Syntheses of mucin-type O-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of Bifidobacterium endo-α-N-acetylgalactosaminidase

Article abstract of DOI:10.1007/s10719-009-9247-8

Endo-α-N-acetylgalactosaminidase catalyzes the release of Galβ1-3GalNAc from the core 1-type O-glycan (Galβ1-3GalNAcα1- Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) α-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-α-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Galβ1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Galβ1-3GalNAcα1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Galβ1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-α-N- acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.

Full text of DOI:10.1007/s10719-009-9247-8

Glycoconj J (2010) 27:125–132
DOI 10.1007/s10719-009-9247-8
Syntheses of mucin-type O-glycopeptides
and oligosaccharides using transglycosylation
and reverse-hydrolysis activities of Bifidobacterium
endo-α-N-acetylgalactosaminidase
Hisashi Ashida & Hayato Ozawa & Kiyotaka Fujita &
Shun’ichi Suzuki & Kenji Yamamoto
Received: 24 April 2009 /Revised: 20 May 2009 /Accepted: 21 May 2009 /Published online: 27 June 2009
#
Springer Science + Business Media, LLC 2009
Abstract Endo-α-N-acetylgalactosaminidase catalyzes
the release of Galβ1-3GalNAc from the core 1-type
O-glycan (Galβ1-3GalNAcα1-Ser/Thr) of mucin glyco-
proteins and synthetic p-nitrophenyl (pNP) α-linked
substrates. Here, we report the enzymatic syntheses of
core 1 disaccharide-containing glycopeptides using the
transglycosylation activity of endo-α-N-acetylgalactosa-
minidase (EngBF) from Bifidobacterium longum. The
enzyme directly transferred Galβ1-3GalNAc to serine or
threonine residues of bioactive peptides such as PAMP-12,
bradykinin, peptide-Tand MUC1a when Galβ1-3GalNAcα1-
pNP was used as a donor substrate. The enzyme was also
found to catalyze the reverse-hydrolysis reaction. EngBF
synthesized the core 1 disaccharide-containing oligosacchar-
ides when the enzyme was incubated with either glucose or
lactose and Galβ1-3GalNAc prepared from porcine gastric
mucin using bifidobacterial cells expressing endo-α-N-
acetylgalactosaminidase. Synthesized oligosaccharides are
promising prebiotics for bifidobacteria.
Introduction
Glycosidases are divided into two types based on their
catalytic mechanisms: retaining and inverting glycosi-
dases. Most of the retaining glycosidases show trans-
glycosylation activity in addition to hydrolysis activity.
The transglycosylation reaction is recognized as a part of
the hydrolysis reaction in which the water molecule is
replaced with a compound containing a hydroxyl-group.
In contrast to many examples of transglycosylation by
exoglycosidases, there is a paucity of information
describing transglycosylation by endoglycosidases acting
on complex carbohydrates. Transglycosylation activities
of endoglycosidases acting on complex carbohydrates are
potentially very powerful tools for the syntheses of a
wide variety of glycosyl compounds. This is because
endoglycosidases are able to conjugate oligosaccharides
en bloc to acceptors. Moreover enzymatic remodeling of
O- and N-glycans in glycoproteins using endoglycosidases
is one of the most promising approaches in the production
of homogeneous glycoproteins [1]. We have reported that
several microbial endoglycosidases showed strong trans-
glycosylation activities and some of these enzymes could
be applicable to the syntheses of bioactive glycoconjugates
[2-7].
.
.
.
Keywords Bifidobacteria Endoglycosidase GH101
.
.
