Fucosylation of chitooligosaccharides by human α1,6-fucosyltransferase requires a nonreducing terminal chitotriose unit as a minimal structure

Article abstract of DOI:10.1093/glycob/cwq064

FUT8, a eukaryotic α1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-β-L-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally

Full text of DOI:10.1093/glycob/cwq064

Glycobiology vol. 20 no. 8 pp. 1021–1033, 2010
doi:10.1093/glycob/cwq064
Advance Access publication on April 20, 2010
Fucosylation of chitooligosaccharides by human
α1,6-fucosyltransferase requires a nonreducing
terminal chitotriose unit as a minimal structure
Hideyuki Ihara2, Shinya Hanashima3, Takahiro Okada2,6
,
could be due to structural factor requirements which are
inherent in FUT8.
Ritsu Ito2, Yoshiki Yamaguchi3, Naoyuki Taniguchi4,5
,
and Yoshitaka Ikeda1,2,6
2Division of Molecular Cell Biology, Department of Biomolecular Sciences,
Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan,
3Structural Glycobiology Team, Systems Glycobiology Research Group,
Chemical Biology Department, RIKEN Advanced Science Institute,
2-1 Hirosawa Wako, Saitama 351-0198, Japan, 4Department of Disease
Glycomics, The Institute of Scientific and Industrial Research, Osaka
University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan, 5Disease
Glycomics Team, Systems Glycobiology Research Group, Chemical Biology
Department, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan, and 6Core Research for Evolutionary Science and
Technology, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi, Saitama 332-0012, Japan
Keywords: chitooligosaccharide/fucosylation/
fucosyltransferase/FUT8/NodZ
Introduction
FUT8, a eukaryotic α1,6-fucosyltransferase, catalyzes the
transfer of a fucosyl residue from guanine nucleotide diphos-
phate (GDP)-β-L-fucose to the reducing terminal GlcNAc of
asparagine-linked oligosaccharides (N-glycan) (Wilson et al.
1976) (Figure 1). This fucose moiety is referred to as a core
fucose residue, and is widely distributed in nature, from insects
to animals (Oriol et al. 1999; Staudacher et al. 1999). As pre-
viously reported, it has been suggested that various biological
events are regulated via the core fucosylation of various glyco-
proteins (Miyoshi et al. 1999; Taniguchi et al. 2006). The
enzymatic properties of FUT8 have been extensively investi-
gated, especially in terms of substrate specificities and the
reaction mechanism (Longmore and Schachter 1982; Voynow
et al. 1991; Shao et al. 1994; Kaminska et al. 1998; Staudacher
and Marz 1998; Paschinger et al. 2005; Ihara et al. 2006).
In the fucose transfer reaction, FUT8 requires the presence
of a β1,2-GlcNAc residue linked to the α1,3-mannose arm
in the tri-mannose core structure of an N-glycan (Longmore
and Schachter 1982; Voynow et al. 1991; Shao et al. 1994;
Kaminska et al. 1998; Paschinger et al. 2005) and acts on
only the β-anomer of the reducing terminal GlcNAc which
is linked to the asparagine residue in the N-glycosylation site
(Voynow et al. 1991). On the other hand, some modifications
of the oligosaccharides function as inhibitory factors for
FUT8 reactions: the galactosylation of the β1,2-GlcNAc res-
idue linked to the α1,3-mannose arm (Voynow et al. 1991),
addition of the bisecting GlcNAc that is catalyzed by
β1,4-N-acetylglucosaminylytransferase (GnT-III) (Longmore
and Schachter 1982) and the α1,3-fucosylation of the innermost
GlcNAc residue, as observed in insects (Staudacher and Marz
1998; Paschinger et al. 2005). Concerning the substrate speci-
ficity for the donor, our previous study indicated that FUT8
strongly recognizes both the base portion and the diphosphoryl
group of the donor nucleotide sugar (Ihara et al. 2006).
