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Originally published In Press as doi:10.1074/jbc.M506874200 on September 1, 2005

J. Biol. Chem., Vol. 280, Issue 45, 37415-37422, November 11, 2005
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Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-{alpha}-N-acetylgalactosaminidase from Bifidobacterium longum*

Kiyotaka Fujita{ddagger}1, Fusako Oura{ddagger}, Noriko Nagamine{ddagger}§, Takane Katayama§, Jun Hiratake||, Kanzo Sakata||, Hidehiko Kumagai¶, and Kenji Yamamoto{ddagger}§

From the {ddagger}Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, the ||Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, and the §Core Research for Evolutional Science and Technology Program of Japan Science and Technology Corporation (CREST-JST), Kawaguchi, Saitama 332-0012, Japan

Received for publication, June 24, 2005 , and in revised form, September 1, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We found endo-{alpha}-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl {beta}1,3-N-acetyl-D-galactosamine (Gal{beta}1,3GalNAc) {alpha}-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590–1381 amino acid residues) exhibited 31–53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Gal{beta}1,3GalNAc disaccharide from Gal{beta}1,3GalNAc{alpha}1pNP and asialofetuin, but did not release GalNAc, Gal{beta}1,3(GlcNAc{beta}1,6)GalNAc, GlcNAc{beta}1,3GalNAc, and Gal{beta}1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by 1H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-{alpha}-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucin-type oligosaccharides (O-glycans) are involved in several important biological events including cell-to-cell communication in higher eukaryotes, bacterial adhesion to host cells, and so on (1). Eight core-type structures of O-glycans have been proposed, and among them, galactosyl {beta}1,3-N-acetyl-D-galactosamine (Gal{beta}1,3GalNAc)2 attached to the serine or threonine residue via an {alpha}-linkage (Core 1, also referred to as T antigen) is one of the most abundant core structures found in mucin glycoproteins that are widely distributed in intestinal tracts of human and animals. This immunogenic structure is normally masked by further glycosylation such as sialylation (sialyl T antigen) and N-acetylglucosaminylation (Core 2 structure). However, it has been shown that T antigen is frequently exposed on the cell surface of carcinoma and T-cell lymphoma, and this unmasked form is thought to be involved in tumor cell adhesion and tissue invasion (2, 3). In addition, incomplete synthesis of the Core 1 structure associates with several autoimmune diseases including IgA nephropathy (4), Tn-syndrome (5), and Henoch-Schönlein purpura (6).

Endo-{alpha}-N-acetylgalactosaminidase (EC 3.2.1.97 [EC] ; endo-{alpha}-GalNA-case, glycopeptide {alpha}-N-acetylgalactosaminidase) is a unique enzyme that hydrolyzes Core 1-type O-glycan from glycoproteins. This enzyme also serves as a powerful tool for elucidating the presence of O-glycans in glycoproteins (7), the role of O-glycan in the biological events (8), and analysis of the O-glycan binding site (9), because the enzyme selectively cleaves and removes the disaccharide from the targets without damaging them. To date, endo-{alpha}-GalNAcases have been purified from Clostridium perfringens (10), Streptococcus pneumoniae (11–15), Alcaligenes sp. (16), and Bacillus sp. (17). All of the enzymes are secretory and high-molecular mass proteins (>110 kDa), and are able to release Gal{beta}1,3GalNAc disaccharide from glycoproteins. A similar enzyme was found in the culture medium of Streptomyces sp., which is capable of releasing longer sugar chains than the disaccharide from porcine mucin (18, 19), although further studies are necessary to validate its action.

Bifidobacteria are strictly anaerobic lactic acid-producing bacteria that constitute a major part of the intestinal microflora of human and animals, and have attracted a great deal of attention because of their many beneficial probiotic effects (20). Recently, we found that bifidobacteria widely have an endo-{alpha}-GalNAcase specific for Core 1 mucin-type O-glycans. Despite its significance and usefulness for research on O-linked oligosaccharides and several related pathogens, no genetic information is available yet. Therefore, we performed molecular cloning of the endo-{alpha}-GalNAcase of Bifidobacterium longum JCM 1217 and characterized the properties of the recombinant enzyme. This is the first report describing isolation of the gene for this enzyme from any organisms.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Gal{beta}1,3GalNAc{alpha}1pNP and GlcNAc{beta}1,3GalNAc{alpha}1pNP were kindly provided by Drs. Murata and Usui, Shizuoka University, and Dr. Kitaoka, National Food Research Institute, respectively. Gal{beta}1,3(GlcNAc{beta}1,6)GalNAc{alpha}1pNP was purchased from Toronto Research Chemicals Inc. GalNAc{alpha}1pNP, calf fetuin, and calf asialofetuin were from Sigma. All other chemicals were of the highest grade available from commercial sources.