Mucin O-glycan Prebiotics
:
:
H. Ashida (*) H. Ozawa K. Yamamoto
Graduate School of Biostudies, Kyoto University,
Kyoto 606-8502, Japan
Recently, we identified the gene engBF encoding an endo-
α-N-acetylgalactosaminidase from Bifidobacterium longum
JCM1217, which belongs to a novel glycoside hydrolase
family GH101 [8]. EngBF preferably released Galβ1-
3GalNAc from the core 1-type O-glycan, also referred to
as the Thomsen-Friedenreich antigen (T-antigen), of mucin
glycoproteins. EngBF also showed transglycosylation acti-
vity of the released disaccharide to other mono- and
disaccharides.
e-mail: ashida@lif.kyoto-u.ac.jp
K. Fujita
Faculty of Agriculture, Kagoshima University,
Kagoshima 890-0065, Japan
S. Suzuki
Ajinomoto Co., Inc.,
Kawasaki, Kanagawa 210-8681, Japan

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Glycoconj J (2010) 27:125–132
In the present paper, we report syntheses of the core 1
EngBF in 50 mM sodium acetate buffer (pH 6.0) at 37°C
for an appropriate period. For transglycosylation to pep-
tides, 5 mM Galβ1-3GalNAcα1-pNP and 15 mM peptide
were incubated with 100 milliunits of EngBF in 50 mM
sodium acetate buffer (pH 6.0) under the same conditions.
For the reverse-hydrolysis reaction, 1 M glucose and
100 mM Galβ1-3GalNAc were incubated with 0.8 units
of EngBF in 50 mM sodium acetate buffer (pH 6.0) at 37°C
for an appropriate period.
disaccharide-containing glycopeptides using the transgly-
cosylation activity of EngBF. Furthermore, EngBF was
found to catalyze the reverse-hydrolysis reaction, and using
this activity we synthesized oligosaccharides without a
synthetic donor substrate. Such an approach may be useful
in the production of bifidus factors, the prebiotics for
bifidobacteria.
Materials and methods
High-performance liquid chromatography
Peptides
Normal phase high-performance liquid chromatography
(HPLC) was performed using a TSK-Gel Amide-80 column
(4.6×250 mm, Tosoh, Japan). Elution was carried out with
acetonitrile:water (3:1, by volume) as the solvent at a flow
rate of 1.0 ml/min at 40°C and detection of GlcNAc-
containing sugars and p-nitrophenol was monitored by the
absorbance at 214 nm. Reversed phase HPLC was
performed using a Cosmosil 5C18-AR-II column (4.6 ×
250 mm Nacalai Tesque, Japan). Elution was carried out
with a linear gradient of acetonitrile containing 0.1%
trifluoroacetic acid at a flow rate of 1.0 ml/min at 40°C
and detection of compounds was monitored by absorbance
at 214 or 280 nm.
PAMP-12, bradykinin and peptide-T were purchased from
the Peptide Institute Inc., Japan. MUC1a and PAMP-12
(S11T) were synthesized by Sigma Genosys (Sigma-
Aldrich Japan). The peptide sequences are shown in Table 1.
Enzyme preparation
The six histidine-tagged EngBF was expressed in E. coli
BL21(λDE3) and purified by affinity chromatography and
gel filtration as previously described [8]. The purified
recombinant EngBF showed no protease and exoglycosi-
dase activities.