Received on March 11, 2010; revised on April 5, 2010; accepted on
April 15, 2010
FUT8, a eukaryotic α1,6-fucosyltransferase, catalyzes
the transfer of a fucosyl residue from guanine nucleotide
diphosphate-β-L-fucose to the innermost GlcNAc of an
asparagine-linked oligosaccharide (N-glycan). The cata-
lytic domain of FUT8 is structurally similar to that of
NodZ, a bacterial α1,6-fucosyltransferase, which acts
on a chitooligosaccharide in the synthesis of Nod factor.
While the substrate specificities for the nucleotide sugar
and the N-glycan have been determined, it is not known
whether FUT8 is able to fucosylate other sugar chains
such as chitooligosaccharides. The present study was
conducted to investigate the action of FUT8 on chitooli-
gosaccharides that are not generally thought to be a
substrate in mammals, and the results indicate that
FUT8 is able to fucosylate such structures in a manner
comparable to NodZ. Surprisingly, structural analyses of
the fucosylated products by high performance liquid
chromatography, mass spectrometry and nuclear mag-
netic resonance indicated that FUT8 does not utilize
the reducing terminal GlcNAc for fucose transfer but
shows a preference for the third GlcNAc residue from
the nonreducing terminus of the acceptor. These findings
suggest that FUT8 catalyzes the fucosylation of chitooligo-
saccharide analogous to NodZ, but that a nonreducing
terminal chitotriose structure is required for the reaction.
The substrate recognition by which FUT8 selects the posi-
tion to fucosylate might be distinct from that for NodZ and
We recently reported on the crystal structure of human FUT8,
and the results showed that the structure of FUT8 includes a
catalytic domain and two additional domains, an N-terminal
coiled-coil structure and a C-terminal SH3 domain (Ihara et
1To whom correspondence should be addressed: Tel: +81-952-34-2190; Fax:
+81-952-34-2189; e-mail: yikeda@med.saga-u.ac.jp
© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org 1021

H Ihara et al.
A FUT8 reaction
+
-
( )
( )
GN
GlcNAcβ1-2Manα1
6
+
( )
Asn
Manβ1-4GlcNAcβ1-4GlcNAcβ-
GN2
-
( )
3
GlcNAcβ1-2Manα1
+
( )
GN3
-
( )
GDP-fucose
+
( )
FUT8
GN4
-
( )
GDP
+
-
( )
( )
GN5
Fucα1
GlcNAcβ1-2Manα1
+
( )
6
6
GN6
-
( )
Asn
Manβ1-4GlcNAcβ1-4GlcNAcβ-
3
10
15
20
25
30
5
GlcNAcβ1-2Manα1
Time (min)
B NodZ reaction
Fig. 3. Fucosylation of chitooligosaccharide by FUT8. FUT8 reactions with
1 mM of various chitooligosaccharides were carried out with (+) or without (−)
GDP-β-L-fucose as donor substrate. GN, GN2, GN3, GN4, GN5 and GN6
denote N-acetylglucosamine, chitobiose, chitotriose, chitotetraose,
GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAc
chitopentaose and chitohexaose, respectively. The reaction products were
analyzed by normal phase HPLC. Elution was monitored by a UV detector at
210 nm. The detailed procedures are described under Materials and methods.
GDP-fucose
NodZ
GDP
Fucα1
al. 2007). In addition, it was found that the enzyme can be struc-
turally classified as members of the GT-B group of
glycosyltransferases (Coutinho et al. 2003; Qasba et al.
2005). The structure of bacterial α1,6-fucosyltransferase, re-
ferred to as NodZ, has also been solved (Brzezinski et al.
2007), and a structural comparison between FUT8 and NodZ
showed that the catalytic domains of these two enzymes are
very similar. NodZ plays a role in the synthesis of the Nod fac-
tor, which is involved in the nodulation of legumes root for
nitrogen-fixing, and is known to catalyze the α1,6-fucosylation
of lipo-chitooligosaccharide and variations thereof, including
chitooligosaccharides (Quesada-Vincens et al. 1997). Both the
mammalian and bacterial fucosyltransferases are classified into
6
GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAc
Fig. 1. Reactions catalyzed by FUT8 (A) and NodZ (B). FUT8 transfers a
fucose residue from GDP-β-L-fucose to the innermost GlcNAc of N-glycan via
an α1,6-linkage. NodZ also transfers fucose to chitooligosaccharide via a
reaction similar to FUT8. Dashed boxes denote the common structure of
products synthesized by enzymes.
