Molecular Characterization of a Novel β-Glucuronidase fromScutellaria baicalensis Georgi
2000; Elsevier BV; Volume: 275; Issue: 35 Linguagem: Inglês
10.1016/s0021-9258(19)61531-0
ISSN1083-351X
AutoresKaori Sasaki, Futoshi Taura, Yukihiro Shoyama, Satoshi Morimoto,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoWe cloned a gene encoding Scutellariaβ-glucuronidase (sGUS) that is involved in the initiation of H2O2 metabolism in skullcap (Scutellaria baicalensis). This gene consists of a 1581-nucleotide open reading frame, the deduced amino acid sequence of which contains an ATP/GTP binding site and a leucine zipper motif. sGUS has apparent similarity to the heparan sulfate-metabolizing β-glucuronidase heparanase but no homology to family 2 β-glucuronidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results suggested that sGUS does not belong to the family 2 β-glucuronidases. We modified several residues predicted to act as the acid-base or nucleophilic residue of sGUS by site-directed mutagenesis. Mutations at Glu212 or Glu329 resulted in much lowerk cat/K m values in the mutants as compared with the wild-type enzyme, indicating that these are the acid-base and nucleophilic residues of the active site, respectively. Moreover, similar site-directed mutagenesis confirmed that Tyr281 is also involved in the β-glucuronidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various structural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family. We cloned a gene encoding Scutellariaβ-glucuronidase (sGUS) that is involved in the initiation of H2O2 metabolism in skullcap (Scutellaria baicalensis). This gene consists of a 1581-nucleotide open reading frame, the deduced amino acid sequence of which contains an ATP/GTP binding site and a leucine zipper motif. sGUS has apparent similarity to the heparan sulfate-metabolizing β-glucuronidase heparanase but no homology to family 2 β-glucuronidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results suggested that sGUS does not belong to the family 2 β-glucuronidases. We modified several residues predicted to act as the acid-base or nucleophilic residue of sGUS by site-directed mutagenesis. Mutations at Glu212 or Glu329 resulted in much lowerk cat/K m values in the mutants as compared with the wild-type enzyme, indicating that these are the acid-base and nucleophilic residues of the active site, respectively. Moreover, similar site-directed mutagenesis confirmed that Tyr281 is also involved in the β-glucuronidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various structural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family. Scutellaria β-glucuronidase polyacrylamide gel electrophoresis rapid amplification of the cDNA ends polymerase chain reaction base pair(s) β-Glucuronidase is a glycosylhydrolase commonly found in animals, plants, and microorganisms. The physiological importance of this enzyme in the metabolism of sulfated glycosaminoglycans is well known, and its genetic deficiency in humans causes a mucopolysaccharide storage disease known as mucopolysaccharidosis type VII (1Sly W.S. Quinton B.A. McAlister W.H. Rimoin D.L. J. Pediatr. 1973; 82: 249-257Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 2Hall C.W. Cantz M. Neufeld E.F. Arch. Biochem. Biophys. 1973; 155: 32-38Crossref PubMed Scopus (80) Google Scholar). On the other hand, molecular characterization of β-glucuronidase has unequivocally established the structure of the active site by x-ray crystallography (3Jain S. Drendel W.B. Chen Z.-W. Mathews F.S. Sly W.S. Grubb J.H. Nat. Struct. Biol. 1996; 3: 375-381Crossref PubMed Scopus (202) Google Scholar) and by site-directed mutagenesis (4Islam M.R. Tomatsu S. Shah G.N. Grubb J.H. Jain S. Sly W.S. J. Biol. Chem. 1999; 274: 23451-23455Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Based on the comparison of amino acid sequences in the active sites, most β-glucuronidases, which have been identified in human (4Islam M.R. Tomatsu S. Shah G.N. Grubb J.H. Jain S. Sly W.S. J. Biol. Chem. 1999; 274: 23451-23455Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 5Oshima A. Kyle J.W. Miller R.D. Hoffmann J.W. Powell P.P. Grubb J.H. Sly W.S. Tropak M. Guise K.