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Cloning, Sequencing, and Expression ofArthrobacter protophormiaeEndo-β-N-acetylglucosaminidase inEscherichia coli
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 338, No. 1, February 1, pp. 22–28, 1997 Article No. BB969803
Cloning, Sequencing, and Expression of Arthrobacter protophormiae Endo-b-N-acetylglucosaminidase in Escherichia coli Kaoru Takegawa,1 Kayo Yamabe, Kiyotaka Fujita, Mitsuaki Tabuchi, Masanori Mita,* Hiroyuki Izu,* Akira Watanabe, Yasuhiko Asada, Mutsumi Sano,* Akihiro Kondo,* Ikunoshin Kato,* and Shojiro Iwahara Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07, Japan; and *Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Ohtsu, Shiga 520-21, Japan
Received August 23, 1996, and in revised form November 1, 1996
The gene encoding endo-b-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology with F01F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-b-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme had no significant homology with any previously reported endo-b-N-acetylglucosaminidases. Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter. The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells. N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity. q 1997 Academic Press Key Words: amino acid sequence; endo-b-N-acetylglucosaminidase; transglycosylation; Arthrobacter protophormiae.
Endo-b-N-acetylglucosaminidase (EC 3.2.1.96) releases N-linked oligosaccharide chains from glycopro1 To whom correspondence and reprint requests should be addressed. Fax: 81-878-98-7295. E-mail: [email protected].
teins by cleaving the di-N-acetylchitobiose unit. This enzyme is useful in glycoprotein research, because it can be used for the recovery of both N-linked oligosaccharides and partially deglycosylated proteins without damaging them. Endo-b-N-acetylglucosaminidase is still widely used by cell biologists to examine aspects of protein sorting, targeting, and the compartmentalization of secreted glycoproteins in the cell. Purification of the enzymes from certain microorganisms has been reported, and substrate specificities of the enzymes have been elucidated (1–6). Using microbial endo-b-Nacetylglucosaminidases, the biological roles of N-linked oligosaccharides in various glycoproteins have been determined (7, 8). Arthrobacter protophormiae produces endo-b-Nacetylglucosaminidase (called Endo-A) when grown in medium containing ovalbumin; the purification and properties of this enzyme have been reported (9). The substrate specificity of Endo-A is very similar to that of Endo-CII from Clostridium perfringens (10), and Endo-A also hydrolyzes primarily high-mannosetype oligosaccharides (9). However, the molecular weight of Endo-A (approximately 72,000) is quite different from those of other endo-b-N-acetylglucosaminidases, such as Endo-H from Streptomyces plicatus (271 amino acids), Endo-Fsp from Flavobacterium sp. (267 amino acids), and Endo-F1 from F. meningosepticum (289 amino acids), all of which show similar substrate specificities (11 – 13). Endo-A, unlike these other enzymes, was shown to have a powerful transglycosylation activity (14). Endo-A can transfer (Man)6GlcNAc to various acceptors, such as glucose, mannose, and gentiobiose (15). Using the transglycosylation activity of the enzyme, we previously es-
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tablished a novel method for the enzymatic synthesis of neoglycoproteins and neoglycoconjugates (16 – 18). Therefore, it is of particular interest to us to determine which part of Endo-A is important for transglycosylation activity. To obtain such information, we performed DNA sequencing of Endo-A in this study. Endo-A has been expressed efficiently in an Escherichia coli host utilizing its own promoter. We isolated the enzyme from the periplasmic space of E. coli cells and characterized the properties of recombinant Endo-A in comparison to those of the native enzyme. MATERIALS AND METHODS Bacterial strains. E. coli strain XL1-Blue (Stratagene, La Jolla, CA) was used for all cloning procedures. E. coli strain P2392 (Stratagene) was used in the screening of an A. protophormiae genomic library containing lEMBL3; A. protophormiae (AKU 0647) was used in the purification of Endo-A and in the construction of a genomic library. Reagents. DNA restriction and modifying enzymes were from Takara Shuzo Co. (Kyoto, Japan). The sequencing kit and Phenyl-Sepharose CL-4B were from Pharmacia Fine Chemical Co. (Piscataway, NJ). DEAE-Toyopearl 650M was purchased from Toso Co. (Tokyo, Japan). All other chemicals not listed above were purchased from Wako Pure Chemicals Co. (Osaka, Japan). Vectors and bacteriophage. The E. coli cloning vectors pBluescript SK and KS (Promega) were used for the construction of an Endo-A gene. The helper phage M13KO7 used for recovery of singlestranded phagemid DNA was from Takara Shuzo Co. Peptide purification and amino acid analyses. Endo-A was purified to homogeneity as previously described (9). For proteolytic cleavage, the purified enzyme (80 mg) was digested with lysylendopeptidase (Wako Pure Chemicals) in 20 mM Tris/HCl buffer containing 1.6 M urea, pH 9.1, for 21 h at 307C at a substrate/enzyme ratio of 400:1. After the reaction was stopped by the addition of formic acid, peptide mixtures obtained by enzymatic cleavages were analyzed on a reversed-phase column using a Pharmacia SMART system (mRPC C2/C18, SC 2.1/10, 2.1 by 100 mm), equilibrated with 0.12% trifluoroacetic acid, and eluted with a linear acetonitrile gradient at a flow rate of 0.1 ml/min. PCR amplification and cloning of the Endo-A gene. Conventional methods were used in the construction and propagation of all plasmids (19). To amplify Endo-A-like sequences from the genomic DNA of the A. protophormiae strain, the following oligonucleotide primers were synthesized and tested: primer 1, 5*-GTTGGATCCTTT(C)CCA(T/G/C)GAA(G)GAA(G)T(C)TA(T/G/C)GCA-(T/G/C)CA-3*, and primer 2, 5*-GTTGAATTCGTA(G)TTA(G)AAA(G)TGA(T/G/ C)GTA(T/G/C) ACA(G)AA-3*. The primers included restriction sites at their 5*-ends to facilitate cloning of the resulting amplified products. All oligonucleotides were synthesized by Bio-synthesis, Inc. The primer 1 encodes amino acids FPEELAQ, while primer 2 encodes FVTHFNT. The PCR (20) mixture contained 3 mg A. protophormiae genomic DNA, 5 mM primers, 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 1.5 mM MgSO4, 0.2 mM dNTPs, and 3 units Takara Taq DNA polymerase (Takara Shuzo Co.), in a total volume of 50 ml. PCR was performed in an Eppendorf tube using 35 repetitions of the following temperature cycle: 947C for 60 s, 497C for 90 s, and 727C for 90 s. The reaction product was digested with BamHI and EcoRI and resolved by electrophoresis in a 1.5% agarose gel. A fragment of approximately 900 bp was recovered and ligated into the pBluescript KS(0) vector that had been digested with BamHI and EcoRI. All nucleotide sequence determination was performed using double-stranded plas-