Bacterial Strains—The bifidobacterial strains used in this study were: B. longum JCM 1217 and JCM 7054, B. bifidum JCM 7004, JCM 1254, and ATCC 29521, B. breve JCM 1192, B. infantis JCM 1222, and B. pseudolongum JCM 1205.

Partial Purification and Molecular Mass Determination of Endo-{alpha}-GalNAcase from B. longum JCM 1217—The bacterium was grown in 2 liters of GAM broth (Nissui Pharmaceutical) for 4 days at 37 °C under anaerobic and static conditions. The cells were harvested by centrifugation and suspended in a final volume of 15 ml of 50 mM potassium phosphate buffer (KPB) (pH 7.0), and were sonicated subsequently using a Branson Sonifier 250 (Branson Ultrasonic Corp.). The extract was applied on a Butyl-Toyopearl 650M column (8 x 100 mm; Tosoh) equilibrated with 1 M ammonium sulfate in 20 mM KPB (pH 7.0), followed by elution with a linear gradient of 1–0 M ammonium sulfate in 20 mM KPB (pH 7.0). The enzyme was eluted at the concentration of 0.6 M ammonium sulfate. The active fractions were combined, desalted using an Amicon Ultra-15 centrifugal filter (Millipore), and then applied to a UNO-Q1 column (Bio-Rad) equilibrated with 20 mM Tris-HCl buffer (pH 7.0), followed by elution with a linear gradient of 0–0.3 M NaCl in 20 mM Tris-HCl buffer (pH 7.0). The enzyme was eluted at the concentration of 0.2 M NaCl. The fractions were then desalted and concentrated using Amicon Ultra-15 centrifugal filter.

The molecular mass of the partially purified enzyme was determined by size exclusion chromatography using a Superose 6 column (Amersham Biosciences). The column was equilibrated in advance with 0.15 M NaCl in 50 mM KPB (pH 7.0). Molecular mass standards used for calibration were: {beta}-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) from Sigma. All fractions were assayed, and the active fractions were loaded on SDS-PAGE, according to the method of Laemmli (21).

Expression and Purification of Recombinant Protein—The putative endo-{alpha}-GalNAcase gene was amplified by high-fidelity PCR using the genomic DNA of B. longum JCM 1217 as a template and a primer pair (forward primer, 5'-TTAACCTCCATGGGCAGCGGGGGAGG-3', and reverse primer, 5'-AACCTGCGGCCGCCAGTTGCTCGCGATTGC-3'). The primers were designed based on nucleotide numbers 58–74 and 5882–5898 of BL0464 of B. longum NCC2705 for the forward and reverse primers, respectively, and contained NcoI and NotI sites (underlined) at the N and C termini, respectively, to facilitate plasmid construction. The amplified gene (hereafter referred to as engBF; encoding amino acid residues 20–1966 eliminating the signal peptide) was digested with NcoI and NotI, and was inserted into the corresponding sites of pET-23d(+) (Novagen) to generate a C-terminal His6-tagged protein. The resulting plasmid, pET23d-engBF, was introduced into Escherichia coli BL21({lambda}DE3), and the cells were grown in LB medium containing ampicillin (150 µg/ml) at 37 °C. After A600 reached 0.4, isopropyl 1-thio-{beta}-D-galactopyranoside was added at the final concentration of 0.4 mM to induce the protein expression. Following additional incubation for 3 h, the cells were harvested by centrifugation, suspended in BugBuster protein extraction reagent (Novagen), and the soluble fraction obtained was applied on a Ni2+-charged HiTrap chelating column (Amersham Biosciences). The protein concentration was determined using the BCA protein assay reagent (Pierce) with bovine serum albumin as a standard.

Isolation and Sequencing of the Endo-{alpha}-GalNAcase Gene—The gene encoding amino acid residues 20–1966 of engBF was amplified as described above, and was directly used for sequencing. The flanking upstream DNA segment was isolated by a standard colony hybridization method (22). The downstream region was amplified by high-fidelity PCR using the genomic DNA of B. longum JCM 1217 as a template and a primer pair (5'-TCAACGGCGATTCCTGG-3', 1686-bp upstream of the stop codon; and 5'-TGCGATTCATCGCCTAGCAG-3', 150-bp downstream of the stop codon). The primers were designed based on the sequence of BL0464 of B. longum NCC2705. The DNA sequence was determined for both strands by the use of the ABI Prism 310 DNA sequencer with a BigDye terminator version 3.1 cycle sequencing ready reaction kit (Applied Biosystems).

