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:
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.
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