Báo cáo khoa học: Synthesis of b-mannosides using the transglycosylation activity of endo-b-mannosidase from Lilium longiflorum pptx

Synthesis of b-mannosides using the transglycosylation activity of endo-b-mannosidase from Lilium longiflorum Akiko Sasaki 1 , Takeshi Ishimizu 1 , Rudolf Geyer 2 and Sumihiro Hase 1 1 Department of Chemistry, Graduate School of Science, Osaka University, Japan 2 Institute of Biochemistry, Faculty of Medicine, University of Giessen, Germany Oligosaccharides containing the Manb structure are found in several natural sources including the core structure of N-linked sugar chains [Mana1-3(Mana1- 6)Manb1-4GlcNAcb1-4GlcNAc] [1], tetrasaccharide in Hyriopsis schlegelii glycosphingolipid (GlcNAcb1- 2Manb1-3Manb1-4Glcb1-Cer) [2], in arthro-series glycosphingolipids (GalNAcb1-4GlcNAcb1-3Manb1- 4Glcb1-Cer) of insects [3] and nematodes [4] and the bacterial O-antigen repeating unit of the Salmonella serogroup E 1 (Manb1-4Rhaa1-3Gal) [5]. As of the het- erogeneous nature of these sugar chains and ⁄ or their low abundance in natural sources, it is crucial to establish a method for construction of oligosaccharides containing the Manb structure for studies on their structure and function. However the Manb structure, one of the 1,2-cis-glycosides, is difficult to synthesize chemically because the vicinal 2-OH group blocks access to the b-face, because of its steric and polar effects [6]. Many attempts have been made to improve the enantio-selectivity on the chemical synthesis of the Manb structure by using silver silicate catalysis [7], oxidation ⁄ reduction at the C2 center of the glucoside [8,9] and configuration inversion at the C2 of the gly- coside [10]. These methods include multiple protection Keywords b-mannoside; endo-b-mannosidase; enzymatic synthesis; N-glycan; transglycosylation Correspondence Sumihiro Hase, Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyamacho, Toyonaka, Osaka 560-0043, Japan Fax: +81 6 6850 5383 Tel: +81 6 6850 5380 E-mail: suhase@chem.sci.osaka-u.ac.jp Note The structures and abbreviations for the sugar chains are listed in Table 1 (Received 8 December 2004, revised 23 January 2005, accepted 31 January 2005) doi:10.1111/j.1742-4658.2005.04587.x Endo-b-mannosidase is an endoglycosidase that hydrolyzes only the Manb1-4GlcNAc linkage of the core region of N-linked sugar chains. Recently, endo-b-mannosidase was purified to homogeneity from Lilium longiflorum (Lily) flowers, its corresponding gene was cloned and import- ant catalytic amino acid residues were identified [Ishimizu T., Sasaki A., Okutani S., Maeda M., Yamagishi M. & Hase S. (2004) J. Biol. Chem. 279, 38555–38562]. In the presence of Manb1-4GlcNAcb1-4GlcNAc-pep- tides as a donor substrate and p-nitrophenyl b-N-acetylglucosaminide as an acceptor substrate, the enzyme transferred mannose to the acceptor sub- strate by a b1-4-linkage regio-specifically and stereo-specifically to give Manb1-4GlcNAcb1-pNP as a transfer product. Further studies indicated that not only p-nitrophenyl b-N-acetylglucosaminide but also p-nitrophenyl b-glucoside and p-nitrophenyl b-mannoside worked as acceptor substrates, however, p-nitrophenyl b-N-acetylgalactosaminide did not work, indicating that the configuration of the hydroxyl group at the C4 position of an acceptor is important. Besides mannose, oligomannoses were also trans- ferred. In the presence of (Man) n Mana1-6Manb1-4GlcNAcb1-4GlcNAc- peptides (n ¼ 0–2) and pyridylamino GlcNAcb1-4GlcNAc, the enzyme transferred (Man) n Mana1-6Man en bloc to the acceptor substrate to pro- duce pyridylamino (Man) n Mana1-6Manb1-4GlcNAcb1-4GlcNAc (n ¼ 0–2). Thus, the lily endo-b-mannosidase is useful for the enzymatic prepar- ation of oligosaccharides containing the mannosyl b1,4-structure, chemical preparations of which have been frequently reported to be difficult. Abbreviations Cer, ceramide; GalNAc, N-acetyl- D-galactosamine; Glc, D-glucose; GlcNAc, N-acetyl-D-glucosamine; Man, D-mannose; PA, pyridylamino; pNP, p-nitrophenyl; Rha, D-rhamnose. 