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 685-689Crossref PubMed Scopus (146) Google Scholar), dog (6Ray J. Bouvet A. Desanto C. Fyfe J.C. Xu D. Wolfe J.H. Aguirre G.D. Patterson D.F. Haskins M.E. Henthorn P.S. Genomics. 1998; 48: 248-253Crossref PubMed Scopus (63) Google Scholar), cat (7Fyfe J.C. Kurzhals R.L. Lassaline M.E. Henthorn P.S. Alur R.P.K. Wang P. Wolfe J.H. Giger U. Haskins M.E. Patterson D.F. Sun H.C. Jain S. Yuhki N. Genomics. 1999; 58: 121-128Crossref PubMed Scopus (73) Google Scholar), rat (8Nishimura Y. Rosenfeld M.G. Kreibich G. Gubler U. Sabatini D.D. Adesnik M. Andy R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7292-7296Crossref PubMed Scopus (53) Google Scholar), mouse (9Gallagher P.M. D'Amore M.A. Lund S.D. Ganschow R.E. Genomics. 1988; 2: 215-219Crossref PubMed Scopus (27) Google Scholar) and Escherichia coli (10Jefferson R.A. Burgess S.M. Hirsh D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8447-8451Crossref PubMed Scopus (823) Google Scholar), are classified as family 2 glycosylhydrolases with two conserved motifs (family 2 glycosylhydrolase signature and family 2 glycosylhydrolase acid-base catalyst). Recently, however, novel β-glucuronidases that do not belong to this family have been identified. For example, heparanases, β-glucuronidases catalyzing the degradation of heparan sulfate, do not show homology to other proteins including family 2 β-glucuronidases (11Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 12Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 13Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar, 14Kussie P.H. Hulmes J.D. Ludwig D.L. Patel S. Navarro E.C. Seddon A.P. Giorgio N.A. Bohlen P. Biochem. Biophys. Res. Commun. 1999; 261: 183-187Crossref PubMed Scopus (182) Google Scholar). In contrast, there have been only a few studies of plant β-glucuronidases. Although the presence of β-glucuronidase has been confirmed in various plants (skullcap, rye, rhubarb, duckweed, sugar beet, and tobacco) (15Levvy G.A. Biochem. J. 1954; 56: 462-469Crossref Scopus (26) Google Scholar, 16Schulz M. Weissenböck G. Phytochemistry. 1987; 26: 933-937Crossref Scopus (32) Google Scholar, 17Hodal L. Bochardt A. Nielsen J.E. Mattsson O. Okkels F.T. Plant Sci. (Limerick). 1992; 87: 115-122Crossref Scopus (46) Google Scholar), the kinetic properties and physiological roles of these β-glucuronidases have remained mostly unclear. However, we recently demonstrated that β-glucuronidase in skullcap (Scutellaria baicalensis), which shows a higher level of activity toward 7-O-β-glucuronides of flavones (18Morimoto S. Harioka T. Shoyama Y. Planta. 1995; 195: 535-540Crossref Scopus (27) Google Scholar), is involved in novel H2O2 metabolism (19Morimoto S. Tateishi N. Matsuda T. Tanaka H. Taura F. Furuya N. Matsuyama N. Shoyama Y. J. Biol. Chem. 1998; 273: 12606-12611Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). WhenScutellaria cells produce a large amount of H2O2 in a response to various forms of stress, baicalein 7-O-β-d-glucuronide, the major flavone of S. baicalensis, is immediately hydrolyzed to baicalein by endogenous β-glucuronidase. The resulting baicalein is then converted to 6,7-dehydrobaicalein by cell wall peroxidases, and a large amount of H2O2 is effectively detoxified during the peroxidase reaction. In vivo inhibition of the β-glucuronidase reaction using saccharic acid 1,4-lactone significantly decreased the rate of H2O2detoxification, resulting in serious damage of Scutellariacells, indicating that Scutellaria β-glucuronidase (sGUS),1 which cannot catalyze the degradation of H2O2, is an important enzyme required for initiation of H2O2 metabolism. Thus, we revealed the physiological roles and biochemical properties of sGUS, although molecular characterization of this enzyme has not been performed. Because the