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mid DNA templates and an AutoRead sequencing kit (Pharmacia), according to the recommendations of the manufacturer. A. protophormiae chromosomal DNA was isolated according to Ausubel et al. (21). The DNA was partially digested with Sau3AI, and fragments of 8 to 15 kb were purified using a Takara EASY trap kit, ligated into BamHI-digested lEMBL3 using a Gigapack III packaging kit (Stratagene), then packaged into phage particles and used to infect E. coli P2392. The entire Endo-A gene was isolated by plaque hybridizing the cloned PCR product. Southern blot analysis and hybridization. Restriction digests of lEMBL3-insert DNA were separated in 1.0% agarose gels and blotted onto Hybond-N/ membranes (Amersham) according to the manufacturer’s recommendation. Detection was carried out with nonradioactive DNA probes. Oligonucleotide probes were labeled using an Amersham ECL nucleotide detection kit. Hybridization and washing were carried out according to the manufacture’s instructions. Endo-A assay and analysis of transglycosylation products. Endob -N-acetylglucosaminidase activity was assayed with (Man)6(GlcNAc)2Asn-dansyl as the substrate (9). One unit was defined as the amount yielding 1 mmol of GlcNAc-Asn-dansyl per minute at 377C under the assay conditions. An asparaginyl oligosaccharide, (Man)6(GlcNAc)2Asn, was prepared from ovalbumin glycopeptides by the method of Huang et al. (22). The transglycosylation reaction of (Man)6(GlcNAc)2Asn to glucose by Endo-A was examined as follows: 1.3 mg of (Man)6(GlcNAc)2Asn was incubated with 10 munits of Endo-A in 0.27 ml of 50 mM ammonium acetate buffer (pH 6.0) in the presence of glucose (final concentration, 0.5 M) for 10 min at 377C. After the incubation was stopped by boiling the reaction mixture for 3 min, the reaction was put on a Sephadex G-15 column (1.5 1 30 cm) to remove the free glucose. The oligosaccharides were spotted on silica gel 60 plates (Merck Art. 5626) and analyzed by thin-layer chromatography. For separation of the oligosaccharides, the following solvent system was used: n-propanol/acetic acid/water Å 3:3:2 (23). Orcinol–H2SO4 reagent was used for the detection of oligosaccharides (23). Analyses of the carbohydrate composition of the oligosaccharides were done as described previously (15). Electrophoresis. Polyacrylamide gel electrophoresis in the presence of SDS was carried out by the standard methods (24). The molecular weight markers were purchased from Pharmacia. Proteins were visualized with Coomassie brilliant blue R-250. Purification of Endo-A from E. coli. Purification of the recombinant Endo-A was carried out as follows. E. coli XLI-Blue strain carrying recombinant plasmid pKY1 was grown aerobically in 3% peptone and 1% NaCl at 377C for 72 h. The cells (a 4-liter-cultivation) were centrifuged, and extract from the periplasmic space of the cells was obtained by the osmotic shock procedure described by Suzuki et al. (25). This extract was applied to a DEAE-Toyopearl 650M (2.5 1 10 cm) previously equilibrated with 10 mM phosphate buffer (pH 7.0). The column was washed with the same buffer and the enzyme eluted in 4-ml fractions with a gradient formed between 10 mM phosphate buffer and 0.5 M NaCl in 10 mM phosphate buffer. The active fractions were combined, and ammonium sulfate was added to the enzyme solution to 1 M. The enzyme solution was applied to a PhenylSepharose CL-4B column (0.8 1 22 cm) equilibrated with 1 M ammonium sulfate in 10 mM phosphate buffer (pH 7.0). The enzyme was eluted in 2-ml fractions with a gradient formed between 1 M ammonium sulfate and 40% ethyleneglycol in 10 mM phosphate buffer. The active fractions were combined and concentrated by the addition of ammonium sulfate to 90% saturation. The precipitate was collected by centrifugation and then resuspended in 10 mM phosphate buffer (pH 7.0).
RESULTS AND DISCUSSION
Determination of partial amino acid sequence of Endo-A. To synthesize oligonucleotide primers for
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FIG. 1. Separation of lysylendopeptidase peptides. The reverse-phase HPLC column (mRPC C2/C18, SC 2.1/10, 2.1.1 100 mm, Pharmacia) was equilibrated with 0.12% trifluoroacetic acid and developed with a linear acetonitrile gradient at a flow rate of 0.1 ml/min.
PCR, the N-terminal amino acid sequence of the purified Endo-A, as well as the amino acid sequence of the major peptide fragments derived by lysyl-endopeptidase, was determined using a protein sequencer. Partial amino acid sequence data were obtained from the N terminus of the mature protein and from nine peptide fragments (Fig. 1). Molecular cloning and sequence analysis of Endo-A. Degenerate oligonucleotides encoding N-terminal and other determined sequences within Endo-A protein were used to PCR amplify a 1-kb gene fragment from A. protophormiae genomic DNA (see Materials and Methods). The complete sequence of the PCR product was determined, and five of the nine peptides were found in the PCR fragment. As this PCR product is therefore part of the Endo-A gene, this gene fragment was used as a probe to recover the Endo-A gene from the A. protophormiae genomic library. To clone the entire Endo-A gene code, chromosomal libraries were constructed using lEMBL3. Screening of the chromosomal phage libraries resulted in six positive clones. Two of these clones were confirmed by hybridization with nonradioactivity labeled PCR fragments to be identical (both containing 9 kb-fragments). To determine the location of the Endo-A gene within the fragment, we prepared various subclones in which ClaI, PstI, KpnI, and HindIII digests of the fragment had been ligated at appropriate sites in pBluescript KS(0) cloning vectors. The plasmid pKY1, a 4.7-kb ClaI–PstI fragment containing the Endo-A gene, was sequenced. The nucleotide sequence of the Endo-A gene is shown in Fig. 2.