Mass spectrometry
Transglycosylation and reverse-hydrolysis reaction of EngBF
For glycopeptides, matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) analy-
ses were carried out using a Bruker Daltonics Autoflex-G
system (MA, USA) with a 337-nm nitrogen laser, using the
positive-ion mode, the reflectron mode and external calibra-
tion with PEG. The sample was dissolved in ethanol at a
For transglycosylation to various monosaccharides or
amino acids as acceptors, 5 mM Galβ1-3GalNAcα1-pNP
(Toronto Research Chemicals, Canada) and 500 mM of the
acceptor substrate were incubated with 80 milliunits of
Table 1 Transglycosylation of Galβ1-3GalNAc to various peptides by EngBF
Peptide
Sequencea
Acceptor peptide
Transglycosylated peptide
RTb
Mass
RTb
Massc
Observed massd
Yielde
PAMP-12
FRKKWNKWALSR-NH2
FRKKWNKWALTR-NH2
RPPGFSPFR
54.8
55.1
58.0
31.1
31.0
1618.91
1632.93
1060.20
1111.17
857.87
52.2
1984.24
1998.26
1425.53
1476.50
1223.20
1984.68 (M+H)+
1999.08 (M+H)+
1425.79 (M+H)+
1476.57 (M+H)+
4.4
1.5
0.47
2.4
9.7
PAMP-12(S11T)
Bradykinin
MUC1a
52.5
52.8
AHGVTSAPDTR
ASTTTNYT
28.5
28.5–29.8f
Peptide T
1245.54 (M+Na)+, 1261.62 (M+K)+
a Possible residues (Ser and Thr) for acceptor are underlined
b Retention times (min) of reversed phase HPLC under the condition described in Materials and method are shown
c Theoretical masses of glycopeptides attached with single disaccharide are shown
d Observed masses in MALDI-TOF-MS analyses are shown
e Yields (%) were calculated from the following formula: yield=peak area of transglycosylation product × 100 / peak areas of acceptor and
transglycosylation product
f A single major peak and two minor peaks were observed

Glycoconj J (2010) 27:125–132
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concentration of 1 mg/ml. An α-cyano-4-hydroxycinnamic
acid solution (25 mg/ml in acetonitrile:0.1% TFA=3:1, by
volume) was used as the matrix. Half-a-microliter of the
mixture of these solutions (matrix:sample=4:1, by volume)
was placed on the AnchorChip target (Bruker, Germany).
During the measurement, 30 laser shots were used and the
data of the mass spectra were collected at different positions of
the crystallized sample spot. For oligosaccharides, MALDI-
TOF-MS analyses were carried out using a Voyager-DE STR
(Applied Biosystems). Samples were analyzed in the positive
ion mode using α-cyano-4-hydroxycinnamic acid, 2,5-
dihydroxybenzoic acid or 2’,4’,6’-trihydroxyacetophenonen-
hydorate as the matrix.
and mono- and disaccharides [8]. To apply this enzyme for
syntheses of O-glycan-containing glycopeptides, we first
examined whether EngBF could transfer the disaccharide to
a hydroxyl group of a free threonine or serine. Galβ1-
3GalNAcα1-pNP and threonine were incubated with the
recombinant EngBF and the reaction mixture was analyzed
by normal phase HPLC. The HPLC results showed a new
peak appearing at the retention time (RT) of 32.25 min.
This peak was considered to be a transglycosylation
product (Fig. 1, middle panel). A new peak with a RT of
37.09 min was detected when serine was used as an
acceptor (bottom panel). No new peaks were observed
when no acceptor was present in the reaction mixture
(upper panel). These two products were isolated and
analyzed by ESI-MS to determine their molecular masses.
On the former product, a major mass ion peak was detected
at m/z 507.75, which corresponded to the sodium adduct of
Thin layer chromatography
Thin layer chromatography (TLC) was performed using
silica-gel TLC plates (Merck 5553) with 1-butanol:acetic
acid:water=2:1:1 (by volume) as the developing solvent.
For separation of Galβ1-3GalNAc and Galβ1-3GlcNAc,
1-propanol:0.2% CaCl2=11:2 (by volume) was used. The
diphenylamine aniline reagent was used to visualize the
sugars [9].
Preparation of Galβ1-3GalNAc from porcine gastric mucin
Bifidobacterium bifidum ATCC29521 was cultured in GAM
broth (Nissui Pharmaceutical, Japan) containing 0.5%
porcine gastric mucin (Sigma-Aldrich) for 16 h at 37°C
under anaerobic conditions using Anaeropack (Mitsubishi
Chemical, Japan). For preparation of Galβ1-3GalNAc, 5 g
porcine gastric mucin was incubated with a cell suspension
of bifidobacteria in 50 mM sodium acetate buffer (pH 6.0)
for 16 h at 37°C. The supernatant was precipitated twice
with 90% ethanol to remove proteins, concentrated and
subsequently applied on to a Sephadex G-15 gel filtration
column (2×50 cm, GE Healthcare). The disaccharide-
containing fraction was concentrated and subjected to normal
phase HPLC with acetonitrile:water=4:1 (by volume) as a
solvent to separate Galβ1-3GalNAc and Galβ1-3GlcNAc.
