Identification of an endo-ß-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli

Toshihiko Kato2, Kiyotaka Fujita2, Makoto Takeuchi3, Kazuo Kobayashi3, Shunji Natsuka4, Koji Ikura4, Hidehiko Kumagai2 and Kenji Yamamoto1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
We report the identification, molecular cloning, and characterization of an endo-ß-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-ß-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-ß-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-ß-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man8GlcNAc2 was the best substrate for Endo-CE, and Man3GlcNAc2 was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-ß-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-ß-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-ß-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.

Key words: Caenorhabditis elegans/endo-ß-N-acetylglucosaminidase/Endo-CE/transglycosylation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Endo-ß-N-acetylglucosaminidase (EC 3.2.1.96, endo-ß-GlcNAc-ase) hydrolyzes the glycosidic bonds in the NN'-diacetylchitobiose moieties of N-linked oligosaccharides of glycoproteins or glycopeptides, releasing oligosaccharides with an N-acetylglucosamine residue at their reducing ends. The occurrence of endo-ß-GlcNAc-ases has been demonstrated in several bacteria, animals, plants, and fungi. The endo-ß-GlcNAc-ases from bacterial sources such as Streptomyces plicatus (Endo-H; Tarentino and Maley, 1974Go) and Streptococcus pneumoniae (Endo-D; Koide and Muramatsu, 1974Go; Muramatsu et al., 2001Go) have been studied in detail and used as tools in studies on N-linked glycans. Their genes have already been cloned. In animals, the enzymes from hen oviduct (Endo-HO; Kato et al., 1997Go), rat liver (Tachibana et al., 1982Go; Lisman et al., 1985Go), and human tissues (Overdijk et al., 1981Go) have been purified and characterized. Among them, the enzyme from hen oviduct has been purified to homogeneity (Kato et al., 1997Go). However, no endo-ß-GlcNAc-ase from an animal source has been cloned to date.

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., 1994aGo). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Presence of endo-ß-N-acetylglucosaminidase activity in C. elegans
At first, we examined whether or not a crude extract of C. elegans exhibits endo-ß-GlcNAc-ase activity. A crude extract of C. elegans was prepared as described under Materials and methods. The enzyme reaction was performed as follows: 0.7 nmol of a dansyl (DNS)-ovalbumin glycopeptide mixture was incubated with 10 µl of the crude C. elegans extract (35 µg protein) in the presence of 100 mM sodium acetate buffer, pH 6.0, in a total volume of 40 µl for 2 h at 20°C. The hydrolysate was separated by thin-layer chromatography (TLC), and DNS-Asn-GlcNAc, the reaction product, was detected as fluorescence on exposure of the plate to UV light (Figure 1). The result confirmed that C. elegans exhibited endo-ß-GlcNAc-ase activity.



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Fig. 1. TLC analysis of the reaction mixture incubated with DNS-ovalbumin glycopeptides and the crude C. elegans extract. DNS-ovalbumin glycopeptides (0.7 nmol) were incubated with the crude C. elegans extract (35 µg protein) and 100 mM sodium acetate buffer, pH 6.0, for 2 h at 20°C. The reaction mixture was applied to a silica gel sheet and developed with the solvent system of n-butanol/acetic acid/water = 3/1/1. Lane 1, DNS-ovalbumin glycopeptide mixture; lane 2, DNS-Asn-GlcNAc; lane 3, reaction mixture.

 
Cloning of a C. elegans gene from a cDNA library
It was found that a C. elegans F01F1.10 gene exhibited homology with the gene of M. hiemalis endo-ß-GlcNAc-ase (Endo-M) in the N-terminal region, as the result of a homology search with the amino acid sequence of Endo-M. This gene encoded 388 amino acids and exhibited 34% homology with the amino acid sequence of Endo-M and 30% homology with that of Arthrobacter protophormiae endo-ß-GlcNAc-ase (Endo-A). This suggests that this C. elegans gene possibly encodes an endo-ß-GlcNAc-ase. Then the gene was amplified by polymerase chain reaction (PCR), a product of approximately 1.5 kbp being obtained. Sequence analysis of this DNA fragment revealed that it was the same as the F01F1.10 gene.

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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Fig. 2. Nucleotide sequence of the Endo-CE gene. The sequence obtained is 135 bps (underscored) longer in the N-terminal region of the gene than the F01F1.10 gene in the database.

