 February 2004 Received by A.M. Campbell Available online 9 April 2004
Abstract An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 jC revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose. D 2004 Elsevier B.V. All rights reserved. Keywords: GDP-mannose pyrophosphorylase; Genome; Mannose metabolism; Cloning; Overexpression
1. Introduction Guanidine diphosphomannose (GDP-Man), the mannosyl donor for most Man-containing polymers, is formed by the transfer of mannose-1-phosphate (Man-1-P) to GTP to form GDP-Man and inorganic pyrophosphate (PPi). This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP) or GTP-a-D-
Man-1-P guanylyltransferase (EC 2.7.7.13), a key enzyme for the formation of mannose-containing glycoconjugates, such as protein N- and C-glycans, some O-glycans, glycosylphosphatidylinositol protein, as well as some glycolipid membrane anchors, known to have a variety of important functions (Varki, 1999). The enzyme GMPP catalyzes the synthesis of GDP-Man from Man-1-P and GTP by the following reversible reaction (Ning and Elbein, 2000): GTP þ Man -1 - P X GDP -Man þ Ppi:
Abbreviations: GDP-Man, guanidine diphosphomannose; GDP-Glc, guanidine diphosphoglucose; GMPP, guanidine diphosphomannose pyrophosphorylase; Man-1-P, mannose-1-phosphate; Glc-1-P, glucose-1-phosphate; PPi, inorganic pyrophosphate; LM, lipomannan; LAM, lipoarabidomannan; GST, glutathione S-tranferase; ORF, open reading frame; GSH, reduced glutathione; IPTG, isopropyl-1-thio-h-D-galactopyranoside; BSA, bovine serum albumin; PBS, phosphate buffered saline. * Corresponding author. Tel.: +39-816132285; fax: +39-816132248. E-mail address: [email protected] (R. Cannio). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.02.033
In eukaryotes, this activated form of mannose is the major mannosyl donor for the glycosylation of proteins in a biosynthetic pathway that produces N-linked oligosaccharides of many membrane and secretory glycoproteins. For this reason, mannose-containing glycans cover also an essential range of indirect functions that include the promotion of correct folding, solubility, stability and intracellular
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sorting of proteins. Particularly in lower eukaryotes, such as the Saccharomyces (Hashimoto et al., 1997) and Candida (Warit et al., 1998) genera of yeast, GMPP is involved in the multistep pathway of covalent linkages between the cell wall proteins and glucans (Warit et al., 2000). Mutations in the gene vig9 have been demonstrated to affect even sugar uptake and/or removal by increased sensitivity to antibiotic aminoglycosides (Hashimoto et al., 1997; Warit et al., 2000). In eukaryotic human pathogens, such as Leishmania, the GMPP gene is a virulence factor since its deletion causes loss of the capacity to infect macrophages and mice (Garami and Ilg, 2001). As another example of its broad biological role, GMPP provides also a key intermediate in the biosynthetic pathway of L-ascorbic acid (Vitamin C) in plants (Conklin et al., 1999). GMPPs have been reported to be the general enzymes catalyzing the formation of the activated sugar nucleotide precursors for both extracellular and capsular polysaccharide biosynthesis of bacteria. The enzyme participates in the elongation of the oligosaccharide moiety of mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), that are potent immunomodulators in tuberculosis and leprosy and hence it is important in light of the role of LAM in phagocytosis and generalized immunosuppression (Besra et al., 1997). Moreover, the GMPP gene is a component of a cluster responsible for the biosynthesis of the Yersinia enterocolitica O:8(YeO8) lipopolysaccharide O-side, the major virulence factor of this pathogen (Zhang et al., 1997). Only a few data are available in the literature about the distribution of this gene in the third domain of life, the Archaea. Recently, Empadinhas et al. (2001) have suggested its involvement in the defence against osmotic stress in the hyperthermophilic euryarchaeon Pyrococcus horikoshii; the compatible solute a-mannosylglycerate has been shown to be one of the most effective stabilizers of biomolecules like proteins (Ramos et al., 1997) and its biosynthetic pathway of mannosylglycerate requires GDP-Man as a substrate for the specific synthase in a fashion similar to the well-characterized mechanism in the thermophilic bacterium Rodothermus marinus (Martins et al., 1999). No data are available for the other