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In this sequence, a single open reading frame (ORF),2 starting at position 1 and ending at position 1937, was found. Putative promoter regions of the Endo-A gene were detected upstream of the Shine–Dalgarno-like sequence: at positions 015 to 011, as well as in the sequence around 025 which was found to be a putative 010 region (see in Fig. 2). The ORF could encode a mature protein of 621 amino acids with a signal peptide of 24 amino acids. All amino acid sequences from the purified Endo-A were discovered within the deduced amino acid sequence. This data suggest that the ORF encodes Endo-A. The amino acid sequences of several endo-b-N-acetylglucosaminidases have been determined; Endo-H from Streptomyces plicatus (11); Endo-F1 (13); F2 (26), and F3 (26) from Flavobacterium meningosepticum; and Endo-Flavo from Flavobacterium sp. (12). We examined sequence comparisons between Endo-A and other endo-b-N-acetylglucosaminidases, but we could not detect any homologous regions. Schumidt et al. have substituted the glutamine, asparagine, and tryptophan residues of Endo-H protein using point mutagenesis and found that Asp-130 and Glu-132 were essential for enzyme activity (27). These two amino acid residues are conserved in other endo-b-N-acetylglucosaminidases and chitinases (28, 29). Van Roey and colleagues analyzed the three-dimensional structures of Endo-H and Endo-F1 using crystallographic studies (30, 31).
2 Abbreviations used: ORF, open reading frame; PNGase F, peptide-N4-(N-acetyl-b-D-glucosaminyl)asparagine amidase F.
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FIG. 2. Nucleotide sequence of the Endo-A gene. The putative ribosome-binding site is indicated by dots. The 010 region of a possible promoter sequence is double underlined. The deduced amino acid sequence is given below the nucleotide sequence. The overlined sequences indicate the amino acid sequences for the PCR primers. The underlined sequences indicate the amino acid sequences determined with a protein sequencer. The arrow indicates the site of cleavage of the signal peptide.
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Both enzymes consisted of a highly irregular a/b-barrel. Asp-130 and Glu-132 of Endo-H are located in a curved cleft over the top of the molecule (31). Although there are two Asp-X-Glu sequences in Endo-A protein (Asp-489 and Asp-550), the surrounding sequences of Endo-A are not conserved among endo-b-N-acetylglucosaminidases. The deduced amino acid sequence was compared with other published sequences in various databases of the National Center for Biotechnology Information using the FASTA Network Service. The N-terminal region of Endo-A is highly similar to that of the Caenorhabditis elegans gene product. This ORF was identified by the genome sequencing project of C. elegans chromosome III (32). Relatively low but significant homology was also observed with peptide-N4-(N-acetyl-bD-glucosaminyl)asparagine amidase F (PNGase F) (33). In contrast to endo- b -N-acetyl-glucosaminidase, PNGase F hydrolyzes N-linked sugar chains by cleaving the amide bond between the proximal GlcNAc and Asn in the peptide chain (34). Crystallographic analysis has shown that Glu-206 is located in a cleft at the interface between the two domains of the PNGase F protein (35, 36). Glu-206 of PNGase F is thought to be important for stabilization of reaction intermediates (36). The similar acidic residue, Asp-301 of Endo-A, may be important for the stabilization of reaction intermediates. In addition, four tryptophan residues are also well conserved between the Endo-A and PNGase F. The C-terminal region of Endo-A had weak homology with Streptomyces olivaceoviridis chitinase (28). This region of the S. olivaceoviridis chitinase also shows sequence similarities to the C-terminal end of chitinase A1 from Bacillus circulans (37) and the type III homology units of fibronectin, a multifunctional extracellular matrix and plasma protein of higher eukaryotes (38). The C-terminal region including this region of chitinase A1 was shown to be essential for its affinity to the substrate chitin (39). These conserved regions of Endo-A and chitinases may be essential for the association of high-molecular-mass substrates. Recently Matsui et al. (40) have reported that transglycosylation activity of a-amylase from Saccharomycopsis increased after changing an aromatic residue in the active center by site-directed mutagenesis. Transglycosylation is a competition against hydrolysis, where the acceptor competes for water at the enzyme active center. Therefore, it is possible to enhance the transglycosylation activity by redesigning the active center of the enzyme to gain stronger affinity for the acceptor. Site-directed mutagenesis is in progress to determine the active site of Endo-A protein. Expression and characterization of recombinant Endo-A in E. coli cells. The 4.7-kb DNA fragment was
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FIG. 3. Profiles of Endo-A produced by E. coli XLI-Blue harboring pKY1. Lane A, molecular weight markers; lane B, purified Endo-A produced by A. protophormiae; lane C, purified Endo-A produced by E. coli XLI-Blue (pKY1).
subcloned into pBluescript KS (pKY1). The transformed E. coli XLI-Blue was cultured at 377C in LB broth for 30 h, and the enzyme activity was measured in the periplasmic space of the cells. Endo-A activity was detected mainly in the periplasmic space of the cells. The plasmid containing the reverse-oriented 4.7kb fragment was also able to degrade dansylated-ovalbumin glycopeptide, indicating that the cloned Endo-A gene is expressed by its own promoter. Endo-A production in E. coli was tested during cultivation at 377C. Until the 72nd hour of cultivation, Endo-A activity was mainly in the periplasmic space, indicating that the Endo-A was exported through the cytoplasmic membrane of E. coli cells. The amount of Endo-A in the periplasmic space increased in the exponential phase and maximized in the early stationary phase, but thereafter Endo-A protein was leaked into the medium. The transformed E. coli XLI-Blue was cultured in 4 liters of 3% peptone and 1% NaCl medium at 377C for 2 days. The cells were then harvested and subjected to osmotic shock. Recombinant Endo-A was purified as described under Materials and Methods, analyzed by SDS–PAGE, and compared with native Endo-A protein. Both enzymes migrated as a single protein band during SDS–PAGE (Fig. 3). The relative molecular weight of each was estimated to be 72,000. The N-terminal sequence of the recombinant Endo-A was determined as Ser-Thr-Tyr-Asn-Gly-Pro-Leu, showing that the signal peptide of the Endo-A produced in E. coli XLI-Blue cells had been removed by cleavage between residues 24 and 25. This data show that the signal peptide of Endo-A was cleaved at the same position in E. coli cells. The enzymatic properties of the recombinant Endo-A enzyme were compared with those of the native enzyme. They showed similar pH-activity profiles (both Endo-A’s showed broad optimum pH and were most active in the range of pH 5.0 to 9.0). Both enzymes were stable up to 607C (after incubation for 10 min) and strongly inhibited by the addition of 1 mM
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2. Koide, N., and Muramatsu, T. (1974) J. Biol. Chem. 249, 4897– 4904. 3. Ito, S., Muramatsu, T., and Kobata, A. (1975) Arch. Biochem. Biophys. 171, 78–86. 4. Elder, J. H., and Alexander, S. (1982) Proc. Natl. Acad. Sci. USA 79, 4540–4544. 5. Yamamoto, K., Kadowaki, S., Takegawa, K., Kumagai, H., and Tochikura, T. (1986) Agric. Biol. Chem. 50, 421–429. 6. Kadowaki, S., Yamamoto, K., Fujisaki, M., and Tochikura, T. (1991) J. Biochem. 110, 17–21. 7. Yamamoto, K. (1994) J. Biochem. (Tokyo) 116, 229–235. 8. Varki, A. (1993) Glycobiology 3, 97–130. FIG. 4. TLC analysis of the transglycosylation products. (Man)6(GlcNAc)2Asn was incubated with native Endo-A (lane C) or recombinant Endo-A (lane D) in the presence of 0.5 M glucose for 10 min at 377C. Lane A, standard (Man)6(GlcNAc)2Asn; lane B, standard (Man)6GlcNAc.
