2 Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; 3 Kirin Brewery, Central Laboratories for Key Technology, Fukuura, Kanazawa-ku, Yokohama 236, Japan; and 4 Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
Received on February 25, 2002; revised on May 13, 2002; accepted on May 14, 2002
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Abstract |
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Key words: Caenorhabditis elegans/endo-ß-N-acetylglucosaminidase/Endo-CE/transglycosylation
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Introduction |
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As to fungi, we already reported about the endo-ß-GlcNAc-ase from Mucor hiemalis (Endo-M), which cleaves not only high-mannose type oligosaccharides but also complex biantennary oligosaccharides (Yamamoto et al., 1994a). Recently, the Endo-M gene was cloned (accession no. AB060586). During a similarity search of the genome database with the Endo-M gene, we found a putative sequence homolog of Endo-M in the genome sequencing project data for Caenorhabditis elegans that exhibited about 34% amino acid identity with the Endo-M amino acid sequence. We attempted to clone the gene because C. elegans is a simple multicellular organism and a good model for investigating the biological roles of endo-ß-GlcNAc-ase in multicellular organisms. We isolated a C. elegans cDNA clone containing the complete open reading frame and expressed it in Escherichia coli. Functional analyses of recombinant worm enzymes were carried out in vitro.
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Results |
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Next, when plaque hybridization was performed using this fragment as a probe, five positive signals were observed. From these plaques, plasmids were obtained by Cre-excision with the ExAssist helper phage, and then the insert fragments of these plasmids (EcoR I/Xho I) were sequenced. The longest clone had an approximately 1.4-kbp insert fragment and an open reading frame encoding 433 amino acids. This amino acid sequence was 45 amino acid residues longer at the N-terminal end than in the case of the F01F1.10 gene in the database (Figure 2).
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Substrate specificity of recombinant Endo-CE
The substrate specificity of recombinant Endo-CE was examined with the various pyridylamino (PA)-sugar chains shown in Table TI. Each PA-sugar chain substrate (40 pmol) was incubated with 0.25 mU recombinant Endo-CE in sodium acetate buffer, pH 6.0, at 20°C for 10 min. The reaction mixture was analyzed by reversed-phase high-performance liquid chromatography (HPLC), the fluorescence of PA groups being monitored at 400 nm with excitation at 320 nm. The results are summarized in Table TI. Concerning the high-mannose type sugar chains of PA-derivatives, the oligomannosides of M9A, M8A, and M6B were more easily hydrolyzed than the oligomannoside of M5A or the trimannosyl core. On the other hand, the complex type sugar chains of PA-derivatives were hardly hydrolyzed. In particular, the PA-asialotriantennary sugar chain could not be cleaved, and the PA-fucosyl trimannosyl core could also not be hydrolyzed, although the PA-trimannosyl core could be cleaved.
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Discussion |
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In this study, we isolated the F01F1.10 gene of C. elegans, expressed it in E. coli and confirmed that the gene product showed endo-ß-GlcNAc-ase activity, which is not only hydrolyzing activity but also transglycosylation activity. Although the presence of endo-ß-GlcNAc-ase in various animal tissues has been reported (Pierce et al., 1979; Overdijk et al., 1981
; Tachibana et al., 1981
, 1982; Lisman et al., 1985
; Weng and Spiro, 1997
), Endo-HO is the only enzyme that has been purified to homogeneity (Kato et al., 1997
) so far. However, no work has been done on the molecular cloning of an animal endo-ß-GlcNAc-ase gene, although some papers have reported that of a microbial endo-ß-GlcNAc-ase gene (Takegawa et al., 1997
; Muramatsu et al., 2001
). We found endo-ß-GlcNAc-ase activity in an extract of C. elegans and cloned the gene of the enzyme from a cDNA library of C. elegans. This is the first report of an endo-ß-GlcNAc-ase gene being isolated from an animal.
Endo-ß-GlcNAc-ases are classified into two glycosyl hydrolase families based on their amino acid sequence homologies. Endo-H and Endo-F from Flavobacterium meningosepticum belong to family 18 containing chitinases. On the other hand, Endo-CE belongs to family 85 including Endo-M, Endo-A, and Endo-D. Figure 5 shows partial multiple alignment of these enzymes and their homologs. Endo-CE exhibits significant homology with these enzymes.