Assay of Endo-{alpha}-GalNAcase Activity—The hydrolytic activity of the enzyme was assayed using Gal{beta}1,3GalNAc{alpha}1pNP as a substrate. The standard reaction mixture contained, in a total volume of 40 µl, 50 mM sodium acetate buffer (pH 5.0), 0.25 mM substrate, and the enzyme. After incubation for an appropriate time at 37 °C, the reaction was stopped by adding 60 µl of 1 M sodium carbonate, and the released p-nitrophenol (pNP) was measured by the absorbance at 400 nm. One unit of enzyme activity was defined as the amount of enzyme releasing 1 µmol of pNP/min. The substrate specificity was determined using various pNP glycosides and natural glycoproteins.

Transglycosylation of Endo-{alpha}-GalNAcase—The transglycosylation reactions were performed according to the procedures of Ashida et al. (36) for 1-alkanols as acceptors and Ashida et al. (17) for monoor disaccharides as acceptors.

Other Assays—The pH dependence of enzyme activity was determined in pH range 2.0–9.0 using the following buffers (50 mM): glycine-HCl (2.0–4.0), sodium acetate (3.5–6.0), sodium phosphate (5.5–8.0), and Tris-HCl (7.0–9.0). Effect of temperature on the enzyme was examined using 50 mM sodium acetate buffer (pH 5.0) at 20–80 °C.

Analytical Methods—The reaction products were analyzed by HPLC and/or TLC. For TLC analysis, a Silica Gel 60 aluminum sheet (Merck) was developed in a solvent system of chloroform/methanol/water, 3/3/1 (v/v/v), and the sugars were visualized by spraying diphenylamine/aniline/phosphate reagent (23). HPLC was carried out using a Hitachi D-7500 chromatograph system equipped with a L-7420 UV-visual detector. Oligosaccharides were fractionated using the TSKgel Amide-80 column (4.6 x 250 mm, Tosoh) under a constant flow (1.0 ml/min) of 75% acetonitrile at 40 °C, and elution was monitored to the absorbance of the N-acetyl group at 214 nm.

Stereochemistry of the Hydrolysis of pNP Substrate by Endo-{alpha}-GalNAcase—1H NMR spectra were recorded on a JEOL JNM-AL300 spectrometer as described previously (24). The reaction mixture, in a total volume of 600 µl, contained 5.0 mM Gal{beta}1,3GalNAc{alpha}1pNP in D2O. After recording the reference spectrum (t = 0 min), 100 milliunits (90 µlinD2O) of the purified enzyme (lyophilized twice from a buffer in D2O prior to use) was added to initiate the reaction. The spectra were recorded at different time intervals (4 min to 16 h).

Mass Spectrometry—Fast atom bombardment-mass spectrometry was performed with a JEOL JMS-700 (MStation) using glycerol as the matrix. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was performed in the negative ion mode using {alpha}-cyano-4-hydroxycinnamic acid as the matrix (PerSeptive Biosystems).

Site-directed Mutagenesis—QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce amino acid substitutions. pET23d-engBF was mutated using the mutagenic forward primers (as shown in TABLE ONE) and these complement primers. After confirmation of the desired mutations by DNA sequencing, the mutant EngBF enzymes were expressed and purified according to the same procedure as for the wild-type enzyme.


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  		TABLE ONE
The forward primers used for site-directed mutagenesis