HgCl2 (final concentration). The recombinant Endo-A caused no changes in kinetic parameters or in the pHactivity–stability profiles. The transglycosylation reaction of (Man)6(GlcNAc)2Asn to glucose by the recombinant Endo-A was carried out as described under Materials and Methods. The transglycosylation products obtained were analyzed by TLC. One oligosaccharide spot was newly generated in the presence of glucose (Fig. 4D). The spot was collected, eluted with water, and lyophilized. The samples were hydrolyzed with 4 M trifluoroacetic acid, and the carbohydrate composition of the transglycosylation product was determined to be Man:GlcNAc:Glc Å 6:1.0:1.0 (relative to Man Å 6). These results show that glucose was attached to (Man)6GlcNAc during cleavage of the chitobiose core and that (Man)6GlcNAcGlc was synthesized by the transglycosylation of Endo-A (15). The recombinant Endo-A was purified to homogeneity by relatively simple procedures: DEAE-Toyopearl and Phenyl-Sepharose 4B column chromatographies. In this study, the cloned Endo-A gene was expressed in E. coli by its own promoter. Therefore, it may be possible to obtain a much higher Endo-A-producing strain utilizing a high-expression promoter.
The authors thank Drs. Hideyuki Suzuki, Kenji Yamamoto (Kyoto University), Jian-Qiang Fan, and Yuan Juan Lee (Johns Hopkins University) for their helpful discussions. We also thank Makiko Shimizu, Satomi Suda, and Tsuyoshi Miyamura for their excellent technical assistance. This work was partly supported by Kagawa Techno Foundation (to K.T.). The Endo-A nucleotide sequence has been deposited in GenBank and can be found using Accession Number U59168.
REFERENCES 1. Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811– 817.
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10. Tai, T., Yamashita, K., Ito, S., and Kobata, A. (1977) J. Biol. Chem. 252, 6687–6694. 11. Robbins, P. W., Trimble, R. B., Wirth, D. F., Hering, C., Maley, F., Maley, G. F., Das, R., Gibson, B. W., Royal, N., and Biemann, K. (1984) J. Biol. Chem. 259, 7577–7583. 12. Takegawa, K., Mikami, B., Iwahara, S., Morita, Y., Yamamoto, K., and Tochikura, T. (1991) Eur. J. Biochem. 202, 175–180. 13. Tarentino, A. L., Quinones, G., Schrader, W. P., Changchien, L.-M., and Plummer, T. H., Jr. (1992) J. Biol. Chem. 267, 3868– 3872. 14. Takegawa, K., Yamaguchi, S., Kondo, A., Iwamoto, H., Nakoshi, M., Kato, I., and Iwahara, S. (1991) Biochem. Int. 24, 849–855. 15. Takegawa, K., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1991) Biochem. Int. 25, 829–835. 16. Takegawa, K., Tabuchi, M., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1995) J. Biol. Chem. 270, 3094–3099. 17. Fan, J. Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., Abeygunawardana, C., and Lee, Y. C. (1995) J. Biol. Chem. 270, 17723–17729. 18. Fan, J. Q., Quesenberry, M. S., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., and Lee, Y. C. (1995) J. Biol. Chem. 270, 17730–17735. 19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 20. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487–491. 21. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology, Wiley, New York. 22. Huang, C.-C., Mayer, H. E., Jr., and Montgomery, R. (1970) Carbohydr. Res. 13, 127–137.
ACKNOWLEDGMENTS
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9. Takegawa, K., Nakoshi, M., Iwahara, S., Yamamoto, K., and Tochikura, T. (1989) Appl. Environ. Microbiol. 55, 3107–3112.
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23. Holmes, E. W., and O’Brien, J. S. (1979) Anal. Biochem. 93, 167– 170. 24. Laemmli, U. K. (1970) Nature 227, 680–685. 25. Suzuki, H., Kumagai, H., and Tochikura, T. (1986) J. Bacteriol. 168, 1332–1335. 26. Tarentino, A. L., Quinones, G., Changchien, L.-M., and Plummer, T. H., Jr. (1993) J. Biol. Chem. 268, 9702–9708. 27. Schmidt, B. F., Ashizawa, E., Jarnagin, A. S., Lynn, S., Noto, G., Woodhouse, L., Estell, D. A., and Lad, P. (1994) Arch. Biochem. Biophys. 311, 350–353. 28. Blaak, H., Schnellmann, J., Walter, S., Henrissat, B., and Schrempf, H. (1993) Eur. J. Biochem. 214, 659–669.
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29. Kuranda, M. J., and Robbins, P. W. (1991) J. Biol. Chem. 266, 19758–19767. 30. Van Roey, P., Rao, V., Plummer, T. H., Jr., and Tarentino, A. L. (1994) Biochemistry 33, 13989–13996. 31. Rao, V., Guan, C., and Van Roey, P. (1995) Structure 3, 449– 457. 32. Wilson, R., et al. (1994) Nature 368, 32–38. 33. Lemp, D., Haselbeck, A., and Klebl, F. (1990) J. Biol. Chem. 265, 15606–15610. 34. Tarentino, A. L., and Plummer, T. H. (1987) Methods Enzymol. 138, 770–778.
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35. Kuhn, P., Tarentino, A. L., Plummer, T. H., Jr., and Van Roey, P. (1994) Biochemistry 33, 11699–11706. 36. Kuhn, P., Guan, C., Cui, T., Tarentino, A. L., Plummer, T. H., Jr., and Van Roey, P. (1995) J. Biol. Chem. 270, 29493–29497. 37. Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K., and Tanaka, H. (1990) J. Biol. Chem. 265, 15659–15665. 38. Rouslathi, E. (1988) Annu. Rev. Biochem. 57, 375–413. 39. Watanabe, T., Oyanagi, W., Suzuki, K., and Tanaka, H. (1990) J. Bacteriol. 172, 4017–4022. 40. Matsui, I., Yoneda, S., Ishikawa, K., Miyairi, S., Fukui, S., Umeyama, H., and Honda, K. (1994) Biochemistry 33, 451–458.