Sugar compositions were confirmed after the treatment with
β1,3-specific galactosidase from Xanthomonas manihotis
(Calbiochem, CA).
Results
Fig. 1 Transglycosylation of Galβ1-3GalNAc to free threonine and
serine by EngBF. The donor substrate Galβ1-3GalNAcα1-pNP was
incubated with EngBF in the presence or absence of threonine or
serine and analyzed by normal phase HPLC. Upper panel, no amino
acid was added; middle panel, threonine was added as an acceptor;
lower panel, serine was added as an acceptor. New peaks appeared at
32.25 min in the middle panel and at 37.09 min in the bottom panel. S,
Galβ1-3GalNAcα1-pNP; NP, p-nitrophenol; DS, Galβ1-3GalNAc
Transglycosylation of Galβ1-3GalNAc to serine
and threonine
We previously demonstrated that EngBF could transfer the
disaccharide Galβ1-3GalNAc from a donor substrate
Galβ1-3GalNAcα1-pNP to acceptors such as 1-alkanols

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Glycoconj J (2010) 27:125–132
Galβ1-3GalNAcα1-Thr (theoretical mass, 484.20). Simi-
larly, on the latter product, a peak at m/z 493.72 was
detected for the sodium adduct of Galβ1-3GalNAcα1-Ser
(theoretical mass, 470.17). These isolated products were
completely re-hydrolyzed by EngBF, indicating that these
compounds were the transglycosylation products (data not
shown).
Enzymatic syntheses of mucin-type glycopeptides by EngBF
We subsequently examined to determine whether EngBF
could transfer the disaccharide to Ser or Thr residues in
peptides. We selected PAMP-12 (FRKKWNKWALSR) as
an acceptor, which is a potent hypotensive peptide processed
from the adrenomedullin precursor and possess one Ser
residue at the penultimate position in the sequence [10].
Incubation of PAMP-12 with EngBF in the presence of the
donor substrate Galβ1-3GalNAcα1-pNP gave rise to two
new peaks in the reversed phase HPLC chromatogram
(Fig. 2a, bottom chart). The RT of the two new peaks were
52.2 and 61.6 min. The new peak eluting at a later RT value
corresponded to p-nitrophenol released from the donor
substrate. Consequently, the peak that eluted at an earlier
RT value was assumed to represent the transglycosylation
peptide product because such a product was not detected in
the reaction mixture using an inactivated enzyme control
(Fig. 2a, top chart). We analyzed this peak by MALDI-TOF-
MS. The singly protonated mass ion peak at m/z 1984.68
corresponded to Galβ1-3GalNAc-attached PAMP-12
(Fig. 2b), indicating that it was the transglycosylation product
formed by EngBF. The product was completely re-hydrolyzed
by EngBF (data not shown), confirming that the disaccharide
from the donor substrate was transferred to the hydroxyl group
of the Ser residue in PAMP-12 via an α-linkage.
Furthermore, to examine whether EngBF could transfer the
disaccharide to a Thr residue in peptides, we used a mutant
PAMP-12 as an acceptor, in which the Ser residue was
changed to a Thr (PAMP-12(S11T)). Similarly in the case of
PAMP-12, a new peak was observed in the reversed phase
HPLC chromatogram. This peak was identified to be a
transglycosylation product by MALDI-TOF-MS analysis
(m/z 1999.08 for (M+H)+) (Fig. 2c). The amount of the
transglycosylation product was about one-third when com-
pared to the amount produced using PAMP-12 as the acceptor.