The positions of the mutated sequences are underlined.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of Endo-{alpha}-GalNAcase Activity in Various Strains of Bifidobacteria and Search for the Gene of the Enzyme—During our studies on various glycosidases of bifidobacteria to analyze an assimilation mechanism of oligosaccharide specific for the growth of bifidobacteria, we found that the endo-{alpha}-GalNAcase activity was widely distributed among various bifidobacterial strains such as B. longum, B. bifidum, and B. breve (Fig. 1). The enzymatic activities were also detected in the culture supernatants and the cell extracts treated with cell wall lytic enzyme, mutanolysin (data not shown). These results suggested that the enzyme probably has a secretion signal, and is secreted and associated with the cell surface. To elucidate the function of this enzyme, we partially purified the enzyme from B. longum JCM 1217 and determined the molecular mass as about 200 kDa. Based on the structural information of the enzyme, i.e. an ORF with more than 1,800 amino acid residues carrying a secretion signal, we scrutinized the genomic sequence of B. longum NCC2705 (25) and chose two hypothetical proteins of unknown functions, BL0464 and BL0421, as the candidate ORFs encoding the enzyme. Data base search using BLAST (26) revealed that BL0464 exhibits high similarity to hypothetical proteins of C. perfringens (CPE0693), S. pneumoniae (SPR0328), Enterococcus faecalis (EF1800), and Streptomyces coelicolor (SCO6348), the former two organisms being known to produce endo-{alpha}-GalNAcase (10–15), whereas BL0421 did not have any homologues in the data base. The molecular mass of endo-{alpha}-GalNAcase from S. pneumoniae was reported to be 190 kDa (15), which corresponded to the predicted molecular mass of SPR0328. Also, the molecular size of endo-{alpha}-GalNA-case of S. coelicolor A3 (2) was in good agreement with the predicted molecular mass of SCO6348.3 These results suggested that BL0464, a "narrowly conserved hypothetical protein," might encode endo-{alpha}-GalNAcase.



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  		FIGURE 1.
TLC analysis of the reaction mixtures incubated with pNP substrate and various bifidobacteria cells. The cell pellets were incubated with 0.5 mM Gal{beta}1,3GalNAc{alpha}1pNP in 10 mM acetate buffer (pH 5.0) for2hat37°C.The bifidobacterium strains incubated with the substrate are as follows: B. breve JCM 1192 (lane 5), B. infantis JCM 1222 (lane 6), B. bifidum JCM 1254 (lane 7), B. bifidum JCM 7004 (lane 8), B. bifidum ATCC 29521 (lane 9), B. pseudolongum JCM 1205 (lane 10), B. longum JCM 1217 (lane 11), and B. longum JCM 7054 (lane 12). Lane 1, Gal{beta}1,3GalNAc{alpha}1pNP; lane 2, Gal; lane 3, GalNAc; lane 4, Gal{beta}1,3GalNAc.

 



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  		FIGURE 2.
SDS-PAGE analysis of the recombinant EngBF. The protein was subjected to SDS-PAGE using a 8% polyacrylamide gel, and visualized by Coomassie Brilliant Blue R-250 staining. Lane 1, crude extract of E. coli BL21({lambda}DE3); lane 2, purified recombinant EngBF; lane M, molecular size markers. The arrow indicates the position of the recombinant EngBF.

 
Molecular Cloning and Expression of Endo-{alpha}-GalNAcase (EngBF) of B. longum JCM 1217—Based on the DNA sequence of BL0464 of B. longum NCC2705, oligonucleotide primers were designed as described under "Experimental Procedures." A 6.0-kilobase pair DNA fragment amplified using a genomic DNA of B. longum JCM 1217 as a template was inserted into expression plasmid pET23d. The recombinant protein was expressed in E. coli BL21({lambda}DE3), and the soluble cell lysate was examined for the endo-{alpha}-GalNAcase activity using Gal{beta}1,3GalNAc{alpha}1pNP as a substrate. The liberation of the disaccharide and pNP were observed in time- and dose-dependent manners (data not shown), indicating that the endo-{alpha}-GalNAcase gene (engBF) was successfully isolated. The details of its enzymatic properties are given below. Furthermore, the upstream and downstream regions of the engBF gene were cloned by Southern hybridization and PCR, respectively. EngBF consisted of 1,966 amino acid residues exhibiting 99.1% identity with that of BL0464. The His-tagged EngBF was purified as described under "Experimental Procedures," and the final preparation migrated as a single band with an apparent molecular size of 200 kDa on SDS-PAGE (Fig. 2), which is in agreement with the calculated molecular mass of 210,269 Da.