archaeal subdomain of Crenarchaeota. Sulfolobus solfataricus, originally isolated from a solfataric field in the area of Naples, Italy, is a hyperthermophilic crenarchaeon able to grow chemoheterotrophically at acidic pH (pH 3 –5) and at high temperatures (80 – 87 jC). The inspection of the S. solfataricus genome, recently fully sequenced (She et al., 2001) and available at the web site http://www-archbac.u-psud.fr/projects/sulfolobus/, revealed an open reading frame (ORF) encoding a putative GDPMan pyrophosphorylase (SsoGMPP) with the predicted gene product showing significant similarity to corresponding proteins from all three domains of life. We describe here the cloning and heterologous expression of the Ssogmpp gene and the enzymatic characterization of its gene product in comparison with the yeast enzyme Vig9 as well as the
detection of the specific wild type protein in S. solfataricus cells. Moreover, we show that SsoGMPP is a thermophilic functional enzyme and that it is able to catalyze both the direct and reverse reaction for the synthesis and pyrophosphorolysis of GDP-Man in a highly specific manner. This is the first GDP-Man pyrophosphorylase identified in Archaea: its molecular characterization has implications for the function in vivo and the evolution of these enzymes.
2. Materials and methods 2.1. Strains, plasmids, enzymes and chemicals Escherichia coli TOP10F’ (purchased from Invitrogen) and Rb791 (Brent and Ptashne, 1981) strains were used as hosts for plasmid propagation and protein expression, respectively, and routinely cultured in Luria –Bertani medium, with ampicillin (100 Ag/ml). S. solfataricus strain P2 (DSM 1617) was supplied by Deutsche Sammlung von Mikroorganismen (DSM) (Braunschweig, Germany) and grown aerobically at 80 jC in Brock’s basal salt medium (Brock et al., 1972) containing 0.1% (w/v) yeast extract, 0.1% (w/v) casamino acids, and buffered at pH 3.7. For mRNA analysis simpler media containing 0.1% yeast extract and alternatively 0.2% (w/v) of sucrose, or mannose, or arabinose or lactose were tested. Nutrients and agar were purchased from Difco. Plasmid vectors pUC18 SmaI/BAP and pGEX-2TK were supplied by Pharmacia Biotech. Restriction/modification enzymes and Taq DNA polymerase were obtained from Roche and Promega, respectively. IPTG, X-Gal, all buffers, organic solvents and reagents were high grade pure and supplied by Sigma. Radioactive materials were from NEN-Dupont. 2.2. Isolation of the Ssogmpp gene from S. solfataricus P2 strain The gene encoding GDP-Man pyrophosphorylase (SsoGMPP) was amplified via PCR; the 5V primer used in the amplification contained the ATG start codon and a NcoI site (bold-faced letters and underlined in the sequence, respectively): manUP (5V-CATTTTTCTTACCATGG TATCCGCAAT-3V). The 3V primer had the following sequence: manDW (5V-CTAAGGATCATAATTGTCGAC TTATAG-3V), and was designed to insert a recognition site for the SalI endonuclease (underlined) immediately upstream of the termination codon (bold faced letters). The reaction was performed in a Perkin Elmer apparatus using as enzyme the Taq polymerase under the following conditions: denaturation at 95 jC for 10 min, followed by 35 cycles of denaturation at 95 jC for 1 min, annealing at 45 jC for 1 min and extension at 72 jC for 1 min. The resulting DNA fragment was ligated into the SmaI cloning site of the plasmid pUC18 and DNA fragments from four independent
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clones were sequenced by MWG-BIOTECH (Ebersberg, Germany). The complete coding sequence obtained was analyzed using Vector NTIk Suite program purchased by InforMax (North Bethesda, MD). Homology comparison and multiple alignment of the SsoGMPP protein with other nucleotidylsugar pyrophosphorylases were performed using Blast and ClustalW programs, respectively, available on the Internet. 