We further attempted to transfer Galβ1-3GalNAc to
various Ser/Thr-containing peptides. The results of
these analyses are summarized in Table 1. Bradykinin
(RPPGFSPFR) is a bioactive peptide inhibitor for the
angiotensin-converting enzyme, and therefore a potent
hypotensive drug [11]. This peptide has a single Ser
residue located four residues from the C-terminus. A
transglycosylation product toward bradykinin was found
by analysis using reversed phase HPLC, but the efficiency
Fig. 2 Transglycosylation of Galβ1-3GalNAc to PAMP-12 by
EngBF. a Reversed phase HPLC chromatograms of the transglycosy-
lation reaction mixtures containing Galβ1-3GalNAcα1-pNP and
PAMP-12 with heat-inactivated (upper chart) or active (bottom chart)
EngBF. S, Galβ1-3GalNAcα1-pNP; NP, p-nitrophenol; TG, possible
transglycosylation product. b MALDI-TOF-MS analysis of the
PAMP-12 transglycosylation product. The theoretical mass of
Galβ1-3GalNAc-attached PAMP-12 is 1984.24. c MALDI-TOF-MS
analysis of the PAMP-12(S11T) transglycosylation product. The
theoretical mass of Galβ1-3GalNAc-attached PAMP-12(S11T) is
1998.26
of the transglycosylation was about one-tenth of the
observed transglycosylation of PAMP-12. MUC1a
(AHGVTSAPDTR) is a part of the repetitive icosapep-
tides (AHGVTSAPDTRPAPGSTAPP) of a mucin MUC1
that is expressed by the normal mammary gland, stomach
and pancreas, and is also highly expressed by cancer cells
[12]. MUC1a contains one Ser and two Thr residues: i.e.,
three transglycosylation sites are available. However, only
a single major peak in the HPLC chromatogram was
observed and assumed to be a transglycosylation product
of MUC1a with a single Galβ1-3GalNAc. Peptide T
(ASTTTNYT) is a potent peptide for anti-HIV infection,
having one Ser and four Thr residues [13]. The analysis of
the transglycosylation products showed that a single major

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129
and two minor peaks were observed in the HPLC
chromatogram. These products were isolated and analyzed
by MALDI-TOF-MS and were found to be modified by
one disaccharide.
with active or heat-inactivated EngBF. After incubation for
3 h at 37°C, the reaction mixtures were analyzed by normal
phase HPLC. In the presence of the active enzyme, a broad
new peak was found at a RT of 28 min (Fig. 4a, top chart),
which was not observed in the chromatogram of the
reaction mixture containing heat-inactivated enzyme (data
not shown). The reaction was subsequently lengthened to
120 h. The peak of the new product reached a maximum
after 72 h incubation, and then slightly decreased until
120 h (Fig. 4a). We collected the fractions corresponding to
the new peak from the 72-h reaction mixture and analyzed
this species by TLC (Fig. 4b, lane 4). The analysis showed
that this compound had the same mobility as the authentic
Galβ1-3GalNAcα1-Glc prepared by the transglycosylation
reaction using Galβ1-3GalNAcα1-pNP and Glc (lane 3)
[8]. The product was completely hydrolyzed to Galβ1-
3GalNAc and Glc after incubating with EngBF (lane 5).
MALDI-TOF-MS analysis of this new product detected
mass ion peaks at m/z 569.18 for (M+Na)+ and m/z 585.13
for (M+K)+, confirming that the product was the trisac-
charide Galβ1-3GalNAcα1-Glc (theoretical mass, 545.49)
(Fig. 4b). The yield of the trisaccharide at 72 h incubation
based on the amount of the substrate Galβ1-3GalNAc was
estimated to be 10.7 % by the peak areas of HPLC
chromatogram. Furthermore, when 100 mM Galβ1-
3GalNAc and 0.5 M lactose were incubated with EngBF,
the peak representing the reverse-hydrolysis product was
found at a RT of 47 min using normal phase HPLC.