standard
70
60
50
40
30
20
10
0
+
( )
pNP-GN
-
( )
+
( )
pNP-GN2
-
( )
+
( )
pNP-GN3
pNP-GN4
pNP-GN5
-
( )
+
( )
-
( )
+
( )
-
( )
0
2
4
6
8
10
12
Time (min)
Fig. 2. Fucosylation of pNP-labeled chitooligosaccharide by FUT8. FUT8
reactions were carried out with various pNP-labeled chitooligosaccharides in the
presence (+) or absence (−) of GDP-β-L-fucose as donor substrate. pNP-GN2,
pNP-GN3, pNP-GN4 and pNP-GN5 represent pNP-chitobiose, pNP-chitotriose,
pNP-chitotetraose and pNP-chitopentaose, respectively. Nonfucosylated and
fucosylated chitooligosaccharides were separated by normal phase HPLC as
described under Materials and methods. The labeled oligosaccharides were
detected by UV absorbance at 300 nm for the pNP group. The profile for STD
indicates the elution of pNP-GN2, pNP-GN3, pNP-GN4 and pNP-GN5 as a
standard sample.
1
2
3
4
5
6
7
Degree of polymerization
Fig. 4. Correlation between the activity and degree of polymerization of
chitooligosaccharide in the fucosylation by FUT8. The closed circles indicate
nonlabeled chitooligosaccharides series. The open circles indicate pNP-labeled
chitooligosaccharides series. The closed square represents 4MU-labeled
chitotriose.
1022

Fucosylation of chitooligosaccharides by FUT8
the GT23 family of Carbohydrate-Active enZYmes (Campbell
et al. 1997) and share GDP-β-L-fucose as the donor substrate
(Figure 1). Although the acceptor substrates are different, a
“common” chitobiose unit is contained in the reducing term-
inals of both substrates. Thus, in addition to the structural
similarities, it is reasonable to expect that these mammalian
and bacterial enzymes are functionally similar in their other en-
zymatic properties. Furthermore, a comparison of the primary
structures of FUT8, NodZ, α1,2- and protein O-fucosyltrans-
ferases indicates that all contain three small, highly conserved
regions (Breton et al. 1998; Oriol et al. 1999; Takahashi et al.
2000; Chazalet et al. 2001; Martinez-Duncker et al. 2003), and
the spatial arrangement of these regions is essentially the same
in the steric structures of FUT8 and NodZ (Brzezinski et al.
2007; Ihara et al. 2007).
acceptor substrate for FUT8. However, it was reported that
NodZ can utilize N-glycan as an acceptor substrate and actual-
ly fucosylate it to a lesser extent, compared to FUT8 (Quinto et
al. 1997). This observation suggests that the bacterial enzyme
displays a broad specificity toward the acceptor, and it appears
that the enzyme does not strictly recognize the substrate. On
the other hand, an earlier study showed that chitobiose and
chitotriose do not serve as active substrates for FUT8, and it
seemed likely that FUT8 acts exclusively on N-glycans
(Voynow et al. 1991). However, because chitooligosacchar-
ides that are sufficiently active substrates for NodZ have
not been examined for fucosylation by FUT8, it is not
known whether the specificity of FUT8 toward the acceptor
is broad or not, as reported for NodZ. In this study, we ex-
amined and characterized the reactions of FUT8 with various
chitooligosaccharides as acceptor substrates. As for the re-
sults, the fucosylation of the chitooligosaccharide acceptors
Chitooligosaccharides are precursors of the Nod factor and
serve as acceptor substrates for NodZ, while an N-glycan is an
GN5F
GN3F
1
2
3
1
2
3
4
5
6
7
4
5
6
7
5
10
15
20
25
30
35
5
15 25 35 45 55 65 75
Time (min)
Time (min)
GN6F
GN4F
1
2
3
1
2
3
4
5
6
7
4
5
6
7
5
15
25
35
45
55
5
15 25 35 45 55 65 75 85 95
Time (min)
Time (min)
Fig. 5. Structural analyses of fucosylated chitooligosaccharides obtained by glycosidase digestion. The fucosylated chitooligosaccharides by FUT8 were
digested by β-N-acetylhexosaminidase and α-fucosidase, and analyzed by normal phase HPLC analysis. The detailed procedures are described in Materials
and methods. Numbers for chromatographs in each panel represent 1, nonfucosylated chitooligosaccharide substrate; 2, β-N-acetylhexosaminidase
digestion of the nonfucosylated chitooligosaccharide; 3, fucosylated chitooligosaccharide product; 4, fucosidase digestion of the fucosylated
chitooligosaccharide; 5, β-N-acetylhexosaminidase digestion of the fucosylated chitooligosaccharide; 6, β-N-acetylhexosaminidase and fucosidase digestion
of the fucosylated chitooligosaccharide; 7, standard oligosaccharides.