structural characteristics of plant β-glucuronidases have not been reported, it is of interest to determine the primary structure of sGUS. Therefore, in the present study we cloned and characterized a cDNA encoding sGUS. Our results demonstrated that sGUS has various structural characteristics in common with heparanase, and therefore we assumed that sGUS and heparanase belong to the same new β-glucuronidase family. Furthermore, to determine the features of this family, we attempted to identify the active site residues by modification using site-directed mutagenesis. We report here the structural characteristics of sGUS and identification of its active site. Reagents for molecular biological procedures were purchased from Amersham Pharmacia Biotech, Promega, Roche Molecular Biochemicals, Takara (Tokyo, Japan), and Toyobo (Osaka, Japan). DE-52 cellulose and hydroxylapatite were obtained from Whatman and Nacalai Tesque (Kyoto, Japan), respectively. Baicalein-7-O-β-d-glucuronide-conjugated Sepharose 6B was prepared using baicalein 7-O-β-d-glucuronide (50 mg) and epoxy-activated Sepharose 6B (3 g, Amersham Pharmacia Biotech) according to the manufacturer's protocol. The callus used in this study was induced from shoot stem segments of S. baicalensisand cultured as described previously (19Morimoto S. Tateishi N. Matsuda T. Tanaka H. Taura F. Furuya N. Matsuyama N. Shoyama Y. J. Biol. Chem. 1998; 273: 12606-12611Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and reagents for callus culture medium were obtained from Wako Pure Chemical Corp. (Osaka, Japan). Baicalein 7-O-β-d-glucuronide and baicalein were purified from the roots of S. baicalensis as described previously (18Morimoto S. Harioka T. Shoyama Y. Planta. 1995; 195: 535-540Crossref Scopus (27) Google Scholar), whereas luteolin 3′-O-β-glucuronide was a gift from Dr. A. Yagi (Fukuyama University, Japan). Because our previous extraction procedures for sGUS resulted in somewhat low specific activity (18Morimoto S. Harioka T. Shoyama Y. Planta. 1995; 195: 535-540Crossref Scopus (27) Google Scholar), we used a modified procedure. Three-week-old callus (500 g) was washed with 1 m NaCl (1,500 ml) to remove cell wall proteins and then homogenized with a 100 mm sodium phosphate buffer (pH 7.0, 1,000 ml) containing 10 mm 2-mercaptoethanol. The homogenate was filtered through nylon screens and centrifuged at 100,000 × g for 15 min. The supernatant was then fractionated with the addition of ammonium sulfate. Proteins precipitating in the 30–70% saturation fraction were collected by centrifugation at 20,000 × g for 15 min, resuspended in 10 mm sodium phosphate buffer (pH 7.0) containing 1 mm 2-mercaptoethanol, and dialyzed overnight against the same buffer. Insoluble materials were removed by centrifugation at 100,000 × g for 15 min, and sGUS was purified from the supernatant by chromatography as described previously (18Morimoto S. Harioka T. Shoyama Y. Planta. 1995; 195: 535-540Crossref Scopus (27) Google Scholar). Peptides for sequencing were prepared by two different procedures (in-gel proteinase digestion and CNBr digestion). In-gel proteinase digestion (20Cleveland D.W. Fischer S.G. Kirschner M.W. Laemmli U.K. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar) was carried out by incubating purified sGUS (50 μg) and endoproteinase Glu-C (2 unit, Promega) for 30 min in polyacrylamide gels. For CNBr digestion, purified sGUS (50 μg) was dissolved in 70%(v/v) formic acid containing 1% (w/v) CNBr and incubated at 30 °C for 10 h in the dark. The resulting peptide fragments were separated by SDS-PAGE (12.5% acrylamide gels), transferred onto polyvinylidene difluoride membranes at 1.5 mA/cm2 for 1 h with a semidry blotting apparatus, and subjected to N-terminal sequencing on an Applied Biosystems 473A protein sequencer. Total RNA was extracted from three-week-old S. baicalensis callus tissue as described previously (21Morimoto S. Tateishi N. Inuyama M. Taura F. Tanaka H. Shoyama Y. J. Biol. Chem. 1999; 274: 