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Vol. 338, No. 1, February 1, pp. 22–28, 1997 Article No. BB969803
Cloning, Sequencing, and Expression of Arthrobacter protophormiae Endo-b-N-acetylglucosaminidase in Escherichia coli Kaoru Takegawa,1 Kayo Yamabe, Kiyotaka Fujita, Mitsuaki Tabuchi, Masanori Mita,* Hiroyuki Izu,* Akira Watanabe, Yasuhiko Asada, Mutsumi Sano,* Akihiro Kondo,* Ikunoshin Kato,* and Shojiro Iwahara Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07, Japan; and *Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Ohtsu, Shiga 520-21, Japan
Received August 23, 1996, and in revised form November 1, 1996
The gene encoding endo-b-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology with F01F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-b-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme had no significant homology with any previously reported endo-b-N-acetylglucosaminidases. Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter. The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells. N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity. q 1997 Academic Press Key Words: amino acid sequence; endo-b-N-acetylglucosaminidase; transglycosylation; Arthrobacter protophormiae.
Endo-b-N-acetylglucosaminidase (EC 3.2.1.96) releases N-linked oligosaccharide chains from glycopro1 To whom correspondence and reprint requests should be addressed. Fax: 81-878-98-7295. E-mail: [email protected].
teins by cleaving the di-N-acetylchitobiose unit. This enzyme is useful in glycoprotein research, because it can be used for the recovery of both N-linked oligosaccharides and partially deglycosylated proteins without damaging them. Endo-b-N-acetylglucosaminidase is still widely used by cell biologists to examine aspects of protein sorting, targeting, and the compartmentalization of secreted glycoproteins in the cell. Purification of the enzymes from certain microorganisms has been reported, and substrate specificities of the enzymes have been elucidated (1–6). Using microbial endo-b-Nacetylglucosaminidases, the biological roles of N-linked oligosaccharides in various glycoproteins have been determined (7, 8). Arthrobacter protophormiae produces endo-b-Nacetylglucosaminidase (called Endo-A) when grown in medium containing ovalbumin; the purification and properties of this enzyme have been reported (9). The substrate specificity of Endo-A is very similar to that of Endo-CII from Clostridium perfringens (10), and Endo-A also hydrolyzes primarily high-mannosetype oligosaccharides (9). However, the molecular weight of Endo-A (approximately 72,000) is quite different from those of other endo-b-N-acetylglucosaminidases, such as Endo-H from Streptomyces plicatus (271 amino acids), Endo-Fsp from Flavobacterium sp. (267 amino acids), and Endo-F1 from F. meningosepticum (289 amino acids), all of which show similar substrate specificities (11 – 13). Endo-A, unlike these other enzymes, was shown to have a powerful transglycosylation activity (14). Endo-A can transfer (Man)6GlcNAc to various acceptors, such as glucose, mannose, and gentiobiose (15). Using the transglycosylation activity of the enzyme, we previously es-
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tablished a novel method for the enzymatic synthesis of neoglycoproteins and neoglycoconjugates (16 – 18). Therefore, it is of particular interest to us to determine which part of Endo-A is important for transglycosylation activity. To obtain such information, we performed DNA sequencing of Endo-A in this study. Endo-A has been expressed efficiently in an Escherichia coli host utilizing its own promoter. We isolated the enzyme from the periplasmic space of E. coli cells and characterized the properties of recombinant Endo-A in comparison to those of the native enzyme. MATERIALS AND METHODS Bacterial strains. E. coli strain XL1-Blue (Stratagene, La Jolla, CA) was used for all cloning procedures. E. coli strain P2392 (Stratagene) was used in the screening of an A. protophormiae genomic library containing lEMBL3; A. protophormiae (AKU 0647) was used in the purification of Endo-A and in the construction of a genomic library. Reagents. DNA restriction and modifying enzymes were from Takara Shuzo Co. (Kyoto, Japan). The sequencing kit and Phenyl-Sepharose CL-4B were from Pharmacia Fine Chemical Co. (Piscataway, NJ). DEAE-Toyopearl 650M was purchased from Toso Co. (Tokyo, Japan). All other chemicals not listed above were purchased from Wako Pure Chemicals Co. (Osaka, Japan). Vectors and bacteriophage. The E. coli cloning vectors pBluescript SK and KS (Promega) were used for the construction of an Endo-A gene. The helper phage M13KO7 used for recovery of singlestranded phagemid DNA was from Takara Shuzo Co. Peptide purification and amino acid analyses. Endo-A was purified to homogeneity as previously described (9). For proteolytic cleavage, the purified enzyme (80 mg) was digested with lysylendopeptidase (Wako Pure Chemicals) in 20 mM Tris/HCl buffer containing 1.6 M urea, pH 9.1, for 21 h at 307C at a substrate/enzyme ratio of 400:1. After the reaction was stopped by the addition of formic acid, peptide mixtures obtained by enzymatic cleavages were analyzed on a reversed-phase column using a Pharmacia SMART system (mRPC C2/C18, SC 2.1/10, 2.1 by 100 mm), equilibrated with 0.12% trifluoroacetic acid, and eluted with a linear acetonitrile gradient at a flow rate of 0.1 ml/min. PCR amplification and cloning of the Endo-A gene. Conventional methods were used in the construction and propagation of all plasmids (19). To amplify Endo-A-like sequences from the genomic DNA of the A. protophormiae strain, the following oligonucleotide primers were synthesized and tested: primer 1, 5*-GTTGGATCCTTT(C)CCA(T/G/C)GAA(G)GAA(G)T(C)TA(T/G/C)GCA-(T/G/C)CA-3*, and primer 2, 5*-GTTGAATTCGTA(G)TTA(G)AAA(G)TGA(T/G/ C)GTA(T/G/C) ACA(G)AA-3*. The primers included restriction sites at their 5*-ends to facilitate cloning of the resulting amplified products. All oligonucleotides were synthesized by Bio-synthesis, Inc. The primer 1 encodes amino acids FPEELAQ, while primer 2 encodes FVTHFNT. The PCR (20) mixture contained 3 mg A. protophormiae genomic DNA, 5 mM primers, 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 1.5 mM MgSO4, 0.2 mM dNTPs, and 3 units Takara Taq DNA polymerase (Takara Shuzo Co.), in a total volume of 50 ml. PCR was performed in an Eppendorf tube using 35 repetitions of the following temperature cycle: 947C for 60 s, 497C for 90 s, and 727C for 90 s. The reaction product was digested with BamHI and EcoRI and resolved by electrophoresis in a 1.5% agarose gel. A fragment of approximately 900 bp was recovered and ligated into the pBluescript KS(0) vector that had been digested with BamHI and EcoRI. All nucleotide sequence determination was performed using double-stranded plas-