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We found that recombinant Endo-CE exhibited transglycosylation activity. It has been reported that a few (but not all) microbial endo-ß-GlcNAc-ases exhibited transglycosylation activity. However, this article is the first to report that an animal endo-ß-GlcNAc-ase exhibited transglycosylation activity. The transglycosylation by Endo-M and Endo-A has been well studied (Yamamoto et al., 1994b; Takegawa et al., 1995
), and various novel products possessing oligosaccharides have been prepared using their transglycosylation activities (Haneda et al., 1996
; Takegawa et al., 1995
). Regarding the important amino acid residues for the transglycosylation activity of Endo-A, it has been reported that a mutant, W216R, of Endo-A exhibited no transglycosylation activity, although it was found to exhibit hydrolyzing activity (Fujita and Takegawa, 2001
). Surprisingly, this amino acid residue, W216, is even conserved in the nematode, human, and fruit fly genes (Figure 5). Therefore, it might be possible that endo-ß-GlcNAc-ases from other organisms have this activity, although there is no evidence that the transglycosylation activity of endo-ß-GlcNAc-ase plays any physiological role in vivo.
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Materials and methods |
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Cultivation of C. elegans and preparation of a crude extract of it
Nematode C. elegans wild-type strain N2 Bristol was cultivated on nematode growing medium agar (Brenner, 1974) plates at 20°C using E. coli (OP50) as the feed. Mixed-stage worms were harvested and washed with sterile M9 buffer (Brenner, 1974
). An appropriate volume of the extraction buffer (10 mM acetate buffer, pH 6.0, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM ethylenediamine tetra acetic acid, 3 µg/ml pepstatin A, and 1 mM DTT) was added. The resulting suspension was subjected to sonication for 5 min at 0°C with an INSONATOR201M (Kubota, Tokyo, Japan). After centrifugation for 20 min at 13,000 x g, the supernatant obtained was used as the crude C. elegans extract.
Cloning of an Endo-CE gene from a C. elegans cDNA library
Oligonucleotides 5'-ATGCGTGGTGGATATTTGGAAG-3' and 5'-TTAATTAGAATCAATGGAAAAGTTTTC-3' were used as forward and reverse primers, respectively, to amplify the F01F1.10 gene (GenBank accession no. U13070, AAC46644) from a C. elegans mixed-stage cDNA library in the Uni-ZAP XR vector. After PCR, the resultant ~1.5-kb product was sequenced. The plaques prepared with the cDNA library were screened by means of the plaque hybridization technique using the PCR product as a probe. Cre-excision was performed with the ExAssist helper phage from positive plaque clones and the nucleotides of the insert fragment (EcoR I/ Xho I) in the plasmid clone were sequenced. One of them (pBXR-CE5) was chosen for subsequent experiments. This cDNA sequence has been deposited in GenBank under accession number AB079783.
Construction of an expression plasmid and its expression
To add restriction enzyme sites to both terminal ends of the open reading frame of the Endo-CE gene, PCR was performed with 25 cycles of 98°C for 10 s, 60°C for 45 s, and 72°C for 90 s, using plasmid pBXR-CE5 as a template. DNA polymerase PYROBEST (TaKaRa, Shiga, Japan) and the following primers were used: 5'-TTCGGCACC ATGGCGATTTCTCCAATTGAC-3' (Nco I-Endo-CE) and 5'-AAATTAGAATCTCGAGATTAGAATC AATGG-3' (Endo-CE-Xho I-(His6)-tag). The amplified fragment was double-digested with Nco I and Xho I, and the resultant restriction fragment was subcloned into the Nco I and Xho I sites of the pET-23d (+) vector. The pET-Endo-CE plasmid was transformed into E. coli BL21(DE3), and then the E. coli cells were grown in Luria-Bertani medium containing 100 µg/ml ampicillin with shaking for 48 h at 20°C. Endo-CE(His6) expressed in the cells was collected, and the protein was extracted by adding 20 mM sodium acetate buffer (pH 6.0) containing 1 mM DTT, 1 mM PMSF, and 1µg/ml pepstatin A, followed by sonication for 5 min at 0°C. The cell extract was centrifuged at 13,000 x g for 20 min at 4°C, and the resulting supernatant was subjected to purification.