1023

H Ihara et al.
GN5F
+
[M+Na]
300
600
900
m/z
1200
1500
Hexosaminidase digest of GN5F
+
[M+Na]
300
600
900
m/z
1200
1500
Fig. 6. MALDI-TOF MS spectra of GN5F and the β-N-acetylhexosaminidase digest. GN5F; calculated as 1202.46 [C46H77N5O30+-Na]+-, found 1202.5. The
β-N-acetylhexosaminidase digest; calculated as GN3F 796.30 [C30H51N2O20+-Na]+-, found 796.3.
was observed, and it was found that FUT8 exhibits a
“NodZ-like” fucosylating activity. However, detailed analyses
showed certain difference in the nature by which the accep-
tors are fucosylated. Furthermore, our findings would also
provide new insights into how FUT8 fucosylates oligosac-
charide substrates at a specific position.
Table 1. 1H- and 13C-chemical shifts of GN5F
1H, 13C chemical shifts (ppm)
Residue
Fucose
1
2
3
4
5
6
102.5
4.87
72.3
3.88
71.0
3.77
74.7
3.79
69.7
4.04
18.2
1.22
GlcNAc (A)
GlcNAc (B)
GlcNAc (C)
GlcNAc (D)
GlcNAc (E)
93.2 (α), 97.6 (β)
5.18 (α), 4.68 (β)
56.4 (α), 58.9 (β)
3.87 (α), 3.68 (β)
72.0 (α), 74.9 (β)
3.87 (α), 3.70 (β)
82.3
3.62
72.6 (α), 77.4 (β)
3.87 (α), 3.53 (β)
62.8
3.65, 3.83
104.3
4.57
57.9
3.77
74.9
3.70
82.3
3.62
77.4
3.53
62.8
3.65, 3.83
104.3
4.57
57.9
3.77
76.1
3.69
81.2
3.77
77.4
3.53
69.7
3.69, 3.91
104.3
4.57
57.9
3.77
74.9
3.70
82.3
3.62
77.4
3.53
62.8
3.65, 3.83
104.3
4.57
57.9
3.77
72.5
3.47
76.2
3.56
78.7
3.48
63.3
3.74, 3.91
Chemical shifts (ppm) of 1H- and 13C-NMR of GN5F were performed in D2O at 25°C, and the chemical shifts were calibrated with DSS (0 ppm). The structure and
residue notation of oligosaccharide analyzed are described as GlcNAc(E)β1-4GlcNAc(D)β1-4 (Fucα1-6)GlcNAc(C)β1-4GlcNAc(B)β1-4GlcNAc(A).