1660 FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS and activation steps and laborious work preparing glycosyl donors and acceptors for coupling, and their products are usually protected forms. Enzymatic methods of constructing the Manb struc- ture using b-mannosidases or b-mannosyltransferases are attractive alternatives to be applied to the synthesis of oligosaccharides containing the Manb structure. Using transglycosylation activity of guinea-pig b-man- nosidase, transfer of Man to p-chlorophenyl and p-nitrophenyl (pNP) b-mannosides by a b-linkage has been accomplished to produce the corresponding aryl b-mannobiosides with 2.4 and 4.7% yield, respectively, although stereo-selectivity has not been achieved [11]. The core trisaccharide of the N-linked sugar chain, Manb1-4GlcNAcb1-4GlcNAc, has been synthesized using the transglycosylation activity of b-mannosidases from either Aspergillus oryzae, with 26% yield based on donor [12], Helix pomatia, with 3% yield [13], and b-mannanase from Aspergillus niger, with 3.7% yield [14]. A b-mannosidase mutated at the active site nucleophile glutamic acid residue catalyzes the synthesis of b-mannosides in good yield (74–99%), although ste- reo-selectivity of b-mannosylation was not achieved [15]. b-Mannosyltransferases were also used to synthe- size Manb1-4GlcNAcb1-4GlcNAc [16] and Manb1- 4Rhaa1-3Gal [17,18], but preparation of phytanyl- or 4-(nitrophenyl)-1-butyl-linked acceptor substrates is laborious. Endo-b-mannosidase found in plant tissues was puri- fied to homogeneity from Lilium longiflorum (Lily) flowers [19,20]. This endoglycosidase hydrolyzes the Manb1-4GlcNAc linkage in (Man) n Mana1-6Manb1- 4GlcNAcb1-4GlcNAc-R (n ¼ 0–2) and Manb1-4Glc- NAcb1-4GlcNAc-R (R ¼ H, pyridylamino group, or peptide) [20,21]. If endo-b-mannosidase can be used for glycosidase-catalyzed synthesis of Manb structures, oligomannose and ⁄ or Man are expected to be building blocks for the formation of Manb structures. In this study, we show that this enzyme had transglycosylation activity. Regio- and stereo-selectivity, oligomannose or Man transfer, and wide specificity for the transglycosy- lation acceptor substrates of this enzyme are described. Results Transglycosylation activity of endo- b-mannosidase Transglycosylation and a reverse hydrolysis reaction of endo-b-mannosidase were investigated. Incubation of purified lily endo-b-mannosidase with 90 mm Man (or Mana1-6Man) and 140 mm GN2-PA (the structures and abbreviations for sugar chains are listed in Table 1), which are the hydrolysis products of M1-PA (or M2B-PA) with this enzyme, did not generate any reaction product, meaning that the reverse hydrolysis reaction of this enzyme does not occur despite the high concentrations of donor and acceptor substrates. Incubation of the enzyme with 90 mm M2B-peptide, which is the best substrate tested to date for lily endo- b-mannosidase [21], and 140 mm GN2-PA generated substantial amounts of a fluorogenic product (peak 1 in Fig. 1). The elution times for this product on reversed-phase and size-fractionation HPLC were iden- tical to those for M2B-PA. MALDI-TOF MS of this product was dominated by a signal at m ⁄ z 826.6 (cal- culated m ⁄ z for M2B-PA: 826.8). a-Mannosidase and subsequent b-mannosidase digestion of this product gave products corresponding to M1-PA and GN2-PA, respectively, by two-dimensional sugar mapping. These results indicated that this product was M2B-PA and that lily endo-b-mannosidase had transglycosylation activity to transfer Mana1-6Man from the M2B-pep- tide to GN2-PA with b-linkage. Regio- and stereo-selectivity of transglycosylation of endo-b-mannosidase When endo-b-mannosidase was incubated with 80 mm M1-peptide as a donor substrate and 140 mm pNP b-GlcNAc as an acceptor substrate at 37 °C for 10 h, two new products were