Purification of recombinant Endo-CE
The recombinant Endo-CE (Endo-CE[His6]) in E. coli was purified on a Hi trapTM chelating HP prepacked column (Amersham Pharmacia, Buckinghamshire, United Kingdom). The E. coli extract was obtained as described. This extract was applied to the chelating HP prepacked column preequilibrated with the binding buffer (20 mM acetate buffer, pH 6.0, and 0.5 M NaCl) including 60 mM imidazole. After the column had been washed with the binding buffer including 80 mM imidazole until no further protein was eluted, Endo-CE(His6) was eluted with the binding buffer including 500 mM imidazole. The fractions exhibiting endo-ß-GlcNAc-ase activity were desalted by ultrafiltration using Ultrafree-15 (Millipore). Subsequently, the solution was subjected to Mono Q chromatography, which was performed with an ÄKTA FPLC system (Amersham Pharmacia) and a Mono Q HR 5/5 column. The column was equilibrated with 20 mM acetate buffer (pH 6.0) at a flow rate 0.8 ml/min. After injection of the enzyme solution, the column was washed with two column volumes of the same buffer. Recombinant Endo-CE was eluted with a linear gradient of NaCl from 0 to 2 M for 25 min. The fractions containing recombinant Endo-CE were stored at 4°C.
Assay for endo-ß-GlcNAc-ase activity
Endo-ß-GlcNAc-ase activity was assayed with oligosaccharide-Asn-DNS or PA-sugar chains as the substrate. One unit was defined as the amount yielding 1 nmol of GlcNAc-PA per min at 20°C with Man6GlcNAc2-PA (M6B-PA) as the substrate. Oligosaccharide-Asn-DNS was prepared from ovalbumin glycopeptides by the method of Huang et al. (1970). PA-sugar chains were obtained from Takara Shuzo.
HPLC
The reaction mixture including PA-sugar chains was analyzed by reversed-phase HPLC with a Hitachi L-6200 chromatograph and an F-1050 fluorescence spectrophotometer. Reversed-phase HPLC was performed on a Cosmosil 5C18-AR column (4.6 x 150 mm) eluted with 0.1 M ammonium acetate buffer, pH 4.0, at a flow rate of 1.5 ml/min with a linear gradient of 1-butanol from 0.025% to 0.5% for 55 min. Elution was monitored as to the fluorescence at 400 nm with excitation at 320 nm.
Transglycosylation of oligosaccharides by recombinant Endo-CE
The transglycosylation of oligosaccharides to glucose by the recombinant Endo-CE was achieved as follows. The reaction mixture was composed of 30 µg of GP4 (Man6GlcNAc2Asn), 25 mM potassium-phosphate buffer (pH 6.0), 100 mM glucose, and an appropriate amount of enzyme, in a total volume of 40 µl. After incubation of the mixture at 20°C for an appropriate time, the reaction was stopped by boiling for 3 min. Aliquots of the reaction mixture were subjected to TLC on silica gel 60 plates (Merck Art. 5626). For the separation of oligosaccharides, the following solvent system was used: n-propanol/acetic acid/water (v/v) = 3/3/2. Orcinol-H2SO4 reagent was used for the detection of oligosaccharides.
MALDI-TOF MS analysis
The following were used for MALDI-TOF MS analysis of the transglycosylation product (Man6GlcNAcGlc); a Kratos Kompact MALDI I (Shimadzu, Kyoto, Japan) as the analysis device, a SPARC-station (Sun Microsystem) for data analysis and control of the device, and a KOMPACT (UNIX software) for spectrum analysis. Measurements were performed in the linear mode using a nitrogen laser (wavelength of laser beam, 337 nm; 3 ns-wide pulses/s) as the ion source. As MW standards, leucine enkephalin (MW 555.6), angiotensin (MW 1046.2), neurotensin fragment 111 (MW 1446.6), and insulin chain A (MW 2531.6) were used; all were purchased from Sigma (St. Louis, MO).
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Acknowledgment |
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Abbreviations |
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Footnotes |
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References |
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Duvet, S., Labiau, O., Mir, A.M., Kmiecik, D., Krag, S.S., Verbert, A., and Cacan, R. (1998) Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end. Biochem. J., 335, 389396.[ISI][Medline]
Fujita, K. and Takegawa, K. (2001) Tryptophan-216 is essential for the transglycosylation activity of endo-ß-N-acetylglucosaminidase A. Biochem. Biophys. Res. Commun., 283, 680686.[CrossRef][ISI][Medline]
Haneda, K., Inazu, T., Yamamoto, K., Kumagai, H., Nakahara, Y., and Kobata, A. (1996) Transglycosylation of intact sialo complex-type oligosaccharides to the N-acetylglucosamine moieties of glycopeptides by Mucor hiemalis endo-ß-N-acetylglucosaminidase. Carbohydr. Res., 292, 6170.[CrossRef][ISI][Medline]
Huang, C.-C., Mayer, H.E. Jr., and Montgomery, R. (1970) Microheterogeneity and paucidispersity of glycoproteins; Part 1. The carbohydrate of chicken ovalbumin. Carbohydr. Res., 13, 127137.[CrossRef][ISI]
Kato, T., Hatanaka, K., Mega, T., and Hase, S. (1997) Purification and characterization of endo-ß-N-acetylglucosaminidase from hen oviduct. J. Biochem. (Tokyo), 122, 11671173.[Abstract]
Koide, N. and Muramatsu, T. (1974) Endo-ß-N-acetylglucosaminidase acting on carbohydrate moieties of glycoproteins. Purification and properties of the enzyme from Diplococcus pneumoniae. J. Biol. Chem., 249, 48974904.