1024

Fucosylation of chitooligosaccharides by FUT8
Table 2. 1H- and 13C-chemical shifts of GN5
1H, 13C chemical shifts (ppm)
Residue
1
2
3
4
5
6
GlcNAc (A)
93.3 (α), 97.6 (β)
5.18 (α), 4.69 (β)
56.5 (α), 59.0 (β)
3.87 (α), 3.68 (β)
72.0 (α), 74.9 (β)
3.87 (α), 3.71 (β)
81.8
3.64
72.9 (α), 77.4 (β)
3.87 (α), 3.54 (β)
62.8
3.65, 3.84
GlcNAc (B)
GlcNAc (C)
GlcNAc (D)
GlcNAc (E)
104.1
4.57
57.9
3.76
74.9
3.71
81.8
3.64
77.4
3.54
62.8
3.65, 3.84
104.1
4.57
57.9
3.76
74.9
3.71
81.8
3.64
77.4
3.54
62.8
3.65, 3.84
104.1
4.57
57.9
3.76
74.9
3.71
81.8
3.64
77.4
3.54
62.8
3.65, 3.84
104.1
4.57
57.9
3.76
72.5
3.47
76.3
3.56
78.7
3.48
63.4
3.74, 3.91
Chemical shifts (ppm) of 1H- and 13C-NMR of GN5 were performed in D2O at 25°C, and the chemical shifts were calibrated with DSS (0 ppm). The structure and
residue notation of oligosaccharide analyzed are described as GlcNAc(E)β1-4GlcNAc(D)β1-4GlcNAc(C)β1-4GlcNAc(B)β1-4GlcNAc(A).
Table 3. 1H- and 13C-chemical shifts of β-N-acetylhexosaminidase digest of GN5F
1H, 13C chemical shifts (ppm)
Residue
Fucose
1
2
3
4
5
6
102.5
4.90
72.2
3.87
71.2
3.74
74.6
3.79
69.7
4.08
18.1
1.22
GlcNAc (A)
93.2 (α), 97.7 (β)
5.18 (α), 4.68 (β)
56.5 (α), 58.8 (β)
3.87 (α), 3.68 (β)
72.2 (α), 75.2 (β)
3.87 (α), 3.69 (β)
82.4
3.62
72.2 (α), 76.2 (β)
3.87 (α), 3.57 (β)
62.8
3.65, 3.77 (α)
3.65, 3.82 (β)
GlcNAc (B)
GlcNAc (C)
103.9
4.60
58.1
3.78
75.2
3.69
82.4
3.62
77.0
3.59
62.8
3.65, 3.82
104.6
4.58
58.1
3.78
72.7
3.54
77.6
3.66
77.4
3.50
70.2
3.75, 4.00
Chemical shifts (ppm) of 1H- and 13C-NMR of β-N-acetylhexosaminidase digest of GN5F were performed in D2O at 25°C, and the chemical shifts were calibrated
with DSS (0 ppm). The structure and residue notation of oligosaccharide analyzed are described as Fucα1-6GlcNAc(C)β1-4GlcNAc(B)β1-4GlcNAc(A).
Table 4. 1H- and 13C-chemical shifts of GN3
1H, 13C chemical shifts (ppm)
Residue
1
2
3
4
5
6
GlcNAc (A)
93.2 (α), 97.7 (β)
5.18 (α), 4.69 (β)
56.4 (α), 58.9 (β)
3.87 (α), 3.68 (β)
72.0 (α), 75.1 (β)
3.87 (α), 3.69 (β)
82.0
3.64
72.9 (α), 77.3 (β)
3.87 (α), 3.78 (β)
62.8
3.66, 3.78 (α)
3.66, 3.84 (β)
62.8
GlcNAc (B)
GlcNAc (C)
104.1
4.58
58.0
3.77
75.0
3.72
82.0
3.64
77.3
3.56
3.66, 3.84
104.1
4.58
58.4
3.74
72.6
3.48
76.3
3.56
78.7
3.49
63.3
3.75, 3.91
Chemical shifts (ppm) of 1H- and 13C-NMR of β-N-acetylhexosaminidase digest of GN3 were performed in D2O at 25°C, and the chemical shifts were calibrated
with DSS (0 ppm). The structure and residue notation of oligosaccharide analyzed are described as GlcNAc(C)β1-4GlcNAc(B)β1-4GlcNAc(A).
1025

H Ihara et al.
Results
matography (HPLC) and electrophoresis. However, these
methods for labeling of N-glycans cannot be used for the assay
of FUT8 because the enzyme requires that the innermost
GlcNAc exists in a closed ring form and cannot act on an
opened form. In fact, most of the oligosaccharides used for
the FUT8 assay are an asparagine-linked form in which the
carboxyl or the amino group of the aglycon is modified by a
chromophore or fluorophore (Uozumi, Teshima et al. 1996;
Roitinger et al. 1998; Martinez-Duncker et al. 2004; Ihara et
al. 2006). Thus, O-p-nitrophenyl (pNP) and O-4-methylumbel-
In general, when the enzyme activities of glycosyltransferases
that act on N-glycans are assayed, oligosaccharides labeled via
reductive amination are often used for sensitive detection. For
example, it is well known that 2-aminopyridine (Hase et al.