2.3. RNA analyses 2.3.1. Total RNA extraction and Northern analysis S. solfataricus P2 cultures were grown in the media described above; cells were harvested when the cultures reached middle exponential phase, namely an absorbance at 600 nm of about 0.5 OD on the average. Total RNA was extracted by the guanidine thiocyanate method (Sambrook and Russel, 2001). For Northern blot analysis, the different RNAs extracted (14 Ag) were electrophoretically separated together with molecular weight RNA standards (MBI fermentas) in 1.2% agarose gel containing 10% formaldehyde and blotted onto a Hybond-XP nylon membrane (Amersham). Hybridization with [a-32P]-labelled Ssogmpp probe (1086-bp coding sequence) was carried out at 65 jC for 16 h in 5 Denhardt’s reagent, 6 SSC, 0.5% SDS, 100 Ag/ ml sonicated and denaturated salmon sperm DNA. After hybridization, blots were washed twice in 1 SSC, 0.1% SDS for 10 min at RT and three times in 0.5 SSC, 0.1% SDS for 15 min at 65 jC and signals visualized by autoradiography and quantified with a densitometric analysis. A replica filter was hybridized under the same conditions with the coding sequence of the Sso10b gene encoding the DNA binding protein ALBA (Wardlerworth et al., 2002) for the normalization of the specific signal quantification. 2.3.2. Cloning of the 5V Ssogmpp gene sequence from S. solfataricus P2 A 468-bp DNA fragment comprising 361-bp upstream of the S. solfataricus P2 Ssogmpp coding sequence was PCR amplified using oligonucleotides designed on the basis of the Sso0317 sequence on the P2 genome. The sequences of the 5V (GMPPup) and 3V (GMPPex) oligonucleotides were: 5 V- G G T G C A C TA G C A G A C T TA ATA A A C A A G - 3 V (corresponding to the region-361 to-335 from the A nucleotide in the translation start codon of the SsoGMPP gene) and 5V-CCCAGTATAGGTTTGTTTAAAATGGGG-3V (complementary to the region from + 81 to + 107 of the SsoGMPP coding sequence). The resulting amplicon was cloned into a pUC18 SmaI/BAP vector, producing the plasmid pUC-GMPPr.
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mmol) and purified on Sep-Pak cartridges (Waters) following the procedure described by Sambrook and Russel (2001). 105 cpm (106 cpm/pmol) of the oligonucleotide were co-precipitated with 50 Ag of total RNA isolated from S. Solfataricus cells grown in the simplified media described and were denatured, before coprecipitating with 1 pmol of the labelled oligonucleotide. The mixture was resuspended in 6 Al of reverse transcriptase buffer supplied by the manufacturer (Roche) and incubated for 3 min at 65 jC, placed on dry ice for 1 min and on ice for 5 min and then incubated for 40 min at 37 jC for the annealing step. Two microliters of deoxynucleotide triphosphates (2 mM each) suspended in reverse transcriptase buffer, 20 units reverse transcriptase (Roche) and 25 units ribonuclease inhibitor from human placenta were added and sample incubated at 48 jC for 30 min to drive cDNA synthesis. The synthesized cDNA was denatured at 100 jC and analyzed by electrophoresis on 6% acrylamide gel. The same primer was used for the DNA sequence performed on the 5V flanking region cloned in pUC18 to produce size standards. Both primer extension cDNA and the sequencing ladder were run on the same gel and the products visualized by autoradiography. 2.4. Gene expression and protein purification The Ssogmpp coding sequence obtained by PCR and cloned in pUC18 was digested with NcoI and HindIII, filled-in by Klenow treatment and inserted into the expression vector pGEX-2TK at the BamHI site made blunt by the same treatment. The cloning was designed to construct a plasmid, pGEX-SsR, in which an uninterrupted hybrid coding sequence was expressed as an unique inducible gene encoding GST-SsoGMPP fusion protein. E. coli RB791 competent cells were transformed with pGEX-SsR and grown at 22 jC in LB and 0.5% NaCl) until the A600 nm reached 2.0. IPTG (0.1 mM) was added, and the incubation was continued for 16 h. The procedure described by Hashimoto et al. (1997) was followed for the gene expression induction and production in E. coli BL21/pTgroE of the yeast Vig9 protein. Lysis for protein extract preparations was performed for both recombinant SsoGMPP and Vig9 as already described for recombinant thioredoxin from Bacillus acidocaldarius by Pedone et al. (1999), using PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) as the lysis buffer, and the cytosol was isolated by centrifugation 13,000 g. GST fusion proteins were loaded onto a Glutathione Sepharose 4B RediPack (Pharmacia) at 0.2 ml/min flow rate; after washing with 10 ml of PBS, the elution was obtained with 50 mM Tris – HCl pH 8.0 containing 10 mM GSH. 2.5. Protein determination and enzyme assays
2.3.3. Primer extension analysis The extension primer, the GMPPex oligonucleotide used for the 5V flanking region amplification, was radiolabelled using T4 polynucleotide kinase and [g-32P]dATP (3000 Ci/
Protein concentration was determined using the BioRad protein staining assay and BSA as the standard, following the instructions of the manufacturer.