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  		FIGURE 3.
Structural analysis of EngBF of B. longum. A, schematic representation of EngBF of B. longum JCM 1217. The numbering starts at a possible initiation codon. Three repetitive sequences are depicted as shaded boxes, and contain so-called FIVAR domains (Pfam 07554). B, a phylogenetic tree was constructed with the ClustalW program using the neighbor-joining method. The gray boxes indicate highly conserved regions among the hypothetical ORFs, which result in the establishment of a new glycoside hydrolase family 101. Numbering starts at a probable initiation codon of each sequence, and the lengths of the sequences are indicated on the right. C, multiple alignment of the amino acid sequences of the conserved regions in B, created with ClustalW and BoxShade 3.21. Identical residues and conserved substitutions are highlighted in black and dark gray, respectively. Asterisks indicate well conserved acidic residues (aspartate and glutamate), and each of them was replaced with a noncharged residue (asparagine and glutamine), as described in the text. The organisms and accession numbers (bracket) are as follows: SC, S. coelicolor A3 (CAA20079 [GenBank] ); EF, E. faecalis V583 (AAO81568 [GenBank] ); BL1, B. longum JCM 1217 (AY836679 [GenBank] ); BL2, B. longum NCC2705 (AAN24297 [GenBank] ); SP, S. pneumoniae R6 (AAK99132 [GenBank] ); and CP, C. perfringens 13 (BAB80399 [GenBank] ).

 
Structural Analysis of EngBF—Sequence analysis of EngBF revealed the presence of a signal peptide and a transmembrane region at the N (amino acid residues 1–29) and C (amino acid residues 1936–1963) termini, respectively (Fig. 3A). The sequences in amino acid residues 1703–1779, 1780–1857, and 1858–1935 are highly homologous to each other, and contain the so-called FIVAR domains that are presumably involved in cell wall binding (Pfam 07554). The primary structure of EngBF showed no similarity to those of any known glycoside hydrolase families, but exhibited significant similarity to hypothetical proteins of C. perfringens, S. pneumoniae, S. coelicolor, and E. faecalis (Fig. 3B), 32–54% identity being observed for amino acid residues 590–1381. From these results, a novel glycoside hydrolase family (GH family 101) was established.

Substrate Specificity and General Properties of the Recombinant EngBF—The substrate specificity of the recombinant enzyme was determined using synthetic pNP substrates and natural glycoproteins. The enzyme was capable of liberating Core 1 disaccharide, but did not release Core 2 trisaccharide (Gal{beta}1,3(GlcNAc{beta}1,6)GalNAc), GalNAc monosaccharide, Core 3 disaccharide (GlcNAc{beta}1,3GalNAc), or Gal{beta}1,3GlcNAc disaccharide from each pNP substrate, as revealed by colorimetric assay and TLC analysis (Fig. 4A). The enzyme did not act on any O-glycosidic linkages of fetuin, but released Gal{beta}1,3GalNAc disaccharide from asialofetuin (Fig. 4A). The ability to liberate the disaccharide from asialofetuin was verified by fast atom bombardmentmass spectrometry analysis, in which the molecular ion peaks of m/z 384 [M + H]+ and m/z 406 [M + Na]+ appeared, but no molecular ion peaks of mono- and trisaccharides appeared (Fig. 4B). High resolution MS of the peak of m/z 384 exactly corresponded to the calculated mass of [M + H]+ (calculated for C14H26NO11 (M + H) 384.1506, found 384.1505). Furthermore, the 1H NMR spectrum of the liberated product from asialofetuin was identical to that of Gal{beta}1,3GalNAc released from its pNP glycoside (data not shown). These results revealed that the enzyme exclusively acts on the unsubstituted Core 1 structure to release Gal{beta}1,3GalNAc disaccharide.



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  		FIGURE 4.
Substrate specificity of the recombinant EngBF. A, TLC analysis of the reaction products. pNP substrates and glycoproteins were incubated without (lane a) or with (lane b) the recombinant enzyme at 30 °C for 12 h. Lane 1, galactose; lane 2, N-acetyl-D-galactosamine; lane 3, Gal{beta}1,3GalNAc{alpha}1pNP; lane 4, asialofetuin; lane 5, fetuin; lane 6, GalNAc{alpha}1pNP; lane 7, Gal{beta}1,3 (GlcNAc{beta}1,6) GalNAc{alpha}1pNP; lane 8, GlcNAc{beta}1,3GalNAc{alpha}1pNP; lane 9, Gal{beta}1,3GlcNAc{alpha}1pNP. B, fast atom bombardment-mass spectrometry analysis of the hydrolysate liberated from asialofetuin by the enzyme.

 
The enzyme was stable up to 37 °C and retained 73% activity after incubation at 45 °C for 30 min in 50 mM acetate buffer (pH 5.0). The optimal temperature and pH were determined as 60 °C and 5.0, respectively. The Km and kcat values for Gal{beta}1,3GalNAc{alpha}1pNP were calculated as 21.8 µM and 17.8 s–1, respectively, which coincide with the values obtained for the Bacillus enzyme (17).