1981), 8-aminonaphthalene-1,3,6-trisulfonic acid (Klockow et
al. 1995) and a hydrazide-derivative of naphthalene (Muramo-
to et al. 1994; Leteux et al. 1998) are very useful for
quantitative analyses involving high performance liquid chro-
B,C,D,E1
NAc
A
Fuc H1
(3JH-H = 3.6 Hz)
A1(α)
A1(β)
Fuc H6
5.2
5.1
5.0
4.9
4.7
4.6
4.5
2.5
HOD
Fuc H5
5.5
5.0
4.5
4.0
3.5
3.0
2.0
1.5
1.0
1H (ppm)
B,C,D,E1
B
NAc
A1(α)
A1(β)
5.2
5.1
5.0
4.9
4.0
4.7
4.6
4.5
HOD
5.5
5.0
4.5
3.5
3.0
2.5
2.0
1.5
1.0
1H (ppm)
Fig. 7. NMR analyses of GN5F. 1H-NMR spectra of GN5F and GN5 are shown in (A) and (B), respectively. The spectra were measured using a 600 MHz
spectrometer and the chemical shifts were calibrated using the DSS singlet adjusted at 0.00 ppm. H1, H5 and H6 signals assigned to Fuc residue are indicated in the
3
spectrum (A). The anomeric configuration of the fucose residue was confirmed as the α-form because the value of the JH1–H2 coupling constant (3.6 Hz) is the
1
typical value for a gauche configuration. The anomeric area of the H-13C HSQC spectra of GN5F and GN5 is shown in (C) and (D), respectively. The anomeric
1H-13C correlation signal of Fuc residue is found in (C). The nonanomeric area of GN5F and GN5 is indicated in (E) and (F), respectively. HSQC signals of
Fuc 2-, 3-, 4- and 5-positions are found in (E). The detailed procedures are described in Materials and methods. The structure and residue notations for the
oligosaccharides analyzed are shown in Tables 1 and 2.
1026

Fucosylation of chitooligosaccharides by FUT8
C
90
D
90
A1(α)
A1(α)
95
100
105
110
95
100
105
110
A1(β)
A1(β)
Fuc1
B1,C1,
D1,E1
B1,C1,
D1,E1
5.4
5.2
5.0
4.8
4.6
5.4
5.2
5.0
4.8
4.6
1H (ppm)
1H (ppm)
E
50
F
50
55
60
65
70
75
80
85
55
60
65
70
75
80
85
A2(α)
B2, C2,
D2, E2
A2(α)
B2, C2,
D2, E2
A2(β)
A2(β)
A6a, B6a,
C6a, D6a
A6a, B6a,
D6a
A6b,
B6b,
D6b
A6b, B6b,
C6b, D6b
E6a
E6b
E6a
E6b
Fuc5
C6b
C6a
Fuc3
A3(α)
A5(α)
A3(α)
E3
E3
A3(β), B3,
C3, D3
Fuc2
A3(β),
B3, D3
E4
A5(α)
A5(β), B5,
C5, D5
E4
Fuc4
A5(β), B5,
C5, D5
C3
E5
E5
C4
A4, B4, C4, D4
A4, B4, D4
4.0
3.8
1H (ppm)
3.6
3.4
4.0
3.8
1H (ppm)
3.6
3.4
Fig. 7. (continued).
liferyl (4MU) forms of various chitooligosaccharides, all of
which are commercially available, were examined for use in
the assay for the fucosylation by FUT8.
The labeled chitooligosaccharides were incubated with puri-
fied recombinant FUT8 in the presence of GDP-β-L-fucose,
and the products of the reaction were analyzed by normal
phase HPLC using an ultraviolet (UV) or fluorescent detector.