generated (peaks 2 and 3 in Fig. 2B). Digestion of the major product (peak 3) by Achatina fulica b-mannosidase gave pNP b-GlcNAc, showing that the enzyme transferred a Man residue to Table 1. Abbreviation of sugar chains used in this study. Abbreviation Structure GN2 M1 M2B M3B M3C M4B M5A A. Sasaki et al. Synthesis of b-mannosides using endo-b-mannosidase FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS 1661 pNP b-GlcNAc by forming a new b-linkage. Methyla- tion analysis of this product was conducted and the partially methylated alditol acetates obtained were ana- lyzed by GLC ⁄ MS (Fig. 3A). The peak at 21.72 min in Fig. 3A was identified as 1,5-di-O-acetyl-2,3,4,6-tetra- O-methyl mannitol from its electron impact ionization EI-MS (data not shown). The 37.55 min in Fig. 3) was identical to that of 2-deoxy-2-(N-methyl)acetamido- 1,4,5-tri-O-acetyl-3,6-di-O-methyl glucitol. This assign- ment was confirmed by corresponding EI-MS obtained after reduction with sodium borohydride (Fig. 3B) or sodium borodeuteride (Fig. 3C). In addition to the major fragment ions at m ⁄ z 116 (117) and 158 (159), found in the EI-MS spectra of all partially methylated HexNAc derivatives, diagnostically relevant signals were registered at m ⁄ z 173 and 233, which clearly iden- tified this compound as a 3,6-di-O-methyl HexNAc derivative [28]. The presence of 4,6 (or 3,4)-di-O- methyl derivatives, reflecting 3 (or 6)-substituted Hex- NAc residues, could be excluded due to the lack of corresponding primary fragment ions at m ⁄ z 161 and 274 (or 189), respectively. Hence, the results suggest that the analyzed product comprised a Man1-4GlcNAc unit. These observations allow us to conclude that lily endo-b-mannosidase is able to transfer a Man residue to pNP b-GlcNAc in a regio- and stereo-selective man- ner, thus generating Manb1-4GlcNAcb1-pNP. A minor product (peak 2 in Fig. 2B) was also di- gested with Achatina fulica b-mannosidase. Partial digestion of peak 2 gave peak 3 (Manb1-4GlcNAc- pNP) in addition to pNP b-GlcNAc (data not shown), showing that peak 2 is Manb-(Manb1-4GlcNAc-pNP). When endo-b-mannosidase was incubated with 80 mm M2B-peptide as a donor substrate and 140 mm pNP b-GlcNAc as an acceptor substrate at 37 °C for 10 h, a new product was generated (peak 4 in Fig. 2C). a-Mannosidase digestion of peak 4 resulted in a prod- uct with a retention time identical to that of peak 3 (Manb1-4GlcNAcb1-pNP), and subsequent b-mannosi- dase digestion gave pNP b-GlcNAc. These observations indicated that peak 4 was Mana1-6Manb1-4Glc- A 265 Retention Time (min) 20 10 0 C B 2 3 4 A Fig. 2. Transglycosylation of endo-b -mannosidase incubated with 140 m M pNP b-GlcNAc as an acceptor substrate. Transglycosylation was conducted with 80 m M M1-peptide (B) or 80 mM M2B-peptide (C) as donor substrates at 37 °C for 10 h. The reactants were ana- lyzed by reversed-phase HPLC as described in Experimental proce- dures. (A) Elution profile of pNP b-GlcNAc. Product peaks 2, 3 and 4 were collected. 30 0 15 Elution time (min) Fluorescence a b 1 Fig. 1. Transglycosylation activity of endo-b-mannosidase. The puri- fied lily endo-b-mannosidase (40 mU) was incubated with 90 m M M2B-peptide and 140 mM GN2-PA at 37 °C for 0 h (A) and 10 h (B). The reactants were analyzed by size-fractionation HPLC as des- cribed in Experimental procedures. The transglycosylation product (peak 1) was collected and analyzed (see text). Arrows show the elution positions of the expected endo-b-mannosidase transglycosy- lation products. Synthesis of b-mannosides using endo-b-mannosidase A. Sasaki et al. 1662 FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS NAcb1-pNP. Obviously, the enzyme transferred Mana1-6Man from M2B-peptide to pNP b-GlcNAc and formed a new b-linkage. Transfer of oligomannose from different glyco- peptides to GN2-PA using the transglycosylation activity of endo-b-mannosidase Endo-b-mannosidase transferred