26192-26198Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and three cDNA pools were prepared. cDNA pools 1 and 2 were synthesized by reverse transcription of the above RNA solution using oligo dT primer (5′-(T)17–3′) and oligo dT primer possessing adapter (5′-GACTCGTCTAGAGGATCCCG(T)17–3′), respectively. Both reactions were carried out with Moloney murine leukemia virus reverse transcriptase according to the manufacturer's protocol (Toyobo, Osaka). cDNA pool 3 was prepared by attaching poly(A) tails to cDNA pool 1 using terminal deoxynucleotidyltransferase according to the manufacturer's protocol (Takara). The following oligonucleotide primers were used in this study: degenerate primers 1 (5′-GGGGTACCGTIAARATHGARGARAAYCC-3′), 2 (5′-CGGGATCCCCIARRTTRTACATIARRTG-3′), and 3 (5′-GGGGTACCGTIGCNCARACIGAYGARAA-3′); gene-specific primers 1 (5′-CCAATCTATCTTGCAAAGTGC-3′), 2 (5′-GGGGTACCCTTTGACGGCATTCCTGATG-3′), 3 (5′-CCAAGATCAGGGTACCATGC-3′), 4 (5′-GGGGTACCTGATTTGGAATGGTATACAG-3′), 5 (5′-GCTCTAGAGGAGTGTGATTATGGAGGAAACAACTATT-3′), and 6 (5′- AAACCTGCTGGGAAGTGATGAAAGCTTAAACTG-3′); and adapter primers 1 (5′-GACTCGTCTAGAGGATCCCG(T)17-3′) and 2 (5′- GACTCGTCTAGAGGATCCCG-3′). All cDNA fragments were amplified in 50-μl reaction mixtures consisting of primers (degenerate primer, 1 μm; gene-specific or adapter-specific primer, 0.2 μm), 0.2 mm dNTPs, 1.5 mmMgCl2, 50 mm KCl, 10 mm Tris-HCl (pH 8.3), and Taq DNA polymerase (1 unit, Roche Molecular Biochemicals). To amplify fragments of cDNA encoding sGUS, nested PCR was employed as the first step. The first round of PCR was conducted with degenerate primers 1 and 2 using template cDNA pool 1 (30 cycles of 1 min at each of 94, 40, and 72 °C). The PCR product (∼800 bp) was amplified by a second round of PCR with degenerate primers 2 and 3 in the presence of the PCR products from the first reaction as template (30 cycles of 1 min at each of 94, 50, and 72 °C). The 3′-terminal and 5′-terminal regions were amplified by RACE. After the first round of PCR with gene-specific primer 3, adapter primer 2, and template cDNA pool 2 (30 cycles of 1 min at each of 94, 55, and 72 °C), a 3′-RACE product (∼1,000 bp) was obtained by a second round of PCR with gene-specific primer 4, adapter-specific primer 3, and the first PCR products as template (30 cycles of 1 min at each of 94, 55, and 72 °C). 5′-RACE was performed in a similar manner. The first round of PCR was performed with gene-specific primer 1 and adapter primer 1 in the presence of template cDNA pool 3 (five cycles of 1 min at each of 94, 50, and 72 °C and then 30 cycles of 1 min at each of 94, 55, and 72 °C). A second round of PCR (30 cycles of 1 min at each of 94, 55, and 72 °C) with gene-specific primer 2, adapter primer 2, and the PCR products from the first reaction resulted in the amplification of the 5′-RACE product (450 bp). PCR products were cloned into the plasmid vector pUC119, transformed into competent E. coli cells (Top10F′), and sequenced on an Applied Biosystems 373S DNA sequencer. The obtained sequences were combined using the SeqEd™ program (Applied Biosystems). To create the appropriate site-directed mutants, two-reaction PCR was employed using four primers as described by Higuchi et al. (22Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2100) Google Scholar). Briefly, two overlapping PCR fragments were generated incorporating the codon for the new amino acid, and then the second PCR reaction amplified the 1.5-kilobase band (15 cycles at 98 °C (15 s), 60 °C (2 s), and 74 °C (5 s)). The PCR products were sequenced as describe above to verify the mutagenesis and to confirm that no additional mutations had arisen spontaneously. This procedure was used in the construction of all site-directed mutants (E194A, E212A, E225A, E272A, and E329A). The mutant oligonucleotides (sense) were as follows: E194A, TTT CTG ATA G C A TAC AGT CTG AAG; E212A, CTT GGT AAC G C A TTA GGA GGA CAC; E225A, GTT TCT CCA G C A GAT TAC GCC AAC; E272A, GAT CGG ACA CCC G C G CTA CAT GTG; E329A, TGG ATT GGT G C A