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mid DNA templates and an AutoRead sequencing kit (Pharmacia), according to the recommendations of the manufacturer. A. protophormiae chromosomal DNA was isolated according to Ausubel et al. (21). The DNA was partially digested with Sau3AI, and fragments of 8 to 15 kb were purified using a Takara EASY trap kit, ligated into BamHI-digested lEMBL3 using a Gigapack III packaging kit (Stratagene), then packaged into phage particles and used to infect E. coli P2392. The entire Endo-A gene was isolated by plaque hybridizing the cloned PCR product. Southern blot analysis and hybridization. Restriction digests of lEMBL3-insert DNA were separated in 1.0% agarose gels and blotted onto Hybond-N/ membranes (Amersham) according to the manufacturer’s recommendation. Detection was carried out with nonradioactive DNA probes. Oligonucleotide probes were labeled using an Amersham ECL nucleotide detection kit. Hybridization and washing were carried out according to the manufacture’s instructions. Endo-A assay and analysis of transglycosylation products. Endob -N-acetylglucosaminidase activity was assayed with (Man)6(GlcNAc)2Asn-dansyl as the substrate (9). One unit was defined as the amount yielding 1 mmol of GlcNAc-Asn-dansyl per minute at 377C under the assay conditions. An asparaginyl oligosaccharide, (Man)6(GlcNAc)2Asn, was prepared from ovalbumin glycopeptides by the method of Huang et al. (22). The transglycosylation reaction of (Man)6(GlcNAc)2Asn to glucose by Endo-A was examined as follows: 1.3 mg of (Man)6(GlcNAc)2Asn was incubated with 10 munits of Endo-A in 0.27 ml of 50 mM ammonium acetate buffer (pH 6.0) in the presence of glucose (final concentration, 0.5 M) for 10 min at 377C. After the incubation was stopped by boiling the reaction mixture for 3 min, the reaction was put on a Sephadex G-15 column (1.5 1 30 cm) to remove the free glucose. The oligosaccharides were spotted on silica gel 60 plates (Merck Art. 5626) and analyzed by thin-layer chromatography. For separation of the oligosaccharides, the following solvent system was used: n-propanol/acetic acid/water Å 3:3:2 (23). Orcinol–H2SO4 reagent was used for the detection of oligosaccharides (23). Analyses of the carbohydrate composition of the oligosaccharides were done as described previously (15). Electrophoresis. Polyacrylamide gel electrophoresis in the presence of SDS was carried out by the standard methods (24). The molecular weight markers were purchased from Pharmacia. Proteins were visualized with Coomassie brilliant blue R-250. Purification of Endo-A from E. coli. Purification of the recombinant Endo-A was carried out as follows. E. coli XLI-Blue strain carrying recombinant plasmid pKY1 was grown aerobically in 3% peptone and 1% NaCl at 377C for 72 h. The cells (a 4-liter-cultivation) were centrifuged, and extract from the periplasmic space of the cells was obtained by the osmotic shock procedure described by Suzuki et al. (25). This extract was applied to a DEAE-Toyopearl 650M (2.5 1 10 cm) previously equilibrated with 10 mM phosphate buffer (pH 7.0). The column was washed with the same buffer and the enzyme eluted in 4-ml fractions with a gradient formed between 10 mM phosphate buffer and 0.5 M NaCl in 10 mM phosphate buffer. The active fractions were combined, and ammonium sulfate was added to the enzyme solution to 1 M. The enzyme solution was applied to a PhenylSepharose CL-4B column (0.8 1 22 cm) equilibrated with 1 M ammonium sulfate in 10 mM phosphate buffer (pH 7.0). The enzyme was eluted in 2-ml fractions with a gradient formed between 1 M ammonium sulfate and 40% ethyleneglycol in 10 mM phosphate buffer. The active fractions were combined and concentrated by the addition of ammonium sulfate to 90% saturation. The precipitate was collected by centrifugation and then resuspended in 10 mM phosphate buffer (pH 7.0).
RESULTS AND DISCUSSION
Determination of partial amino acid sequence of Endo-A. To synthesize oligonucleotide primers for
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FIG. 1. Separation of lysylendopeptidase peptides. The reverse-phase HPLC column (mRPC C2/C18, SC 2.1/10, 2.1.1 100 mm, Pharmacia) was equilibrated with 0.12% trifluoroacetic acid and developed with a linear acetonitrile gradient at a flow rate of 0.1 ml/min.
PCR, the N-terminal amino acid sequence of the purified Endo-A, as well as the amino acid sequence of the major peptide fragments derived by lysyl-endopeptidase, was determined using a protein sequencer. Partial amino acid sequence data were obtained from the N terminus of the mature protein and from nine peptide fragments (Fig. 1). Molecular cloning and sequence analysis of Endo-A. Degenerate oligonucleotides encoding N-terminal and other determined sequences within Endo-A protein were used to PCR amplify a 1-kb gene fragment from A. protophormiae genomic DNA (see Materials and Methods). The complete sequence of the PCR product was determined, and five of the nine peptides were found in the PCR fragment. As this PCR product is therefore part of the Endo-A gene, this gene fragment was used as a probe to recover the Endo-A gene from the A. protophormiae genomic library. To clone the entire Endo-A gene code, chromosomal libraries were constructed using lEMBL3. Screening of the chromosomal phage libraries resulted in six positive clones. Two of these clones were confirmed by hybridization with nonradioactivity labeled PCR fragments to be identical (both containing 9 kb-fragments). To determine the location of the Endo-A gene within the fragment, we prepared various subclones in which ClaI, PstI, KpnI, and HindIII digests of the fragment had been ligated at appropriate sites in pBluescript KS(0) cloning vectors. The plasmid pKY1, a 4.7-kb ClaI–PstI fragment containing the Endo-A gene, was sequenced. The nucleotide sequence of the Endo-A gene is shown in Fig. 2.