Mana1-6Man from M2B-peptide to GN2-PA with concomitant formation of a new b-linkage, hence producing M2B-PA via its transglycosylation activity (Fig. 1). This enzyme also transferred a Man residue from M1-peptide to GN2-PA to give M1-PA (Table 2). The product was identified by two-dimensional sugar mapping in combi- nation with exoglycosidase digestion (data not shown). Interestingly, M3B-peptide did not act as a donor sub- strate for endo-b-mannosidase-mediated transglyco- sylation (Table 2). When a mixture of glycopeptides containing M2B-, M3C-, M4B- and M5A-peptides as potential donor substrates and GN2-PA as an acceptor substrate were incubated with the enzyme, M2B-, M3C- and M4B-peptides worked as donor sub- strates for transglycosylation and led to the corres- ponding PA derivatives (Table 2). These structures were also identified by two-dimensional sugar mapping (data not shown). In contrast, the M5A-peptide did not work as a donor substrate. Thus, the donor sub- strate specificity of endo-b-mannosidase for transglyco- sylation corresponded to that for hydrolysis as this enzyme does not hydrolyze sugar chains containing the Mana1-3Manb structure (Table 2) [19–21]. Reaction yields for the transglycosylation activity of endo- b-mannosidase against these glycopeptides were relat- ively higher than those for the transglycosylation of other b-mannosidases or b-mannanase (Table 2) [11– 14]. When M2B-peptide was used as a donor substrate, transglycosylation yield was up to 67% based on the donor concentration. Table 3 shows the donor concen- tration dependency of transglycosylation when M2B- peptide and GN2-PA were used as the donor and acceptor substrate, respectively. Even though the con- centration of M2B-peptide was changed, transglycosy- lation yields were not much altered. Transfer of Man residue from M1-peptide to various pNP monosaccharides using the trans- glycosylation activity of endo-b-mannosidase The acceptor substrate specificity of transglycosylation by endo-b-mannosidase was investigated. M1-peptide was used as a donor substrate and pNP b-GlcNAc, pNP b-GalNAc, pNP b-Glc and pNP b-Man were used as acceptor substrates. In the presence of 140 mm M1-peptide and 80 mm of each pNP monosaccharide, transmannosylation by endo-b-mannosidase occurred as shown in Fig. 4 and Table 4. pNP b-GlcNAc 20 30 40 Retention Time (min) 37.55 21.72 36.05 Total ion intensity A B m / z 100 300200 m / z 100 300200 116 158 233 173 117 159 233 173 74 75 Ion intensity Ion intensity C Fig. 3. Methylation analysis of Manb1-4GlcNAcb1-pNP. (A) Partially methylated alditol acetates obtained from the product eluting as peak 3 in Fig. 2B were analyzed by GLC ⁄ MS. 1,5-Di-O-acetyl- 2,3,4,6-tetra-O-methyl mannitol (21.72 min) and 2-deoxy-2-(N-met- hyl)acetamido-1,4,5-tri-O-acetyl-3,6-di-O-methyl glucitol (37.55 min) were registered. The peak at 36.05 min is due to contamination by glucitol hexaacetate. (B) and (C) show EI-MS of partially methylated HexNAc derivatives (peak at 37.55 min) after reduction with sodium borohydride and sodium borodeuteride, respectively. A. Sasaki et al. Synthesis of b-mannosides using endo-b-mannosidase FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS 1663 (Fig. 2B), pNP b-Glc (Fig. 4B) and pNP b-Man (Fig. 4C) worked as acceptor substrates in endo- b-mannoside-catalyzed transglycosylation, whereas pNP b-GalNAc (Fig. 4A) did not. b-Mannosylation of these pNP derivatives was confirmed by Achatina fulica b-mannosidase digestion of the products. Among the four pNP monosaccharides applied, only pNP b-Gal- NAc has an axial hydroxyl group at the C4 position. This means that transglycosylation by endo-b-manno- sidase requires an equatorial hydroxyl group at the C4 position of an acceptor substrate. In contrast, the type of substituent and its orientation at C2 are different among pNP b-GlcNAc, pNP b-Glc and pNP b-Man. This indicates that transglycosylation by this enzyme