Stereochemistry of the Hydrolysis Catalyzed by EngBF—The stereochemical course of the hydrolysis of EngBF was determined by 1H NMR using Gal{beta}1,3GalNAc{alpha}1pNP as the substrate (Fig. 5). Prior to the experiment, we assigned the chemical shifts of two anomers of Gal{beta}1,3GalNAc using the completely hydrolyzed and equilibrated product of the enzyme. After recording a reference spectrum (t = 0 min), the enzyme was added to the reaction mixture. At 4 min, the signal (Ha, {delta} 5.82, J = 3.9 Hz) derived from the anomeric equatorial proton of GalNAc residue of the substrate was slightly decreased, and instead, a new doublet appeared at {delta} 5.21 (J = 3.6 Hz), which corresponds to Ha'eq of the {alpha}-GalNAc moiety of the liberated disaccharide. The intensity of this signal was increased as the hydrolysis proceeded until a small doublet ({delta} 4.69, J = 8.4 Hz) appeared at 67 min, which corresponds to the axial proton of the {beta}-GalNAc residue (Ha'ax) of the anomer of the product disaccharide after mutarotation. At this time, the {alpha}/{beta} anomer ratio of the GalNAc moiety of the liberated disaccharide was 82/18. As the reaction proceeded (t = 94, 140 min, and 4 h), the signals ({delta} 5.21 and 4.69) for the liberated products were increased, and the ratio of the {alpha}/{beta} anomer of the liberated Gal{beta}1,3GalNAc gradually changed by mutarotation ({alpha}/{beta} ratios of 78/22, 73/27, and 63/37, respectively) to reach equilibrium ({alpha}/{beta} 53/47) after 16 h, when hydrolysis was completed. The results clearly indicate that hydrolysis proceeds with retention of the anomeric configuration. Therefore, we concluded that the enzyme was a retaining glycoside hydrolase.



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  		FIGURE 5.
1H NMR spectra showing the hydrolysis of Gal{beta}1,3GalNAc{alpha}1pNP by EngBF. A, the reaction scheme and assignment of the anomeric protons. B, reaction was monitored by 1H NMR at different times, and the signals for the region of the anomeric protons (4.62–5.88 ppm) are shown. The signal for the equatorial anomeric proton of the GalNAc residue of Gal{beta}1,3GalNAc (Ha'eq; {delta} 5.21, J = 3.6 Hz) appeared first (t = 4 and 38 min), followed by the appearance of the signal of the axial anomeric proton of the GalNAc residue of the disaccharide (Ha'ax; {delta} 4.69, J = 8.4 Hz) (t = 67 min) as a consequence of mutarotation. The large signal around {delta} 4.8 is from HDO.

 
Critical Amino Acid Residues of the Enzyme for Hydrolysis—The hydrolysis of retaining glycoside hydrolases normally proceeds through a double-displacement mechanism. This mechanism requires the actions of two amino acid residues; one functioning as a general acid/base and the other as a nucleophile, and generally these residues are carboxylic acids. As this enzyme was thought to be a retaining glycoside hydrolase as described above, we attempted to identify the catalytically important residues of EngBF by replacing each of 15 aspartate and 7 glutamate residues that are highly conserved among the EngBF homologues (indicated as asterisks in Fig. 3C) with asparagine and glutamine residues, respectively. The mutant enzymes with amino acid substitutions were expressed in E. coli under control of the T7 promoter. Cellfree extracts of E. coli cells expressing D601N, D612N, D665N, E679Q, D700N, D839N, D864N, D898N, D1058N, E1302Q, and E1350Q mutants exhibited endo-{alpha}-GalNAcase activity almost comparable with that of the cells carrying wild-type EngBF, whereas strains expressing D605N, D655N, E723Q, E828Q, D1106N, D1248N, and E1276Q mutants remained at 10–30% activity. However, a significant decrease in the activity (below 2%) was observed for E. coli cells expressing D682N, D762N, D789N, and E822Q mutants. Alanine substitutions were introduced singly at positions Asp-682, Asp-762, Asp-789, and Glu-822, and the mutant enzymes were purified for determination of the kinetic parameters. As shown in TABLE TWO, the D682A and D789A mutant enzymes had lost the activity for the hydrolysis of Gal{beta}1,3GalNAc{alpha}1pNP, whereas the D762A mutant exhibited activity almost comparable with that of the wild-type enzyme. A significant decrease in the kcat value and a slight increase in the Km value were observed for the E822A mutant, but the enzyme was still active toward Gal{beta}1,3GalNAc{alpha}1pNP.