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In this sequence, a single open reading frame (ORF),2 starting at position 1 and ending at position 1937, was found. Putative promoter regions of the Endo-A gene were detected upstream of the Shine–Dalgarno-like sequence: at positions 015 to 011, as well as in the sequence around 025 which was found to be a putative 010 region (see in Fig. 2). The ORF could encode a mature protein of 621 amino acids with a signal peptide of 24 amino acids. All amino acid sequences from the purified Endo-A were discovered within the deduced amino acid sequence. This data suggest that the ORF encodes Endo-A. The amino acid sequences of several endo-b-N-acetylglucosaminidases have been determined; Endo-H from Streptomyces plicatus (11); Endo-F1 (13); F2 (26), and F3 (26) from Flavobacterium meningosepticum; and Endo-Flavo from Flavobacterium sp. (12). We examined sequence comparisons between Endo-A and other endo-b-N-acetylglucosaminidases, but we could not detect any homologous regions. Schumidt et al. have substituted the glutamine, asparagine, and tryptophan residues of Endo-H protein using point mutagenesis and found that Asp-130 and Glu-132 were essential for enzyme activity (27). These two amino acid residues are conserved in other endo-b-N-acetylglucosaminidases and chitinases (28, 29). Van Roey and colleagues analyzed the three-dimensional structures of Endo-H and Endo-F1 using crystallographic studies (30, 31).
2 Abbreviations used: ORF, open reading frame; PNGase F, peptide-N4-(N-acetyl-b-D-glucosaminyl)asparagine amidase F.
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FIG. 2. Nucleotide sequence of the Endo-A gene. The putative ribosome-binding site is indicated by dots. The 010 region of a possible promoter sequence is double underlined. The deduced amino acid sequence is given below the nucleotide sequence. The overlined sequences indicate the amino acid sequences for the PCR primers. The underlined sequences indicate the amino acid sequences determined with a protein sequencer. The arrow indicates the site of cleavage of the signal peptide.
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Both enzymes consisted of a highly irregular a/b-barrel. Asp-130 and Glu-132 of Endo-H are located in a curved cleft over the top of the molecule (31). Although there are two Asp-X-Glu sequences in Endo-A protein (Asp-489 and Asp-550), the surrounding sequences of Endo-A are not conserved among endo-b-N-acetylglucosaminidases. The deduced amino acid sequence was compared with other published sequences in various databases of the National Center for Biotechnology Information using the FASTA Network Service. The N-terminal region of Endo-A is highly similar to that of the Caenorhabditis elegans gene product. This ORF was identified by the genome sequencing project of C. elegans chromosome III (32). Relatively low but significant homology was also observed with peptide-N4-(N-acetyl-bD-glucosaminyl)asparagine amidase F (PNGase F) (33). In contrast to endo- b -N-acetyl-glucosaminidase, PNGase F hydrolyzes N-linked sugar chains by cleaving the amide bond between the proximal GlcNAc and Asn in the peptide chain (34). Crystallographic analysis has shown that Glu-206 is located in a cleft at the interface between the two domains of the PNGase F protein (35, 36). Glu-206 of PNGase F is thought to be important for stabilization of reaction intermediates (36). The similar acidic residue, Asp-301 of Endo-A, may be important for the stabilization of reaction intermediates. In addition, four tryptophan residues are also well conserved between the Endo-A and PNGase F. The C-terminal region of Endo-A had weak homology with Streptomyces olivaceoviridis chitinase (28). This region of the S. olivaceoviridis chitinase also shows sequence similarities to the C-terminal end of chitinase A1 from Bacillus circulans (37) and the type III homology units of fibronectin, a multifunctional extracellular matrix and plasma protein of higher eukaryotes (38). The C-terminal region including this region of chitinase A1 was shown to be essential for its affinity to the substrate chitin (39). These conserved regions of Endo-A and chitinases may be essential for the association of high-molecular-mass substrates. Recently Matsui et al. (40) have reported that transglycosylation activity of a-amylase from Saccharomycopsis increased after changing an aromatic residue in the active center by site-directed mutagenesis. Transglycosylation is a competition against hydrolysis, where the acceptor competes for water at the enzyme active center. Therefore, it is possible to enhance the transglycosylation activity by redesigning the active center of the enzyme to gain stronger affinity for the acceptor. Site-directed mutagenesis is in progress to determine the active site of Endo-A protein. Expression and characterization of recombinant Endo-A in E. coli cells. The 4.7-kb DNA fragment was
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FIG. 3. Profiles of Endo-A produced by E. coli XLI-Blue harboring pKY1. Lane A, molecular weight markers; lane B, purified Endo-A produced by A. protophormiae; lane C, purified Endo-A produced by E. coli XLI-Blue (pKY1).
subcloned into pBluescript KS (pKY1). The transformed E. coli XLI-Blue was cultured at 377C in LB broth for 30 h, and the enzyme activity was measured in the periplasmic space of the cells. Endo-A activity was detected mainly in the periplasmic space of the cells. The plasmid containing the reverse-oriented 4.7kb fragment was also able to degrade dansylated-ovalbumin glycopeptide, indicating that the cloned Endo-A gene is expressed by its own promoter. Endo-A production in E. coli was tested during cultivation at 377C. Until the 72nd hour of cultivation, Endo-A activity was mainly in the periplasmic space, indicating that the Endo-A was exported through the cytoplasmic membrane of E. coli cells. The amount of Endo-A in the periplasmic space increased in the exponential phase and maximized in the early stationary phase, but thereafter Endo-A protein was leaked into the medium. The transformed E. coli XLI-Blue was cultured in 4 liters of 3% peptone and 1% NaCl medium at 377C for 2 days. The cells were then harvested and subjected to osmotic shock. Recombinant Endo-A was purified as described under Materials and Methods, analyzed by SDS–PAGE, and compared with native Endo-A protein. Both enzymes migrated as a single protein band during SDS–PAGE (Fig. 3). The relative molecular weight of each was estimated to be 72,000. The N-terminal sequence of the recombinant Endo-A was determined as Ser-Thr-Tyr-Asn-Gly-Pro-Leu, showing that the signal peptide of the Endo-A produced in E. coli XLI-Blue cells had been removed by cleavage between residues 24 and 25. This data show that the signal peptide of Endo-A was cleaved at the same position in E. coli cells. The enzymatic properties of the recombinant Endo-A enzyme were compared with those of the native enzyme. They showed similar pH-activity profiles (both Endo-A’s showed broad optimum pH and were most active in the range of pH 5.0 to 9.0). Both enzymes were stable up to 607C (after incubation for 10 min) and strongly inhibited by the addition of 1 mM
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2. Koide, N., and Muramatsu, T. (1974) J. Biol. Chem. 249, 4897– 4904. 3. Ito, S., Muramatsu, T., and Kobata, A. (1975) Arch. Biochem. Biophys. 171, 78–86. 4. Elder, J. H., and Alexander, S. (1982) Proc. Natl. Acad. Sci. USA 79, 4540–4544. 5. Yamamoto, K., Kadowaki, S., Takegawa, K., Kumagai, H., and Tochikura, T. (1986) Agric. Biol. Chem. 50, 421–429. 6. Kadowaki, S., Yamamoto, K., Fujisaki, M., and Tochikura, T. (1991) J. Biochem. 110, 17–21. 7. Yamamoto, K. (1994) J. Biochem. (Tokyo) 116, 229–235. 8. Varki, A. (1993) Glycobiology 3, 97–130. FIG. 4. TLC analysis of the transglycosylation products. (Man)6(GlcNAc)2Asn was incubated with native Endo-A (lane C) or recombinant Endo-A (lane D) in the presence of 0.5 M glucose for 10 min at 377C. Lane A, standard (Man)6(GlcNAc)2Asn; lane B, standard (Man)6GlcNAc.