does not require strictly defined structural features at the C2 position of an acceptor substrate, although the substituent at C2 may partly influence the yield of transglycosylation products (Table 4). Successive transfer of two Man residues was observed when pNP b-GlcNAc and pNP b-Glc were used as acceptor substrates (Figs 2B and 4B and Table 4). It was not observed when pNP b-Man was used as an acceptor substrate under the same conditions. Discussion Lily endo-b-mannosidase has been shown to possess transglycosylation activity to produce b1-4-linkages in a regio- and stereo-selective way. One of the unique characteristics of the transglycosylation activity of this enzyme is the transfer of (Man) n Mana1-6Man from (Man) n Mana1-6Manb1-4GlcNAcb1-4GlcNAc- peptide (n ¼ 0–2) to GN2-PA or pNP b-GlcNAc (Figs 1 and 2C; Table 2). This finding is relevant because (Man) n Mana1-6Man is a useful building block for the synthesis of N-linked sugar chains. The trans- glycosylation features of this enzyme reflect its sub- strate specificity for hydrolysis [20,21]. The fact that endo-b-mannosidase did not transfer oligosaccharides containing Mana1-3Manb (Table 2), for example, par- allels its substrate specificity for hydrolysis, because endo-b-mannosidase does not hydrolyze oligosaccha- rides containing Mana1-3Manb [20,21]. The other unique characteristic of this transglycosy- lation is the transfer of the Man residue to various acceptor substrates including GN2-PA, pNP b-Glc- NAc, pNP b-Glc and pNP b-Man (Figs 2B and 4, Tables 2 and 4). Because the nonreducing end residues of all of these compounds have an equatorial hydroxyl group at their C4 position, monosaccharides with sim- ilar features, such as xylose and rhamnose, might also work as acceptor substrates. Thus, the enzyme may be used to synthesize various glycoconjugates containing different b-mannosyl linkages. Table 2. Transglycosylation activity of endo-b-mannosidase incubated with glycopeptides and GN2-PA. Reaction yields for transglycosylation with endo-b-mannosidase are shown. Relative hydrolysis rate of endo-b-mannosidase against PA-derivatives of donor substrate are also shown. Donor substrate Acceptor substrate Product Yield based on donor (%) Yield based on acceptor (%) Relative hydrolysis rate against PA- derivatives of donor substrate M1-peptide (90 m M) GN2-PA (140 mM) M1-PA 23 15 4 M2B-peptide (90 m M) GN2-PA (140 mM) M2B-PA 67 43 100 M3B-peptide (90 m M) GN2-PA (140 mM) –0 0 0 M3C-peptide (100 m M) GN2-PA (140 mM) M3C-PA 19 13 48 M4B-peptide (60 m M) GN2-PA (140 mM) M4B-PA 11 5 42 M5A-peptide (70 m M) GN2-PA (140 mM) –0 0 0 Table 3. Donor substrate (M2B-peptide) concentration dependency on transglycosylation of endo-b-mannosidase. Transglycosylation was conducted in the presence of 140 m M GN2-PA as an acceptor substrate. Concentration of M2B-peptide used as a donor substrate (m M) Yield based on donor (%) Yield based on acceptor (%) 10 67 5 50 64 23 90 67 43 Synthesis of b-mannosides using endo-b-mannosidase A. Sasaki et al. 1664 FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS The donor substrate for transmannosylation used in this study is M1-peptide. This glycopeptide can be easily prepared from glycoprotein on a synthetic scale by complete a-mannosidase digestion of glycopeptides derived from pronase digests. Acceptor substrates and products in this study were PA-oligosaccharides. PA-oligosaccharides can be converted to the corres- ponding oligosaccharide as reported previously [29]. In addition, an expression system of the Arabidopsis endo-b-mannosidase in Escherichia coli has been constructed [20]. Therefore, large quantities of endo- b-mannosidase are available for the large-scale synthe- sis of b1-4 mannosides by transglycosylation. Experimental procedures Materials pNP b-GlcNAc, pNP b-GalNAc, pNP b-Glc, pNP b-Man and Mana1-6Man were purchased from Sigma (St. Louis, MO, USA). Jack bean a-mannosidase