As shown in Figure 2, shifts in the peaks were observed, ex-
cept for derivatives of GlcNAc and chitobiose, and the shifted
peaks were not produced in the absence of the donor. No peak
shift was found when the enzyme was omitted from the reac-
tion (data not shown). Because fucose is a deoxy sugar, an
oligosaccharide containing a fucose residue is not retained as
much as a GlcNAc in normal phase HPLC. Therefore, peaks
corresponding to fucosylated chitooligosaccharides wedged
between those of GlcNAc polymers as the substrates in the elu-
tion profiles. These properties allowed separation of the
substrates and products. The results indicate that FUT8 is
1027

H Ihara et al.
clearly able to catalyze the fucosylation of chitooligosacchar-
ides that contain three or more GlcNAc, in addition to N-
glycans. As shown by the HPLC analyses in Figure 2, only
a single shifted peak was observed in the elution profiles of
the chitooligosaccharides that were fucosylated by FUT8,
which suggests that the oligosaccharides are mono-fucosylated
regardless of the degree of polymerization of the substrate.
Although the findings show that FUT8 is able to glycosylate
pNP or other derivatives of chitooligosaccharides as well as N-
glycans, the actual substrates for NodZ contain a free reducing
terminal. Thus, while labeling of the oligosaccharides with a
chromophore or fluorophore is useful for sensitive detection,
but the possibility that such an aglycon might play a role in as-
sisting the enzymatic reaction cannot be excluded. Hence, in
order to more appropriately evaluate the activity of FUT8 on
chitooligosaccharides, unlabeled substrates were examined for
the fucosylation by FUT8. In these substrates, the reducing
group is present as both α and β anomers, which are in equilib-
rium and this could complicate the assay as compared to the use
of a labeled substrate.
As shown in Figure 3, when FUT8 was incubated with
GDP-β-L-fucose and the chitooligosaccharides as acceptors,
peaks corresponding to fucosylated oligosaccharides were
found in the cases of chitotriose (GN3), chitotetraose (GN4),
chitopentaose (GN5) and chitohexaose (GN6). In these analy-
ses, using the normal phase mode, each sugar chain, including
the fucosylated one, was successfully detected as a combination
of two separate peaks that are based on anomeric configuration,
A
NAc
Fuc H1
(3JH-H = 3.6 Hz)
B, C1
A1(α)
A1(β)
Fuc H6
5.2
5.1
5.0
4.9
4.7
4.6
4.5
2.5
Fuc H5
HOD
5.5
5.0
4.5
4.0
3.5
3.0
2.0
1.5
1.0
1H (ppm)
A1(β)
4.7
B
B, C1
NAc
A1(α)
5.2
5.1
5.0
4.9
4.0
4.6
4.5
2.5
HOD
5.5
5.0
4.5
3.5
3.0
2.0
1.5
1.0
1H (ppm)
Fig. 8. NMR analysis of the β-N-acetylhexosaminidase digest of GN5F. 1H-NMR spectra of the β-N-acetylhexosaminidase digest (A) are shown as well as a
standard sugar, GN3, (B) to compare. The H1, H5 and H6 signals assigned to Fuc residue are indicated in the spectrum (A). The 3JH1–H2 coupling constant of the
Fuc residue was 3.6 Hz, which was identical to the corresponding value of fucose in GN5F. The anomeric area of the 1H-13C HSQC spectra is shown in (C) and (D),
respectively. The nonanomeric area of the digest and GN3 is indicated in (E) and (F), respectively. The HSQC signals assigned to Fuc residue are shown in (C) and
(E). The detailed procedures are described in Materials and methods. The structure and residue notations of oligosaccharide analyzed are shown in Tables 3 and 4.