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  		TABLE TWO
Kinetic parameters of wild-type and mutants

 
Transglycosylation Activity of EngBF—Transglycosylation should serve as a powerful tool for creating neo-glycoconjugates having biological activities. In particular, retaining endoglycosidases are useful in creating neo-glycoconjugates because transglycosylation proceeds with retention of the anomeric configuration. We examined the transglycosylation activity of the recombinant enzyme using Gal{beta}1,3GalNAc{alpha}1pNP as a donor and D-glucose as an acceptor. Gal{beta}1,3GalNAc{alpha}1pNP was incubated in the presence of the purified recombinant enzyme with and without glucose, and then the reaction mixture was analyzed by HPLC. As shown in Fig. 6A, when glucose was added to the reaction mixture, a new peak appeared at the retention time of 28 min. The fraction of this peak was collected and subjected to MALDI-TOF MS analysis. The molecular ion peaks appeared at m/z 568.11 and 584.09 (Fig. 6B), which exactly coincided with the sodium and potassium adducts of a transglycosylation product, Gal{beta}1,3GalNAc-Glc, respectively. The transglycosylation products were also detected when D-galactose, D-mannose, L-arabinose, and maltose were used as acceptors (TABLE THREE). These results indicated that the enzyme is able to transfer Gal{beta}1,3GalNAc disaccharide to various mono- and disaccharides. Various 1-alkanols were also found to be good acceptors for transglycosylation by this enzyme (TABLE THREE).


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  		TABLE THREE
Acceptor specificity of EngBF transglycosylation

 



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  		FIGURE 6.
Analysis of the transglycosylation product with EngBF. A, HPLC profile of the reaction mixture incubated in the presence (a) and absence (b) of D-glucose as an acceptor. B, MALDI-TOF mass spectrometry analysis of the transglycosylation product.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the complex intestinal microflora, bifidobacteria are considered to be key commensals that promote a healthy intestinal tract because of their many beneficial effects on the host, such as regulation of the state of the intestine, reduction of harmful bacteria and toxic compounds, immunomodulation, and anticarcinogenic activity (27–30). Bifidobacteria naturally colonize the lower intestinal tract, an environment poor in mono- and disaccharides because such sugars are preferentially consumed by the host and microbes present in the upper intestinal tract. Therefore, to survive in the lower intestinal tract, bifidobacteria produce various kinds of exo- and endoglycosidases in surface-bound and/or extracellular forms, by which they can utilize diverse carbohydrates (31, 32). Recent genome sequence analysis of B. longum NCC2705 revealed that more than 8.5% of the total predicted proteins were involved in the degradation of oligo- and polysaccharides, perhaps reflecting the superior ability of this organism to adapt to its environment (25). In particular, there are several predicted endoglycosidases, such as endo-{beta}-xylanase, endo-{beta}-N-acetylglucosaminidase, endo{alpha}-arabinosidase, and arabinogalactan endo-{beta}-galactosidases. These enzymes may be useful for the intake of extracellular carbohydrate polymers. Because mucin glycoproteins abundantly exist in the intestinal tract, it is not surprising that bifidobacteria have an endoglycosidase that acts on O-glycan.

In their habitat colon, mucin glycoprotein could be a good oligosaccharide supplier for intestinal bacteria (33), and Core 1 disaccharide is most abundant in the environment. Therefore, an enzyme degrading the Core 1 structure might give an organism an advantage to prevail in that ecosystem. Derensy-Dron et al. (34) found the presence of {beta}1,3-galactosyl-N-acetylhexosamine phosphorylase (lacto-N-biose phosphorylase, EC 2.4.1.211 [EC] ), which catalyzes the phosphorolytic cleavage of Core 1 disaccharide to produce galactose 1-phosphate and GalNAc in B. bifidum DSM 20082 (same strain as JCM 1254), which exhibited endo-{alpha}-GalNAcase activity as shown in this study. Recently, Kitaoka et al. (35) also found Lacto-N-biose phosphorylase activity and isolated its gene from B. longum JCM 1217 in which we isolated the endo-{alpha}-GalNAcase gene. Considering that these two strains were originally isolated from a human intestine, it is highly likely that the Core 1 structure-degrading pathway plays a significant role for bifidobacteria inhabiting the colon.