and Achatina fulica b-mannosidase were purchased from Seikagaku Kogyo (Tokyo, Japan). A Shodex Asahipak NH2-P column (0.46 · 25 cm) was obtained from Showa Denko (Tokyo, Japan), and we used an Inertsil ODS column (0.46 · 25 cm) from GL Science (Tokyo, Japan). Endo-b-mannosidase was purified from lily flowers as described previously [20]. M3B-peptides were prepared by pronase digestion of ovomucoid from Japanese quail egg white and then purified by Sephadex G-25 gel chromatogra- phy [21]. Analysis of this sample, using MALDI-TOF MS and pyridylamination of the carbohydrate portion, showed it to consist of a mixture of glycopeptides of 4–6 amino acid residues with M3B structure (data not shown). M2B- and M1-peptides were prepared by partial and complete digestion, respectively, of M3B-peptides with jack bean a-mannosidase [21]. The amount of glycopeptide was quan- tified by analyzing PA- derivatives of the hydrazynolysate of glycopeptides. M5A-peptide (Val-Ser-Asn) was prepared by exhaustive thermolysin digestion of Taka-amylase A. A mixture of glycopeptides containing M2B-, M3C-, M4B- and M5A-peptides was prepared by partial digestion of M5A-peptide with jack bean a-mannosidase [22]. Prepar- ation of standard GN2-PA, M1-PA, M2B-PA, M3B-PA, 20 10 0 Retention time (min) A 265 20 10 0 Retention time (min) A 265 20 Retention time (min) A 265 A B C a b a b a b 7 5 6 10 0 Fig. 4. Acceptor substrate specificity of endo-b-mannosidase trans- glycosylation incubated with 80 m M M1-peptide as a donor sub- strate. Transglycosylation was conducted in the presence of 140 m M pNP b-GalNAc (A), pNP b-Glc (B) and pNP b-Man (C). pNP monosaccharides (a) and reactants (b) were analyzed by reversed- phase HPLC. Transglycosylation products (peaks 5, 6 and 7) were collected and submitted for structural analysis (see text). A. Sasaki et al. Synthesis of b-mannosides using endo-b-mannosidase FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS 1665 M3C-PA, M4B-PA and M5A-PA has been reported previ- ously [23]. Transglycosylation activity of endo- b-mannosidase A donor substrate and an acceptor substrate in 50 mm sodium phosphate buffer (pH 6.0) were incubated with the purified endo-b-mannosidase (40 mU) at 37 °C for 10 h. One unit of enzyme activity was defined as the amount of enzyme that released 1 nmol of GN2-PA from 12.5 lm of M2B-PA per minute in 0.16 m ammonium acetate buffer, pH 5.0 at 37 °C. The donor substrates used were Man (90 mm), Mana1-6Man (90 mm), M1-pep- tide (80 or 90 mm), M2B-peptide (80 or 90 mm), M3B- peptide (90 mm) or a mixture of glycopeptides. The glycopeptide mixture obtained by partial digestion of M5A-peptide with a-mannosidase consisted of M2B-, M3C-, M4B- and M5A-peptides at concentrations of 10, 100, 60 and 70 mm, respectively. The acceptor substrates used were GN2-PA (140 mm), pNP b-GlcNAc (140 mm), pNP b-GalNAc (140 mm), pNP b-Glc (140 mm)orpNP b-Man (140 mm). The reaction was stopped by heating at 100 °C for 3 min. The reactant was diluted and the enzyme was removed by filtering through a Microcon YM-10 membrane (Millipore, Billerica, MA, USA). The resultant PA-sugar chains were analyzed by size-fraction- ation HPLC. HPLC A Hitachi L-6200 pump equipped with a Hitachi F-1050 fluorescence spectrophotometer was used. Size-fractionation HPLC was performed on a Shodex Asahipak NH2-P col- umn at a flow rate of 0.8 mLÆmin )1 at 25 °C. Two eluents used were 0.3% (v ⁄ v) acetic acid in acetonitrile ⁄ water (93 : 7, v ⁄ v) (Eluent A) and 0.3% (v ⁄ v) acetic acid in aceto- nitrile ⁄ water (20 : 80, v ⁄ v) (Eluent B) adjusted to pH 7.0 with aqueous ammonia. The column was equilibrated with 5% Eluent B. After injecting a sample, the proportion of Eluent B was increased linearly to 80% in 35 min. PA derivatives were detected by their fluorescence using an excitation and an emission wavelength at 310 and 380 nm, respectively. The resultant pNP derivatives were analyzed