Lisman, J.J., van der Wal, C.J., and Overdijk, B. (1985) Endo-N-acetyl-ß-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification. Biochem. J., 229, 379385.[ISI][Medline]
Moore, S.E.H. (1999) Oligosaccharide transport: pumping waste from the ER into lysosomes. TCB, 9, 441446.
Moore, S.E.H. and Spiro, R.G. (1994) Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis. J. Biol. Chem., 269, 1271512721.
Muramatsu, H., Tachikui, H., Ushida, H., Song, X., Qiu, Y., Yamamoto, S., and Muramatsu, T. (2001) Molecular cloning and expression of endo-ß-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides. J. Biochem. (Tokyo), 129, 923928.[Abstract]
Ohashi, S., Iwai, K., Mega, T., and Hase, S. (1999) Quantitation and isomeric structure analysis of free oligosaccharides present in the cytosol fraction of mouse liver: detection of a free disialobiantennary oligosaccharide and glucosylated oligomannosides. J. Biochem. (Tokyo), 126, 852858.[Abstract]
Overdijk, B., van der Kroef, W.M., Lisman, J.J., Pierce, R.J., Montreuil, J., and Spik, G. (1981) Demonstration and partial characterization of endo-N-acetyl-ß-D-glucosaminidase in human tissues. FEBS Lett., 128, 364366.[CrossRef][ISI][Medline]
Pierce, R.J., Spik, G., and Montreuil, J. (1979) Cytosolic location of an endo-N-acetyl-ß-D-glucosaminidase activity in rat liver and kidney. Biochem. J., 180, 673676.[ISI][Medline]
Tachibana, Y., Yamashita, K., Kawaguchi, M., Arashima, S., and Kobata, A. (1981) Digestion of asparagine-linked oligosaccharides by endo-ß-N-acetylglucosaminidase in the human skin fibroblasts obtained from fucosidosis patients. J. Biochem. (Tokyo), 90, 12911296.[Abstract]
Tachibana, Y., Yamashita, K., and Kobata, A. (1982) Substrate specificity of mammalian endo-ß-N-acetylglucosaminidase: study with the enzyme of rat liver. Arch. Biochem. Biophys., 214, 199210.[ISI][Medline]
Takegawa, K., Tabuchi, M., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1995) Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-ß-N-acetylglucosaminidase. J. Biol. Chem., 270, 30943099.
Takegawa, K., Yamabe, K., Fujita, K., Tabuchi, M., Mita, M., Izu, H., Watanabe, A., Asada, Y., Sano, M., Kondo, A., and others. (1997) Cloning, sequencing and expression of Arthrobacter protophormiae endo-ß-N-acetylglucosaminidase in Escherichia coli. Arch. Biochem. Biophys., 338, 2228.[CrossRef][ISI][Medline]
Tarentino, A.L. and Maley, F. (1974) Purification and properties of an endo-ß-N-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem., 249, 811817.
Verbert, A. and Cacan, R. (1999) Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum. Biochim. Biophys. Acta, 1473, 137146.[ISI][Medline]
Weng, S. and Spiro, R.G. (1997) Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver. Biochem. J., 322, 655661.[ISI][Medline]
Yamamoto, K., Kadowaki, S., Fujisaki, M., Kumagai, H., and Tochikura, T. (1994a) Novel specificities of Mucor hiemalis endo-ß-N-acetylglucosaminidase acting complex asparagine-linked oligosaccharides. Biosci. Biotechnol. Biochem., 58, 7277.[ISI][Medline]
Yamamoto, K., Kadowaki, S., Watanabe, J., and Kumagai, H. (1994b) Transglycosylation activity of Mucor hiemalis endo-ß-N-acetyl-glucosaminidase which transfers complex oligosaccharides to the N-acetylglucosamine moieties of peptides. Biochem. Biophys. Res. Commun., 203, 244252.[CrossRef][ISI][Medline]