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 accept
Trang 1activity of endo-b-mannosidase from Lilium longiflorum Akiko Sasaki1, Takeshi Ishimizu1, Rudolf Geyer2 and Sumihiro Hase1
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 E1 (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 (n¼ 0–2) and pyridylamino GlcNAcb1-4GlcNAc, the enzyme transferred (Man)nMana1-6Man en bloc to the acceptor substrate to pro-duce pyridylamino (Man)nMana1-6Manb1-4GlcNAcb1-4GlcNAc (n¼ 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.
Trang 2and 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 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 (n¼ 0–2) and
Manb1-4Glc-NAcb1-4GlcNAc-R (R¼ 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 37C 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
Trang 3pNP 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 37C 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-A265
Retention Time (min)
20 10
0
C
B
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 m M 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
Elution time (min)
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 m M 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.
Trang 4NAcb1-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
Retention Time (min)
37.55
21.72
36.05
A
B
m / z
m / z
116
158
233 173
117
159
233 173
74
75
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.
Trang 5(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)nMana1-6Man from (Man)n Mana1-6Manb1-4GlcNAcb1-4GlcNAc-peptide (n¼ 0–2) to GN2-PA or pNP b-GlcNAc (Figs 1 and 2C; Table 2) This finding is relevant because (Man)nMana1-6Man 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
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 m M )
M2B-peptide
(90 m M )
GN2-PA (140 m M )
M3B-peptide
(90 m M )
GN2-PA (140 m M )
M3C-peptide
(100 m M )
GN2-PA (140 m M )
M4B-peptide
(60 m M )
GN2-PA (140 m M )
M5A-peptide
(70 m M )
GN2-PA (140 m M )
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 (%)
Trang 6The 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
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 M2B-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)
A265
20 10
0
Retention time (min)
A265
20 Retention time (min)
A265
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).
Trang 7M3C-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
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
used were Man (90 mm), Mana1-6Man (90 mm),
M1-pep-tide (80 or 90 mm), M2B-pepM1-pep-tide (80 or 90 mm),
M3B-peptide (90 mm) or a mixture of glycoM3B-peptides 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) or pNP
b-Man (140 mm) The reaction was stopped by heating
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
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
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
b-man-nosidase A product (200 pmol) was digested with 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 m M M1-peptide as a donor substrate and a 140 m M pNP sugar as an acceptor substrate Peak number in Figs 2 and 4 are listed.
Acceptor
Yield based
on donor (%)
Yield based
on acceptor (%)
Trang 8analyzed 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
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
gradient in which the temperature was increased from 50 to
acetates were identified with a mass spectrometer (Finnigan
Polaris Q) at an ionization potential of 70 eV A mass
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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