by reverse- phase HPLC performed on an Inertsil ODS column at a flow rate of 1.5 mLÆmin )1 at 25 °C. A mixture of 50 mm ammonium acetate, pH 5.0 and acetonitrile (87 : 13, v ⁄ v) was used as the eluent [24]. A Hitachi L-6200 pump equipped with a Hitachi L-4200 UV-VIS spectrophotometer was used. pNP derivatives were detected by their absorb- ance at 265 nm. Two-dimensional sugar mapping The structures of the PA-oligosaccharides were analyzed by two-dimensional sugar mapping. The elution positions of more than 100 standard PA-N-linked sugar chains have already been reported, and the introduction of a reverse-phase scale made it possible to predict the elution positions even if standard PA-N-linked sugar chains were not available [23]. PA-oligosaccharides were separated by reverse-phase HPLC and size-fractionation HPLC, and the elution position of each oligosaccharide was compared with those of standard PA-oligosaccharides on the two-dimen- sional sugar mapping. Each PA-oligosaccharide was then digested with exoglycosidases, and the structures of the products were analyzed on the two-dimensional sugar map- ping as reported previously [23,25]. Exoglycosidase digestion and MALDI-TOF MS of transglycosylation products Transglycosylation products purified by HPLC were digested with mannosidases, a-mannosidase and ⁄ or b-man- nosidase. A product (200 pmol) was digested with the a-mannosidase (10 mUÆlL )1 )in50mm ammonium acetate buffer, pH 4.5, at 37 °C for 10 h. The product was further digested with the b-mannosidase (0.3 mUÆlL )1 )in50mm ammonium acetate buffer, pH 4.5, at 37 °C for 4 h. The glycosidase digests were analyzed using two-dimensional sugar mapping as described above. For MALDI-TOF MS, transglycosylation products were cocrystallized in a matrix of 2,5-dihydroxybenzoic acid and Table 4. Transglycosylation of endo-b-mannosidase incubated with 80 mM M1-peptide as a donor substrate and a 140 mM pNP sugar as an acceptor substrate. Peak number in Figs 2 and 4 are listed. Acceptor substrate Peak no. Product Yield based on donor (%) Yield based on acceptor (%) pNP b-GlcNAc 3 Manb1–4GlcNAcb1-pNP 21 12 2Manb-Manb-GlcNAcb1-pNP 11 6 pNP b-GalNAc 00 pNP b-Glc 6 Manb-Glcb1-pNP 40 23 5Manb-Manb-Glcb1-pNP 14 8 pNP b-Man 7 Manb-Manb1-pNP 17 10 Synthesis of b-mannosides using endo-b-mannosidase A. Sasaki et al. 1666 FEBS Journal 272 (2005) 1660–1668 ª 2005 FEBS analyzed with a Voyager-DE RP Biospectrometry Work- station (PerSeptive Biosystems, Framingham, MA, USA), employing delayed extraction technology and operated in the reflector mode. Methylation analysis The transglycosylation product was permethylated as out- lined elsewhere [26], and the permethylated sample was purified on a Sephadex LH-20 column [27]. The product was hydrolyzed in 4 m trifluoroacetic acid at 100 °C for 4 h. Released methylated sugar derivatives were reduced with sodium borohydride or sodium borodeuteride prior to peracetylation. Resulting partially O-methylated alditol ace- tates were separated by GLC using a FactorFour (VF 5 ms; Varian, Darmstadt, Germany) capillary column (0.25 mm · 60 m, film thickness 0.1 lm) and a temperature gradient in which the temperature was increased from 50 to 130 °Cby40°C per min, from 130 to 210 °Cby2°C per minute and from 210 to 280 °Cby6°C per minute. Alditol acetates were identified with a mass spectrometer (Finnigan Polaris Q) at an ionization potential of 70 eV. A mass range of m ⁄ z 40–400 was scanned within 0.5 s. Acknowledgements The expert technical assistance of W. Mink and P. Kaese is gratefully acknowledged. This work was supported in part by the 21st century COE program (Creation of Integrated Ecochemistry), Protein 3000 program, and the Japan Health Science Foundation. References 1 Lee YC & Scocca JR (1972) A common structural unit in asparagines–oligosaccharides of several glycoproteins from different sources. 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