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2.5.1. Assay in the forward direction The formation of [32P]GDP-Man was determined using [a-32P]GTP and Man-1-P as starting material. The incubation mixture (50 Al) contained: 40 mM HEPES pH 7.6, 8 mM MgCl2, 0.1 mM Man-1-P, 0.2 mM GTP, 0.1 mCi/ ml [a- 32 P]GTP (3000 Ci/mmol) and an appropriate amount of enzyme (affinity-purified GST-SsoGMPP, or crude extracts from S. solfataricus, as well as Vig9 and BSA, as positive and negative control, respectively). The assay was performed at 35 jC for Vig9 and in the range 35 – 60 jC for SsoGMPP and BSA; the reaction was stopped by mixing the samples with equal amounts of stop solution containing 100 mM EDTA and frozen in dry ice-ethanol. As a control of enzyme specificity, the assay was also performed on glucose-1P under the same conditions and using the same substrate concentrations and detection method. The formation of [32P]GDP-Man was detected and quantified by photostimulated luminescence, using a Phosphor Imager apparatus (BioRad) after separating the GDP-Man by thin layer chromatography on polyethyleneimine cellulose (Merck) performed with 1 M LiCl, 1 M HCOOH, as the solvent. Alternatively, for identification of the GDP-Man product, the reaction was performed in 50 Al of 20 mM Tris – HCl, 8.0 mM MgCl2, 4.0 mM GTP and 4.0 mM Man-1-P, pH 7.6 and 1.0 –2.0 Ag of the purified enzyme. Reaction reagents and products were separated by thin layer chromatography on silica gel with a solvent system composed of n-propanol and 25% ammonia (1:1, vol/vol) and visualized by spraying with a-naphthol-sulphuric acid solution followed by charring at 120 jC (Sampaio et al., 2003). Authentic standards of mannose-1-P and GDP-mannose were used for substrate and product band assignment, respectively. 2.5.2. Assay in the reverse direction The formation of [14C]Man-1-P was detected using GDP-[14C]Man and sodium pyrophosphate as starting material. The reaction mixture (50 Al) contained: 100 mM Tris – HCl, pH 7.5, 5 mM sodium pyrophosphate, 10 AM GDP-[14C]Man (330 mCi/mmol), 4 mM MgCl2 and appropriate amounts of GST-SsoGMPP or BSA. The samples were incubated for 30 min at 60 jC, and the reactions were stopped by mixing the samples with equal amounts of a stop solution consisting of 100 mM EDTA and frozen in dry ice-ethanol. The formation of [14C]Man1-P was analyzed by autoradiography after separating the GDP-Man by chromatography on a polyethyleneimine cellulose thin layer performed as described for the assay in the forward direction. 2.5.3. Electrophoretic analysis Electrophoretic runs were performed in a Bio-Rad Mini Protean II cell unit, at room temperature on 12% SDS polyacrylamide gels. Proteins were revealed by staining with Coomassie brilliant blue R250 (Bio-Rad). After the
staining reaction, gels were rinsed with water and fixed by storage in 10% acetic acid. For N-terminal sequences, protein samples after SDS/ PAGE electrophoresis were blotted onto a polyvinylidene fluoride membrane. Edman degradation was carried out on a pulsed liquid-phase sequencer model 477A (Applied Biosystems) equipped with a 120A analyzer for the on-line detection of phenylthiohydantoin amino acids.