GCT GGA GGA GCT; Y281A, ACC CAC CAT ATG GC T AAC CTG GGT; Y376A, ACT GGT GGT AAC GC T GGA CTA CTC. cDNA encoding the deduced mature sGUS was amplified using gene-specific primers 5 and 6 in the presence of template cDNA pool 1 (30 cycles of 1 min at each of 94, 50, and 72 °C). The reaction was conducted with KOD polymerase (Toyobo, 1 unit) according to the manufacturer's instructions. The amplified cDNA was ligated into the plasmid vector pUC119/sGUS and sequenced as described above to confirm that its nucleotide sequence was identical to that of sGUS. The cDNA encoding mature sGUS was excised from pUC119/sGUS atXbaI and HindIII sites and recloned into the expression vector pET28a to give pET28a/sGUS. The vector was transformed into E. coli BL21DE3, and the cells were cultured on LB medium containing 25 μg/ml kanamycin (Sigma). The cells were inoculated into LB broth (50 ml) containing 25 μg/ml kanamycin. When the optical density of culture at 660 nm reached 0.6, isopropyl-β-d-thiogalactoside (final concentration 0.4 mm) was added to the culture to induce the recombinant sGUS. After incubation at 37 °C for 2 h, the cells were harvested by centrifugation at 5,000 rpm at 4 °C for 3 min. The collected cells were washed with lysis buffer (20 mmTris-HCl (pH 8.0) containing 1 mm EDTA) and resuspended in the same buffer (6.8 ml). Lysozyme was added to this solution, and the mixture was incubated for 30 min on ice, then sonicated. Homogenates were centrifuged at 20,000 × g for 20 min, and the supernatant was used for Western blotting analysis and the determination of enzyme activity. E. colitransformed with mutant cDNAs or wild-type cDNA were cultured in LB broth (1, 500 ml) as described above, harvested, and homogenized with 100 mm phosphate buffer (pH 7.0, 20 ml) containing 10 mm 2-mercaptoethanol. The homogenate was centrifuged at 20,000 × g for 20 min, and the supernatant was dialyzed against 10 mm sodium phosphate buffer (pH 7.0) containing 1 mm 2-mercaptoethanol. The dialysate was applied to a DE-52 cellulose column (1.5 × 20.0 cm). The column was washed with three column volumes of the same buffer, and bound proteins were eluted with a 400-ml linear gradient of NaCl (0–0.4m) at a flow rate of 1 ml/min. Fractions containing β-glucuronidase were collected, concentrated, and dialyzed against 10 mm sodium phosphate buffer (pH 7.0) containing 1 mm mercaptoethanol. The dialysate was applied to a hydroxylapatite column (1.0 × 20.0 cm). The column was washed with three column volumes of the same buffer, and sGUS was eluted with a 250-ml gradient of the ionic strength of phosphate buffer (from 10 mm to 200 mm). The most active fractions were pooled, concentrated, and dialyzed against 10 mm sodium phosphate buffer (pH 7.0) containing 1 mmmercaptoethanol. The dialyzed sample was loaded onto a baicalein-7-O-β-d-glucuronide-conjugated Sepharose 6B column (1.0 × 20.0 cm). After washing with 30 ml of the same buffer, a 100-ml linear gradient from 0 to 150 mmsodium glucuronate was passed through the column at a flow rate of 0.1 ml/min. The fractions containing β-glucuronidase activity were pooled, concentrated, and used for the kinetic studies. Polyclonal antiserum against sGUS was generated in female white rabbits by injection of 250 μg (first injection) and 125 μg (second and third injections) of purified sGUS. The antiserum obtained after the third booster was used in this study. For Western blotting analysis, protein samples were resolved by SDS-PAGE (12.5% acrylamide gels) and transferred onto polyvinylidene difluoride membranes at 1.5 mA/cm2 for 1 h with a semidry blotting apparatus. The membranes were soaked overnight in blocking buffer (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% gelatin) and then incubated at room temperature for 1 h with anti-sGUS diluted in blocking buffer. Antibody binding was visualized using horseradish peroxidase-conjugated secondary antibodies (Wako, Tokyo) and 1-chloro-4-naphthol (Sigma). The standard assays consisted of 100 mm sodium