 
Expression of Endo-CE(His6) and its purification
The DNA fragment encoding the complete Endo-CE was cloned into the pET-23 expression vector and then expressed as a C-terminal His6-tagged fusion protein (Endo-CE[His6]) in E. coli BL21(DE3). The recombinant E. coli was cultivated for 48 h at 20°C without isopropyl-1-thio-ß-D-galactoside (IPTG), because induction by IPTG was not effective for expression of the Endo-CE gene. The cell extract of the recombinant E.coli (1.4 g wet weight) was confirmed to exhibit endo-ß-GlcNAc-ase activity by TLC analysis. This extract (including 6.4 units) was applied to a Ni2+ chelating column, and endo-ß-GlcNAc-ase activity was found in the Ni2+-binding fractions. These fractions gave one major protein band and a few minor ones on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The fractions exhibiting endo-ß-GlcNAc-ase activity were desalted and concentrated to 1 ml by centrifugation with an Ultrafree 10K NMWL membrane (Millipore, Bedford, MA). Then the solution was applied to a Mono Q anion exchange column. The purified enzyme exhibited 17.4 U/mg of specific activity, and the final yield was 42%. This only gave a single protein band on SDS–PAGE at a position corresponding to around 50 kDa (Figure 3), which is consistent with the calculated molecular mass of Endo-CE(His6). To confirm that this purified protein was the product of the F01F1.10 gene, N-terminal analysis was performed; its sequence, MAISPIDTLEEL, was found to be the same as that of the recombinant Endo-CE. Therefore, the 50-kDa protein was confirmed to be the product of the F01F1.10 gene.



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Fig. 3. SDS–PAGE of recombinant Endo-CE. A 10% gel was used. Lane 1, crude extract; lane 2, proteins binding to the Ni2+ chelating column; lane 3, purified enzyme with the Mono Q-column. M, molecular weight markers.

 
Various properties of recombinant Endo-CE
We examined the optimal pH and temperature of recombinant Endo-CE, which were 5.0–7.0 and 20°C, respectively. These results were almost the same as those for the enzyme in the crude C. elegans extract. Both the enzyme in the crude C. elegans extract and recombinant Endo-CE were relatively unstable, and the addition of reducing reagents such as dithiothreitol (DTT) and 2-mercaptoethanol caused the recovery of their activities (data not shown).

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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Table I. Relative activity of Endo-CE toward various PA-oligosaccharides

*The value obtained with M8A was taken as 100.

 
Transglycosylation activity of recombinant Endo-CE
We next examined the transglycosylation activity of Endo-CE. The transglycosylation reaction was performed with Man6GlcNAc2Asn as a donor and glucose as an acceptor. The reaction mixture was incubated at 20°C and then subjected to TLC (Figure 4A). The transglycosylation product was concentrated by lyophilization after elution with water from the TLC plate and then subjected to matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS). The molecular weight (MW) of the transglycosylation product was determined to be 1357.82 (Figure 4B), which corresponds to the calculated value for Man6GlcNAcGlc. These results showed that Man6GlcNAc was attached to glucose.



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Fig. 4. Analysis of the transglycosylation activity of recombinant Endo-CE. (A) TLC analysis of the transglycosylation reaction mixture. The reaction was carried out with Man6GlcNAc2Asn as the substrate and the recombinant Endo-CE in the presence of 0.1 M glucose at 20°C for 0, 5, 10, 20, 30, 60, and 120 min. (B) MALDI-TOF MS analysis of the transglycosylation product.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
It is known that two free oligosaccharide species are found in animal cells: OS-GN1 and OS-GN2 (for reviews, see Verbert and Cacan, 1999Go; Moore, 1999Go). OS-GN2 is the oligosaccharide possessing two N-acetylglucosamine residues at its reducing end, and OS-GN1 possesses one N-acetylglucosamine residue at its reducing end. It has been reported that OS-GN1 was exclusively located in the cytosol (Moore and Spiro, 1994Go; Duvet et al., 1998Go; Ohashi et al., 1999Go). This oligosaccharide might be the product of endo-ß-GlcNAc-ase. There have been some reports that endo-ß-GlcNAc-ase exists in the cytosol but not in the endoplasmic reticulum (Pierce et al., 1979Go; Weng and Spiro, 1997Go). Furthermore, it has been described that OS-GN2 was rapidly converted to OS-GN1 in the cytosol (Moore and Spiro, 1994Go; Duvet et al., 1998Go). From these facts, it is assumed that endo-ß-GlcNAc-ases of animal cells participate in the conversion of OS-GN2 to OS-GN1 in the cytosol.