MALDI-TOF-MS analysis gave a mass ion peak at m/z
730.99 (M+Na)+ and the product was assumed to be
Galβ1-3GalNAcα1-lactose (theoretical mass, 707.63). The
yield at 72 h incubation was approximately 2 %.
Preparation of Galβ1-3GalNAc from porcine gastric mucin
using bifidobacterial cells
To examine the reverse-hydrolysis reaction of EngBF, we
first attempted to prepare the free disaccharide Galβ1-
3GalNAc from porcine gastric mucin using the recombinant
EngBF. However, only a slight amount of released Galβ1-
3GalNAc was observed even in the presence of sialidase and
α-N-acetylglucosaminidase [14]. This observation may be
due to the modification of the core 1-type O-glycan (Galβ1-
3GalNAcα1-) by several other sugars such as α-Fuc, β-Gal
and β-GlcNAc. We then used bifidobacterial cells to prepare
the disaccharide because bifidobacteria possess many types
of glycosidases. Among the various bifidobacteria, we found
that B. bifidum ATCC29521 could effectively release the
disaccharide from porcine gastric mucin as well as a few
monosaccharides such as Gal and sialic acid (Fig. 3). We
purified the released disaccharide and confirmed that this
disaccharide was Galβ1-3GalNAc by treatment with β1,3-
specific galactosidase from X. manihotis and MALDI-TOF-
MS (m/z 407.45 for (M+Na)+). Finally, approximately
120 mg of the pure Galβ1-3GalNAc was obtained from
5 g of porcine gastric mucin.
Reverse-hydrolysis reaction of EngBF
To assess the reverse-hydrolysis reaction of EngBF,
100 mM Galβ1-3GalNAc and 1 M glucose were incubated
Discussion
In mammalian cells, the biosyntheses of mucin-type
oligosaccharides are initiated by attachment of α-GalNAc
to Ser/Thr residues of polypeptides by polypeptide:N-
acetylgalactosaminyltransferases located on the luminal
side of the Golgi apparatus. Following this initial attach-
ment, other glycosyltransferases are used for elongation of
the sugar chains. In lower eukaryotes and prokaryotes, this
kind of protein modification is missing. Recently, geneti-
cally engineered yeast Saccharomyces cerevisiae capable of
producing core 1 O-glycan-containing peptides was devel-
oped [15]. In this strain, human polypeptide:N-acetylgalac-
tosaminyltransferase-1, Drosophila melanogaster core 1
β1,3-galactosyltransferase and several other genes were
introduced. However, this approach may be complicated
because there are twenty polypeptide:N-acetylgalactosami-
nyltransferases with different acceptor specificities to be
selected [16]. Chemical synthesis of these glycopeptides in
Fig. 3 Production of Galβ1-3GalNAc from porcine gastric mucin
using the cells of Bifidobacterium bifidum ATCC29521. The cells
grown in the GAM medium were washed with phosphate buffered
saline and incubated with porcine gastric mucin. The supernatant of
the reaction mixture was precipitated by ethanol to remove proteins
and then analyzed by TLC. Lane 1, bifidobacterial cells only; lane 2,
mucin incubated with bifidobacterial cells; lane 3, mucin only; lane 4,
standard Gal; lane 5, standard Galβ1-3GalNAc

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Glycoconj J (2010) 27:125–132
Fig. 4 Reverse-hydrolysis reac-
tion of EngBF. a Normal phase
HPLC chromatogram of the
reverse-hydrolysis reaction
product. Galβ1-3GalNAc and
glucose were incubated with
EngBF for indicated periods and
then analyzed by HPLC. A new
product (P) appeared with a RT
of 28 min. b TLC analysis of the
reverse-hydrolysis reaction
product. Lane 1, standard
Galβ1-3GalNAc; lane 2,
standard Glc; lane 3, Galβ1-
3GalNAcα1-Glc produced by
transglycosylation using EngBF;
lane 4, purified reverse-
hydrolysis reaction product of
EngBF; lane 5, purified reverse-
hydrolysis reaction product
incubated with EngBF. c
MALDI-TOF-MS analysis of
the reverse-hydrolysis reaction