phosphate buffer (pH 6.5), 1 mm substrate (baicalein 7-O-β-d-glucuronide or luteolin 3′-O-β-d-glucuronide), 1 mmmercaptoethanol, 0.1% (w/v) Triton X-100, and 50 μl of enzyme solution in a final volume of 600 μl. Samples were incubated for 30 min at 28 °C, and the reaction was terminated by the addition of 600 μl methanol. The level of aglycone as a reaction product was quantified by high performance liquid chromatography in a system composed of a CCPM pump and a UV 8000 absorbance detector equipped with a Cosmosil 5C18 AR-II column (Nacalai Tesque, 0.46 × 10.0 cm). Baicalein or luteolin was eluted at a flow rate of 1 ml/min with 35% (v/v) aqueous acetonitrile containing 50 mm phosphoric acid. The column effluent was monitored by absorption at 280 nm, and each peak intensity was determined with a Chromatocorder 21 (Tosoh). The amounts of baicalein and luteolin were calculated from standard curves obtained with authentic samples. The enzyme activity (katal) was defined as the amount (moles) of aglycone liberated from corresponding substrate per second. The kinetic parameters were determined by assaying β-glucuronidase activity under the conditions as described above. TheK m and V max values for baicalein 7-O-β-d-glucuronide were calculated by Lineweaver-Burk double-reciprocal plots of the velocity curves of the β-glucuronidase reaction with increasing concentration of substrate. The Michaelis-Menten equation was fitted to the data by nonlinear least-squares regression analysis. Isolation of the gene encoding sGUS was carried out by reverse transcription-PCR. As a first step, mRNA was extracted from 3-week-oldScutellaria callus, which showed the maximum β-glucuronidase activity, and a cDNA template was then synthesized by reverse transcription. Furthermore, to design degenerate PCR primers, we analyzed the N-terminal and internal amino acid sequences of sGUS. After purification of sGUS to homogeneity as reported previously (18Morimoto S. Harioka T. Shoyama Y. Planta. 1995; 195: 535-540Crossref Scopus (27) Google Scholar), its N-terminal amino acid sequence (25 amino acid residues) was determined by Edman degradation (Table I). Concerning the internal sequence, the purified enzyme was digested with endoproteinase Glu-C or CNBr, and three (Glu-C-1, Glu-C-2, and CNBr-1) of the resulting peptide fragments were then subjected to N-terminal sequence analysis (Table I).Table IN-terminal amino acid sequences of the intact purified sGUS and peptides obtained by Glu-C-endoproteinase or CNBr digestionProtein/peptidesReagent for digestionN-terminal amino acid sequenceIntact sGUSEETTIVKIEENPVAQTDENYVXATLDLXPPGlu-C-1Endoproteinase Glu-CLHVATHHMYNLGSGGDGlu-C-2Endoproteinase Glu-CVGTKNVYIYAXXAKCNBr-1CNBrLVLNXDGEXXVKISLDPThe peptides derived from endoproteinase-Glu-C or CNBr digestion of purified sGUS were resolved by SDS-PAGE and transferred onto polyvinylidene difuluoride membranes. Sequencing of each peptide was carried out as described under "Experimental Procedures." The letter X indicates that the identity of the residue was ambiguous. Open table in a new tab The peptides derived from endoproteinase-Glu-C or CNBr digestion of purified sGUS were resolved by SDS-PAGE and transferred onto polyvinylidene difuluoride membranes. Sequencing of each peptide was carried out as described under "Experimental Procedures." The letter X indicates that the identity of the residue was ambiguous. When nested PCR was performed using degenerate primers designed from the N-terminal amino acid sequences of the peptide Glu-C-1 and intact β-glucuronidase, amplification of an ∼800-bp cDNA fragment was observed. Moreover, cDNA fragments containing the 3′- and 5′-end regions (∼1000 bp and ∼450 bp, respectively) were obtained by 3′- and 5′-RACE using gene-specific primers designed from the degenerate PCR product. Finally, the cDNA fragment, which was assumed to contain the entire coding region, was amplified using gene-specific primers from 3′- and 5′-RACE products. After all amplified cDNAs were cloned