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., 1979Go; Overdijk et al., 1981Go; Tachibana et al., 1981Go, 1982; Lisman et al., 1985Go; Weng and Spiro, 1997Go), Endo-HO is the only enzyme that has been purified to homogeneity (Kato et al., 1997Go) 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., 1997Go; Muramatsu et al., 2001Go). 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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Fig. 5. Comparison of the amino acid sequences of various endo-ß-GlcNAc-ases and their homologs. Filled and open triangles show the corresponding positions of the important amino acids for the hydrolyzing activity and transglycosylation activity, respectively. (A) The protein lengths of endo-ß-GlcNAc-ases from human (H. sapiens, Genbank accession no. AK025518), the fruit fly (D. melanogaster, Genbank accession no. AE003505), a nematode (C. elegans), a fungus (M. hiemalis), and a bacterium (A. protophormiae). The black boxes indicate comparatively conserved regions. (B) Multiple alignment of the amino acid sequences shown in (A) in black boxes. Completely conserved residues are shaded black, and conserved substitutions are shaded gray.

 
Regarding the substrate specificity of Endo-CE, high-mannose type PA-derivatives with six to nine mannose residues were more easily hydrolyzed than ones with three to five mannose residues. As shown in Table TI, the relative activity of the enzyme for M6B is significantly higher than that for M5A, although the only structural difference between M6B and M5A is the existence of a Man{alpha}1-residue in the branch. Oligosaccharide possessing six to nine mannose residues are mainly found in the endoplasmic reticulum (ER), and the M5A oligomannoside first occurs in the Golgi through processing of N-linked glycans derived from ER. These facts suggest that endo-ß-GlcNAc-ase recognizes the structural differences of oligomannosides and exclusively acts on oligomannosides derived from ER. This idea is consistent with the hypothesis that endo-ß-GlcNAc-ase participates in the conversion of OS-GN2 species derived from ER to OS-GN1 species. The substrate specificity of recombinant Endo-CE was almost the same as those of Endo-M (Yamamoto et al., 1994aGo), Endo-HO (Kato et al., 1997Go), and endo-ß-GlcNAc-ase from human skin fibroblasts (Tachibana et al., 1981Go). For this reason, it might be possible that the protein of Homo sapiens shown in Figure 5 corresponds to the endo-ß-GlcNAc-ase from human skin fibroblasts.

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., 1994bGo; Takegawa et al., 1995Go), and various novel products possessing oligosaccharides have been prepared using their transglycosylation activities (Haneda et al., 1996Go; Takegawa et al., 1995Go). 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, 2001Go). 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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Bacterial strains and media
The following E. coli strains were used: OP50 as the feed for C. elegans, DH5{alpha} for plasmid construction and as a host for plasmid vector pBluescript SK, BL21(DE3) as a host for plasmid pET-23d (Novagen, Madison, WI) to express Endo-CE, XL1-Blue MRF' as a host for the Uni-ZAP XR vector, and E. coli SOLR as a host for the ExAssist helper phage (Stratagene, La Jolla, CA). All E. coli strains were cultivated in Luria-Bertani medium.

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, 1974Go) plates at 20°C using E. coli (OP50) as the feed. Mixed-stage worms were harvested and washed with sterile M9 buffer (Brenner, 1974Go). 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)Go. 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 1–11 (MW 1446.6), and insulin chain A (MW 2531.6) were used; all were purchased from Sigma (St. Louis, MO).


    Acknowledgment
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
We wish to thank Mr. Kazuhiro Aoki for providing the excellent MALDI-TOF MS analysis data.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
DNS, dansyl; DTT, dithiothreitol; ER, endoplasmic reticulum; HPLC, high-performance liquid chromatography; IPTG, isopropyl-1-thio-ß-D-galactoside; MALDI-TOF MS, matrix-assisted laser desorption ionization–time-of-flight mass spectrometry; MW, molecular weight; PA, pyridylamino; PCR, polymerase chain reaction; PMSF, phenylmethanesulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TLC, thin-layer chromatography.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: yamamotk@kais.kyoto-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
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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, 61–70.[CrossRef][ISI][Medline]

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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, 1167–1173.[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, 4897–4904.[Abstract/Free Full Text]

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, 379–385.[ISI][Medline]

Moore, S.E.H. (1999) Oligosaccharide transport: pumping waste from the ER into lysosomes. TCB, 9, 441–446.

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, 12715–12721.[Abstract/Free Full Text]

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, 923–928.[Abstract]

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