Analysis of O-linked sugar chains is important for biological and medicinal studies. To date, Core 1 mucin type O-glycan-specific endo-{alpha}-GalNAcase is the sole enzyme that is commercially available for the releases of O-linked sugar chains. Although a recombinant endo-{alpha}-Gal-NAcase of S. pneumoniae has been commercially released, the genetic information of this enzyme has not yet been defined. In this study, we revealed for the first time the genetic and primary structures of endo-{alpha}-GalNAcase, and also identified that the enzyme was a retaining glycoside hydrolase with strict substrate specificity for Core 1 mucin-type O-glycan. Homologues of the B. longum engBF gene were found in the genomic sequences of C. perfringens, S. pneumoniae, Streptomyces coelicolor, and E. faecalis. The former two bacteria are known to produce Core 1 mucintype O-glycan-specific endo-{alpha}-GalNAcases (10–15), and we found that S. coelicolor A3 (2) also produced an endo-{alpha}-GalNAcase.3 The properties of the recombinant enzyme, EngBF from B. longum, were essentially the same as those of the enzymes from C. perfringens and S. pneumoniae, suggesting that these homologous ORFs might encode endo-{alpha}-GalNAcase. These highly conserved homologous ORFs showed no similarity to any known glycoside hydrolase families. Considering these data, a novel glycoside hydrolase family (GH family 101) has been established.

EngBF was mostly localized in the cell wall fraction of B. longum, but the cell wall-sorting signals like LPXTG and GW repeats were not identified in the C-terminal domain of EngBF, but instead, the so-called FIVAR domains that are presumably involved in cell wall binding were present. A C-terminal FIVAR domain is also found in other possible extracellular glycosidases of B. longum such as BL0421, BL0420, BL0257, BL1543, and BL1544. Thus, it seems that the repetitive FIVAR sequences as well as the transmembrane region of EngBF are involved in tethering the enzyme to the cell wall.

In the site-directed mutagenesis study toward almost all the conserved acidic residues, we found two critical amino acids for the activity: Asp-682 and Asp-789. The catalytic reaction of glycoside hydrolase normally involves two catalytic acidic residues, catalytic nucleophile and acid/base residues. Because the sequences adjacent to Asp-682 and Asp-789 residues are highly conserved among homologues, it is likely that these residues might be essential for the catalytic reaction of EngBF. We currently attempt to determine the catalytic residues by the chemical rescue technique, which will provide valuable information about the reaction mechanism.

In addition to the hydrolytic activity, EngBF has the ability to transglycosylate Core 1 disaccharide to other compounds with hydroxyl groups. Ashida et al. (17) previously reported that bacillus endo-{alpha}-GalNAcase had a transglycosylation activity, transferring Core 1 disaccharide to various saccharides (17) and 1-alkanols (36) as acceptors. Its acceptor specificity is basically similar to that of EngBF. We attempt to enhance the transglycosylation activity by the site-directed mutagenesis technique for use as otherwise unavailable tools to prepare various kinds of neo-glycoconjugates.


   FOOTNOTES
 
* This work was supported by Grants-in-aid for Scientific Research from Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technol-ogy Agency, Scientific Research (B), No. 14360055, from the Japan Society for the Promotion of Science, and by the Ajinomoto Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence of engBF has been submitted to the GenBankTM/EBI Data Bank with accession number AY836679 [GenBank] . Back

1 To whom correspondence should be addressed: Dept. of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan. Tel.: 81-75-753-4298; Fax: 81-75-753-9228; E-mail: kfujita{at}lif.kyoto.u.ac.jp.

2 The abbreviations used are: Gal{beta}1,3GalNAc, galactosyl {beta}1,3-N-acetyl-D-galactosamine; endo-{alpha}-GalNAcase, endo-{alpha}-N-acetylgalactosaminidase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; pNP, p-nitrophenyl; HPLC, high performance liquid chromatography; ORF, open reading frame. Back

3 K. Fujita, F. Oura, N. Nagamine, T. Katayama, J. Hiratake, K. Sakata, H. Kumagai, and K. Yamamoto, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Drs. T. Murata, T. Usui, and M. Kitaoka for generously donating p-nitrophenyl substrates. We also thank Dr. T. Yamanoi, Noguchi Institute, Tokyo, Japan, for providing the MALDI-TOF MS analysis data, and Dr. B. Henrissat for valuable comments on the EngBF sequence.



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 DISCUSSION
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