product. The theoretical mass
of Galβ1-3GalNAcα1-Glc
is 545.49
vitro is not easy, because this synthesis needs a multistep
process. In this paper, we employed the transglycosylation
activity of EngBF from B. longum to achieve a one-step
synthesis of the core 1 disaccharide-containing glycopep-
tides. Transglycosylation of Galβ1-3GalNAc to a Ser-
containing hexapeptide has already been reported using a
similar enzyme, endo-α-N-acetylgalactosaminidase S par-
tially purified from the culture supernatant of Streptomyces
sp. OH-11242 [17], though transglycosylation to Thr
residues was not reported. In this study, EngBF was shown
to have the ability to transfer Galβ1-3GalNAc to both Ser
and Thr residues using the bioactive peptide PAMP-12 and
its derivative. This enzyme seems to prefer a Ser residue
rather than a Thr residue as an acceptor, though further
experiments are necessary to conclude. We also showed
that other three bioactive peptides could be acceptors for
this enzyme. Therefore, a simple method to synthesize core
1 O-glycan-containing peptides using EngBF with broad
acceptor specificity has been presented. The addition of
sugar chain(s) to bioactive peptides may furnish some
positive effects, such as increasing resistance to proteases,
increasing stability and extending the half-life in the
bloodstream. In the case of the mucin repetitive peptide
MUC1a, sugar-conjugate could be a promising anti-tumor
vaccine for blocking metastasis [18]. Enzymatic trans-
glycosylation require donor substrates such as synthetic
substrates which are usually expensive. Recently, an
efficient chemical synthesis of α-GalNAc glycosides using
the di-tert-butylsylene-directed method was developed [19-
21] and this method is expected to lower the cost of
production of the donor substrate. Although this enzymatic
approach present in this study provides a simple and
powerful tool for syntheses of O-glycopeptides, it is
currently not easy to control the number and position of
the disaccharide modification on the peptides with multiple
Ser/Thr residues. Incomplete glycosylation may be due to
the low efficiency of the transglycosylation by EngBF. The
improvement of the transglycosylation efficiency of EngBF

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by mutagenesis and/or the use of higher amount of the
donor substrate may overcome the weakness of this
approach.
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We also achieved the syntheses of oligosaccharides by the
reverse-hydrolysis reaction of EngBF, which does not require
donor substrates such as Galβ1-3GalNAcα1-pNP. Since the
synthesized oligosaccharides are degraded by endo-α-N-
acetylgalactosaminidases that are widely distributed in the
Bifidobacterium species, these oligosaccharides could be
assimilated by those bacteria and may function as a bifidus
factor. We have previously described that bifidobacteria are
able to incorporate the Galβ1-3GalNAc into the cells
through an ABC-type transporter specific for Galβ1-
3GalNAc and Galβ1-3GlcNAc [22, 23]. The disaccharide
is phosphorolyzed by intracellular Galβ1-3GalNAc/Galβ1-
3GlcNAc phosphorylase to further degradation [24-26].
Since the β1,3-linked Gal is hardly hydrolyzed by entero-
bacterial β-galactosidases, Galβ1-3GalNAc may be specif-
ically utilized by bifidobacteria.
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Acknowledgments We thank Dr. H. Ishida and Dr. M. Kiso (Gifu
University), Dr. M. Yamaguchi (Wakayama University) and Dr. T.
Yamanoi (the Noguchi Institute) for MS analyses. This work was
supported in part by grant-in-aids from the Promotion of Basic
Research Activities for Innovative Biosciences (PROBRAIN) and
from Ajinomoto Co., Inc.
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