3. Results 3.1. Identification and sequence analysis of SsoGMPP The sequence analysis of the S. solfataricus strain P2 genome (She et al., 2001) allowed the identification of an open reading frame (Sso0317), which was named Ssogmpp, potentially coding for a putative GDP-Man pyrophosphorylase. The derived amino acid sequence predicted a polypeptide of 361 residues with a calculated molecular mass of 40,519 Da. All non-redundant data bases were screened for entries showing similarity to this ORF with the BLASTP program, available at the site www.ncbi.nlm.nih.gov/ BLAST/. The predicted gene product deduced from the 1086-bp DNA sequence exhibits high similarity to several (deoxy)ribonucleotide diphosphate-sugar pyrophosphorylases, indicating that the protein is putatively involved in sugar activation-transfer mechanisms. In fact, the search for structural motifs involved in known biochemical functions, performed with the Protein Families database of Alignments and HMMs (PFAM) Program available at the web site www.sanger.ac.uk, pointed out the presence of a generally conserved N-terminal moiety (positions 3– 230) present also in bifunctional bacterial phosphomannose isomerase/guanosine diphospho-D-mannose pyrophosphorylase and, particularly, a block sequence (positions 4 to 28) presumably responsible for the sugar-1-P binding (Fig. 1, panel A). Moreover, the identification of a hexapeptide present in three repeats (located at 270 – 287, 310 –327 and 334– 351, Fig. 1, panels B, C and D), typical for a variety of bacterial transferases and shown to form a left-handed parallel h helix, is indicative of the nucleotide binding domain of the enzyme (Raetz and Roderick, 1995). Therefore, the new protein sequence was compared to orthologs that have been characterized biochemically (Fig. 1) and significant similarity was found with eukaryal and other archaeal representatives. In particular, the alignment with the GMPP from the yeast S. cerevisiae (Vig9) and from the plant Arabidopsis thaliana, produced the highest identity score (32%). Interestingly, this value is higher than that obtained in the comparison with the other archaeal GMPP from the hyperthermophile P. horikoshii (31%), confirming the evolutionary distance between the crenarchaeal and euryarchaeal sub-domains, when widespread and conserved sequences are considered as phylogenetic markers.
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Fig. 1. Multiple alignment of the Sulfolobus solfataricus GDP-Man pyrophosphorylase amino acid sequence with GMPPs from different sources. The sequences are ordered from the top to the bottom with decreasing degree of identity: Sulfolobus solfataricus (S.so, strain P2), S.ce (Vig9 from Saccharomyces cerevisiae), Arabidopsis thaliana (A.th), Pyrococcus horikoshii (P.ho), Leishmania mexicana (L.me). Conserved residues are boxed and the putative amino acid stretches involved in the sugar and nucleotide binding are separately indicated. Conserved amino acid stretches in the SsoGMPP sequence: Man-1-P binding Nterminal sequence in Panel A; GTP binding motif consisting of a three-fold hexapeptide repeat in Panels B, C and D.