HgCl2 (final concentration). The recombinant Endo-A caused no changes in kinetic parameters or in the pHactivity–stability profiles. The transglycosylation reaction of (Man)6(GlcNAc)2Asn to glucose by the recombinant Endo-A was carried out as described under Materials and Methods. The transglycosylation products obtained were analyzed by TLC. One oligosaccharide spot was newly generated in the presence of glucose (Fig. 4D). The spot was collected, eluted with water, and lyophilized. The samples were hydrolyzed with 4 M trifluoroacetic acid, and the carbohydrate composition of the transglycosylation product was determined to be Man:GlcNAc:Glc Å 6:1.0:1.0 (relative to Man Å 6). These results show that glucose was attached to (Man)6GlcNAc during cleavage of the chitobiose core and that (Man)6GlcNAcGlc was synthesized by the transglycosylation of Endo-A (15). The recombinant Endo-A was purified to homogeneity by relatively simple procedures: DEAE-Toyopearl and Phenyl-Sepharose 4B column chromatographies. In this study, the cloned Endo-A gene was expressed in E. coli by its own promoter. Therefore, it may be possible to obtain a much higher Endo-A-producing strain utilizing a high-expression promoter.
The authors thank Drs. Hideyuki Suzuki, Kenji Yamamoto (Kyoto University), Jian-Qiang Fan, and Yuan Juan Lee (Johns Hopkins University) for their helpful discussions. We also thank Makiko Shimizu, Satomi Suda, and Tsuyoshi Miyamura for their excellent technical assistance. This work was partly supported by Kagawa Techno Foundation (to K.T.). The Endo-A nucleotide sequence has been deposited in GenBank and can be found using Accession Number U59168.
REFERENCES 1. Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811– 817.
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10. Tai, T., Yamashita, K., Ito, S., and Kobata, A. (1977) J. Biol. Chem. 252, 6687–6694. 11. Robbins, P. W., Trimble, R. B., Wirth, D. F., Hering, C., Maley, F., Maley, G. F., Das, R., Gibson, B. W., Royal, N., and Biemann, K. (1984) J. Biol. Chem. 259, 7577–7583. 12. Takegawa, K., Mikami, B., Iwahara, S., Morita, Y., Yamamoto, K., and Tochikura, T. (1991) Eur. J. Biochem. 202, 175–180. 13. Tarentino, A. L., Quinones, G., Schrader, W. P., Changchien, L.-M., and Plummer, T. H., Jr. (1992) J. Biol. Chem. 267, 3868– 3872. 14. Takegawa, K., Yamaguchi, S., Kondo, A., Iwamoto, H., Nakoshi, M., Kato, I., and Iwahara, S. (1991) Biochem. Int. 24, 849–855. 15. Takegawa, K., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1991) Biochem. Int. 25, 829–835. 16. Takegawa, K., Tabuchi, M., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1995) J. Biol. Chem. 270, 3094–3099. 17. Fan, J. Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., Abeygunawardana, C., and Lee, Y. C. (1995) J. Biol. Chem. 270, 17723–17729. 18. Fan, J. Q., Quesenberry, M. S., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., and Lee, Y. C. (1995) J. Biol. Chem. 270, 17730–17735. 19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 20. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487–491. 21. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology, Wiley, New York. 22. Huang, C.-C., Mayer, H. E., Jr., and Montgomery, R. (1970) Carbohydr. Res. 13, 127–137.
ACKNOWLEDGMENTS
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9. Takegawa, K., Nakoshi, M., Iwahara, S., Yamamoto, K., and Tochikura, T. (1989) Appl. Environ. Microbiol. 55, 3107–3112.
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23. Holmes, E. W., and O’Brien, J. S. (1979) Anal. Biochem. 93, 167– 170. 24. Laemmli, U. K. (1970) Nature 227, 680–685. 25. Suzuki, H., Kumagai, H., and Tochikura, T. (1986) J. Bacteriol. 168, 1332–1335. 26. Tarentino, A. L., Quinones, G., Changchien, L.-M., and Plummer, T. H., Jr. (1993) J. Biol. Chem. 268, 9702–9708. 27. Schmidt, B. F., Ashizawa, E., Jarnagin, A. S., Lynn, S., Noto, G., Woodhouse, L., Estell, D. A., and Lad, P. (1994) Arch. Biochem. Biophys. 311, 350–353. 28. Blaak, H., Schnellmann, J., Walter, S., Henrissat, B., and Schrempf, H. (1993) Eur. J. Biochem. 214, 659–669.
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29. Kuranda, M. J., and Robbins, P. W. (1991) J. Biol. Chem. 266, 19758–19767. 30. Van Roey, P., Rao, V., Plummer, T. H., Jr., and Tarentino, A. L. (1994) Biochemistry 33, 13989–13996. 31. Rao, V., Guan, C., and Van Roey, P. (1995) Structure 3, 449– 457. 32. Wilson, R., et al. (1994) Nature 368, 32–38. 33. Lemp, D., Haselbeck, A., and Klebl, F. (1990) J. Biol. Chem. 265, 15606–15610. 34. Tarentino, A. L., and Plummer, T. H. (1987) Methods Enzymol. 138, 770–778.
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35. Kuhn, P., Tarentino, A. L., Plummer, T. H., Jr., and Van Roey, P. (1994) Biochemistry 33, 11699–11706. 36. Kuhn, P., Guan, C., Cui, T., Tarentino, A. L., Plummer, T. H., Jr., and Van Roey, P. (1995) J. Biol. Chem. 270, 29493–29497. 37. Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K., and Tanaka, H. (1990) J. Biol. Chem. 265, 15659–15665. 38. Rouslathi, E. (1988) Annu. Rev. Biochem. 57, 375–413. 39. Watanabe, T., Oyanagi, W., Suzuki, K., and Tanaka, H. (1990) J. Bacteriol. 172, 4017–4022. 40. Matsui, I., Yoneda, S., Ishikawa, K., Miyairi, S., Fukui, S., Umeyama, H., and Honda, K. (1994) Biochemistry 33, 451–458.
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