into the vector pUC119 and transformed into E. coli, their nucleotide sequences were determined from both strands to ensure accuracy. To our knowledge, isolation of cDNA encoding plant β-glucuronidase has not been reported previously. The sGUS gene consisted of a 1581-nucleotide open reading frame encoding a protein of 527 amino acid residues (Fig.1). The deduced primary structure contained amino acid sequences of all fragments derived by partial digestion with endoproteinase Glu-C or CNBr. PSORT and signalP programs indicated cleavage between Gly25 and Glu26, which was identical to the results obtained by N-terminal sequencing of the intact enzyme (Table I). Hence, we concluded that mature sGUS consists of 502 amino acid residues. The theoretical values of molecular weight (56,027 Da) and pI (5.52) calculated from the amino acid composition of the deduced mature protein were consistent with the values (M r∼55,000 Da and pI 5.5) obtained by SDS-polyacrylamide gel electrophoresis and isoelectric focusing, respectively (18Morimoto S. Harioka T. Shoyama Y. Planta. 1995; 195: 535-540Crossref Scopus (27) Google Scholar). To determine the structural characteristics of sGUS, we first examined the motifs in this enzyme. The amino acid sequences (Gly61 to Ser68 and Leu459 to Leu480) of the two boxed regions in Fig. 1 demonstrated that sGUS possesses two novel motifs (an ATP/GTP binding site and a leucine zipper pattern, respectively) that are not found in other β-glucuronidases. In addition, an N-glycosylation site was predicted from the sequence from Asn118 to Asn121. We analyzed the sugar moiety by treatment of the purified enzyme with phenol-sulfuric acid, but the presence of a sugar moiety was not confirmed. Although most β-glucuronidases possess family 2 glycosylhydrolase signature and family 2 acid-base catalyst (4Islam M.R. Tomatsu S. Shah G.N. Grubb J.H. Jain S. Sly W.S. J. Biol. Chem. 1999; 274: 23451-23455Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 5Oshima A. Kyle J.W. Miller R.D. Hoffmann J.W. Powell P.P. Grubb J.H. Sly W.S. Tropak M. Guise K.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 685-689Crossref PubMed Scopus (146) Google Scholar, 6Ray J. Bouvet A. Desanto C. Fyfe J.C. Xu D. Wolfe J.H. Aguirre G.D. Patterson D.F. Haskins M.E. Henthorn P.S. Genomics. 1998; 48: 248-253Crossref PubMed Scopus (63) Google Scholar, 7Fyfe J.C. Kurzhals R.L. Lassaline M.E. Henthorn P.S. Alur R.P.K. Wang P. Wolfe J.H. Giger U. Haskins M.E. Patterson D.F. Sun H.C. Jain S. Yuhki N. Genomics. 1999; 58: 121-128Crossref PubMed Scopus (73) Google Scholar, 8Nishimura Y. Rosenfeld M.G. Kreibich G. Gubler U. Sabatini D.D. Adesnik M. Andy R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7292-7296Crossref PubMed Scopus (53) Google Scholar, 9Gallagher P.M. D'Amore M.A. Lund S.D. Ganschow R.E. Genomics. 1988; 2: 215-219Crossref PubMed Scopus (27) Google Scholar, 10Jefferson R.A. Burgess S.M. Hirsh D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8447-8451Crossref PubMed Scopus (823) Google Scholar), neither motif was found in the deduced sequence of sGUS. A protein sequence homology search based on the FASTA program established that sGUS shows homology to rat heparanase (26.8% identity in 487 amino acid overlap) (13Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar) and human heparanase (26.7% identity in 479 amino acid overlap) (11Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 12Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. 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Taken together with the lack of motifs characteristic of family 2 glycosylhydrolases, these results indicated that sGUS is not a family 2 glycosylhydrolase. As sGUS showed similarities to heparanases, we compared the primary structure of sGUS with those of all heparanases including mouse heparanase (13Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar). As shown in Fig. 2, sGUS contained four regions with substa
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