3.2. RNA analysis The regulation of the expression of Ssogmpp was analyzed in S. solfataricus P2 cells grown in yeast extract media supplemented with different mono- and di-saccharides, evaluating RNA synthesis levels in exponential grown cultures; the Northern blot analysis using the Ssogmpp coding sequence as the probe revealed a single hybridization band, showing a signal intensity independent from the growth conditions (Fig. 2, panel A), namely from the specific sugar source in the single cultures. The molecular size, when compared to molecular weight RNA standards,
was calculated to be about 700 nt larger than the value of about 1100 expected for the length (1086 nt) of the gene coding sequence. The location of the transcription initiation site was determined by primer extension analysis using the same RNA samples extracted from S. solfataricus P2 cells in the mannose and arabinose cultures (Fig. 2, panel B). A single initiation site was identified for Ssogmpp corresponding to an A residue, belonging to the ATG translation start codon. Therefore, potential promoter sequences could be assigned: the most confident putative TATA box
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Fig. 2. Northern analysis and 5V mapping of the Ssogmpp transcript. (A) RNA extracted from cells harvested at middle exponential phase of growth and cultured in yeast media supplemented with different carbon sources (M: mannose; A: arabinose; L: Lactose; S: Sucrose) was analysed by electrophoresis on denaturing agarose gel stained with ethidium bromide (panel 1) and Northern blot using the Ssogmpp coding sequence as the probe (panel 2). A replica filter was hybridised with Sso10b gene encoding the DNA binding protein ALBA for signal normalization (panel 3). (B) Total RNAs from mannose and arabinose cultured cells were analysed by primer extension (lanes M1, M2, A1 and A2). The cDNA products were electrophoresed in increasing amounts with the sequence ladder generated by the same primer on the non coding strand of the Ssogmpp gene. The mapped transcription start site (the adenine nucleotide in the ATG translation start codon) and the identified TATA box consensus are indicated on the sequence by a circle and a box, respectively.
(TTTATC), not perfectly matching the archaeal consensus (T/CTTAT/AA), could be centered at 25 nt from the transcription/translation start site and an A/T-rich sequence located in between the TATA box and the ATG start codon, very frequent in archaeal promoters and indicated as ‘‘proximal promoter’’. No typical terminator T-rich sequence downstream of the stop codon, neither a sequence immediately upstream of the TATA box matching significantly the TFB-responsive element (BRE), which is involved in the orientation of the transcription preinitiation complex in archaea (Bell et al., 1999), could be identified. 3.3. Heterologous gene expression and purification of recombinant GST-SsoGMPP In order to demonstrate that the putative GDP-Man pyrophosphorylase encoded by the isolated Ssogmpp se-
quence catalyzes the formation of GDP-Man, a plasmid that produces a GST-SsoGMPP fusion protein was constructed as described in Materials and methods. E. coli Rb791 cells transformed with the hybrid gene were grown in liquid culture under different temperature, inducer concentrations and induction times in order to test their ability as a good source of the recombinant enzyme. Standard conditions, namely growth at 37 jC, sorted the recombinant protein almost exclusively to inclusion bodies (Fig. 3) at any IPTG concentration (0.1, 0.5 and 1.0 mM) tested. Therefore, lower temperature cultures were performed to increase the soluble cytosolic fraction of the enzyme and growth at 22 jC up to 2.0 OD, 0.1 mM IPTG and incubation over night resulted the best conditions for this purpose. Purification of the fusion protein, with free GST as the only contaminant, could be obtained by Glutathione Sepharose affinity chromatography as judged by Coomas-
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lane 9) and electrophoretic separation of the tag GST. The molecular mass of the protein (42.00 F 1.0 kDa) as determined by SDS-PAGE analysis was significantly close to the value calculated for the polypeptide translated from the isolated Ssogmpp gene, taking into account the additional amino acids left at the N-terminus after the thrombin cleavage. 3.4. Specificity of the enzyme activity
Fig. 3. SDS-PAGE analysis of insoluble fractions of E. coli transformed with the hybrid gene expressing the GST-SsoGMPP fusion. Growth of transformed cells and induction of the gene expression were performed at 22 and 37 jC. Solubilized pellets after lysis were loaded onto 12% gel. Lane M: molecular weight standards; lane 1: solubilized proteins from cells grown and induced at 22 jC; lane 2: cytosolic fraction from cells transformed with only the GST gene and grown at 37 jC; lane 3: cytosolic proteins from cells grown at 22 jC; lane 4: insoluble fraction from cells grown at 37 jC; lane 5: protein soluble fraction from cells grown at 37 jC. The expected molecular size of chimeric GST-SsoGMPP protein is indicated by the arrow.
sie staining analysis on SDS-PAGE gel. Fig. 4 shows the results of growth-induction and purification of the enzymes under the different conditions tested. Definitive identification of the GMPP expression product (indicated by the white arrow) was obtained by specific cutting in vitro performed with thrombin on the fusion protein eluted from the affinity column (Fig. 4,
The specific Man-1P guanylyl transferase activity direct assay allowed us to ascertain the biochemical function of the SsoGMPP and hence to confirm sequence analysis prediction by the identification of the specific GDP-Man product, performed with reference standards both on polyethyleneimine cellulose and silica plates thin layer chromatography. In fact, the affinitypurified GST-SsoGMPP protein had activity to synthesize [32P]GDP-Man from [a-32P]GTP and Man-1-P with values increasing in the range 35 –60 jC, whereas the fusion partner GST did not. More interestingly, the enzyme exhibited higher catalytic efficiency at 60 jC, when compared to the corresponding mesophilic Vig9 protein from yeast, assayed at its own optimum temperature of 35 jC. The enzyme specificity was definitively demonstrated in the assay of the catalytic efficiency in the reverse reaction: [14C]Man-1-P was specifically formed in the presence of the enzyme starting from GDP-[14C]Man and sodium pyrophosphate. The enzyme was also highly specific for GDP-Man synthesis as demonstrated by the barely detectable activity
Fig. 4. GST-SsoGMPP purification by affinity chromatography on glutathione sepharose. E. coli Rb791 cells transformed with the plasmid pGEX-SsR were grown and subjected to induction of the gene expression at 17 and 22 jC. The fusion protein (indicated by the black and white arrow) was detectable after affinity chromatography of the cleared protein extracts (lanes 1 and 5) and recovered in two fractions (lanes 3, 4, 7, and 8). Definitive identification of the GMPP expression product (indicated by the white arrow) was obtained by specific cut in vitro with thrombin (lane 9) and electrophoretic separation of the tag GST (indicated by a black arrow). Lane 2 and 6: flow-through fractions of the affinity chromatography; lane M: molecular weight standards.
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Fig. 5. Sugar-1-P specificity assay of SsoGMPP. Sugar-GDP synthesis activity was assayed using [a-32P]GTP and Man-1-P or Glc-1-P as starting material. The labelled products, formed in the reaction performed at 75 jC as described under Material and methods, were detected by chromatography on polyethyleneimine cellulose and imager analyzer. The arrow indicates the nucleotidyl-sugar products of the reactions. (A) Reaction performed using bovine serum albumin on [a-32P]GTP and Man-1-P as a negative control reaction. (B and C) GDP-Man and GDP-Glc formation. (D) Reaction performed at 35 jC using yeast Vig9 on [a-32P]GTP and Man-1-P as a positive control reaction.
when Glc-1-P instead of Man-1-P was used as the substrate (Fig. 5).
4. Discussion In the framework of a more general project aimed at the study of glycosylation mechanisms of biomolecules in S. solfataricus, a gene (Ssogmpp) encoding a Man-1-P guanylyl transferase/GDP-Man pyrophosphorylase was identified on the available analysis data from the recently sequenced genome of the P2 strain. In fact, the amino acid sequence assigned SsoGMPP to the general family of sugar-1-P (deoxy)ribonucleotidyl transferase, a group of enzymes that includes representatives from all three domains of life, namely from Archaea, Eukarya and Bacteria. Interestingly, the highest identity score (32%) was found in the comparison with the sequence of two eukaryal GMPPs from A. thaliana and S. cerevisiae. For this reason, the protein Vig9 from yeast was used along all this work as a positive control for both the heterologous gene expression in E. coli under lower temperature growth and induction conditions and for the identification of the enzyme specificity. The expression as a recombinant GST-fusion protein in E. coli was at fairly high levels and allowed the successful purification in only one affinity chromatography step and the production of enough material for further study. Therefore, the biochemical function of the enzyme could be confirmed by specific assays performed at high temperature, the activity showing thermophilicity as expected for a protein from S. solfataricus (Ladenstein and Antranikian, 1998). Differently from other well-characterized GMPPs
(Ning and Elbein, 2000), the enzyme showed substrate specificity towards both Man-1-P and GTP: barely detectable activity could be revealed when Glc-1-P was used or other nucleotides (ATP, CTP and UTP) were tested under the same conditions. The present study for the first time describes an enzyme from Archaea potentially involved in glycosylation of cell components such as proteins, lipids, and oligosaccharides, namely in those processes requiring GDP-Man as an activated form of mannosyl precursors and there are some evidences strongly supporting this hypothesis. For example, the annotation of the SsoGMPP sequence on the S. solfataricus P2 genome project indicates, on the basis of homology comparison performed by the authors themselves, that this gene could participate in the cell envelope biogenesis, as an outer membrane nucleoside-diphosphate-sugar pyrophosphorylase involved in lipopolysaccharide biosynthesis. In addition, three similar genes, encoding putative polysaccharide glycosyl transferases (Sso0837, Sso1624 and Sso1780), present on the genome of S. solfataricus P2, show significant similarity with genes of the rfb family, namely genes involved in the biosynthesis of lipid-anchored polysaccharides in Bacteria. Members of this family are integral membrane proteins and includes RfbX, part of the O antigen biosynthesis operon in pathogenic bacteria (Marolda et al., 1999) and SpoVB from Bacillus subtilis, which is involved in spore cortex biosynthesis (Popham and Stragier, 1991). These biosynthetic pathways require, among others, GDPMan as a sugar elongation precursor and hence its synthesizing enzyme GMPP. In plant pathogens such as Xanthomonas campestris, exopolysaccharides (xanthans) mediate cell– cell interaction by connecting cell wall components of the microbe and colonized cells (Ielpi et al., 1993). In Sulfolobus, the function of these cell-associated polysaccharides is still unknown but they likely serve as similar mediators for specific interactions of the cell envelope with environmental macromolecules or complex structures, such as plant components. The results of Northern analysis of the SsoGMPP transcript seem to provide a possible role for SsoGMPP in vivo; the specific messenger RNA detected was found to exceed the expected size of about 700 nt and this extra sequence can be explained with the existence of a polycistronic messenger RNA. In fact, a 711-bp ORF, indicated as Sso0318, is located at only 2-bp downstream of the Ssogmpp gene on the S. solfataricus genome sequence. Intriguingly, the translated protein showed significant identity with a chinase specific for the elongation of oligo-/poly-saccharide components of the cell surface. Therefore, this study reasonably strengthens the hypothesis of a specific involvement of SsoGMPP in the biogenesis of cell envelope. Similarly, only a few data are available on protein glycosylation in Sulfolobus but the study performed by Za¨hringer et al. (2000) on post-translational modifications of cytochrome b558/556 from the species acidocaldarius, indicates a specific pattern of Asn-glycosylation, with a
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highly branched hexasaccharide containing two mannose residues, again suggesting the essential contribution of GMPP in the oligosaccharide formation. Differently from the euryarchaeon P. horikoshii (Empadinhas et al., 2001) and as a confirmation of the relative evolutionary distance between the two archaeal subdomains, the involvement of GMPP in mannosylglycerate biosynthesis and more generally in the osmotic stress response can be definitively excluded since the specific compatible solute cannot be found in S. solfataricus cells both under normal (da Costa et al., 1998) or salt stress conditions (data not shown). This work highlights the power of using post-genomics techniques coupled with the S. solfataricus genome initiative to rapidly clone physiologically important genes. In this respect, the assignment of the specific functional role in vivo of the Ssogmpp and its involvement, if any, in the specific transglycosylation processes mentioned, is at present under investigation.
Acknowledgements We thank Prof. Koji Yoda (University of Tokyo) for providing us with the Vig9 expressing E. coli strain and Dr. Giuseppe Ruggiero for technical assistance in the S. solfataricus cell cultures. This work was supported by European Union project ‘‘Hypersolutes’’ (QLRT-2000-00640).
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