1028

Fucosylation of chitooligosaccharides by FUT8
C
90
D
90
A1(α)
A1(α)
95
100
105
110
95
100
105
110
A1(β)
A1(β)
Fuc1
B1
C1
B1,C1
5.4
5.2
5.0
1H (ppm)
4.8
4.6
5.4
5.2
5.0
1H (ppm)
4.8
4.6
E
50
F
50
55
60
65
70
75
80
85
55
60
65
70
75
80
85
A2(α)
A2(α)
B2, C2
C2
B2
A2(β)
A2(β)
A6a(β),
B6a
A6a(α)
A6a(β),
B6a
A6b, B6b
A6b, B6b
C6a
A6a(α)
C6b
Fuc5
C6b
C6a
A3(α)
A3(α)
A5(α)
Fuc3
C3
C3
C5
A5(α)
Fuc2
A3(β), B3
A5(β)
B3
A3(β)
C4
Fuc4
A5(β), B5
B5
C5
C4
A4, B4
A4, B4
4.2
4.0
3.8
1H (ppm)
3.6
3.4
4.2
4.0
3.8
1H (ppm)
3.6
3.4
Fig. 8. (continued).
while the pNP and 4MU-labeled substrates were eluted as
single peaks due to the fact that β-linkage at the reducing
end is fixed. Thus, the analytical system used allowed the
separation of the α- and β-anomer forms of chitooligosac-
charides, as reported previously (Abdel-Banat et al. 2002;
Kabir et al. 2006). The fucosylated products also showed on-
ly a pair of peaks that were based on two anomers as
observed for the substrates, indicating that the substrates that
underwent mono-fucosylation on the FUT8-conducted reac-
tions. On the other hand, no product was detected when
GlcNAc (GN) and chitobiose (GN2) were used as the acceptor
(Figure 3). These results are essentially the same as those
obtained for the labeled oligosaccharides and also suggest
that the aglycon does not interfere with the reaction of
FUT8 with chitooligosaccharides.
In order to evaluate the substrate preference of FUT8, the
activities were assayed using various chitooligosaccharides or
related substrates. No activity was observed in the cases of
GlcNAc and chitobiose, either labeled or unlabeled (Figures
2 and 3). The specific activities for nonlabeled GN3, GN4,
1029

H Ihara et al.
GN5 and GN6 were 25, 40, 55 and 62 nmol/h/mg protein, re-
spectively. The specific activities for pNP-GN3, pNP-GN4,
pNP-GN5 and 4MU-GN3 were also 33, 60, 65 and 31 nmol/
h/mg protein, respectively. These values are in the range of
about 5–6% of the activity of FUT8 for N-glycans. As shown
in Figure 4, a longer oligosaccharide is more active as the sub-
strate, at least, in the range of the chain length examined. In
addition, the activity appeared to reach a plateau in both the
nonlabeled and pNP-labeled GNs, suggesting that an oligosac-
charide with a length of 6–7 residues is sufficient for maximal
activity. While the activity for nonlabeled GN3 was slightly
lower than for the labeled samples, no significant difference
was found between 4MU and pNP derivatives, suggesting that
the β-anomeric configuration of the reducing terminal is im-
portant rather than the presence of an aglycon moiety. As
shown by a comparison of the activities for nonlabeled and
pNP-labeled acceptors, it appears that FUT8 prefers a fixed
anomeric configuration to a free reducing terminal that is in
anomeric equilibrium.
Previous reports showed that NodZ transfers a fucosyl resi-
due to the reducing terminal GlcNAc of a chitooligosaccharide
(Sanjuan et al. 1992; Bec-Ferté et al. 1994). In order to deter-
mine which GlcNAc residue in the chitooligosaccharide is
fucosylated by FUT8, the reaction products were analyzed
by exo-glycosidase digestions. The digestions were carried
out using β-N-acetylhexosaminidase from jack bean and α-fu-
cosidase from bovine kidney, and the digested samples were
applied to normal phase HPLC analysis (Figure 5). When the
samples were treated with fucosidase, the peaks in the elution
profiles were shifted to the positions corresponding to the re-
spective substrates that were used for the FUT8 reaction, as
indicated by the fact that they were identical to nonfucosylated
standards. These results confirm the α-fucosylation of GNs by
FUT8, as expected from the aforementioned normal phase
HPLC analyses. In the fucosidase digestion, while fucosylated
chitotriose (GN3F) and tetraose (GN4F) were readily digested
to nonfucosylated forms by α-fucosidase, fucosylated chito-
pentaose (GN5F) and hexaose (GN6F) were only slightly
digested under the same conditions as were used for GN3F
and GN4F, suggesting their fucosidase-resistant characteristics.
β-N-Acetylhexosaminidase treatment liberated free GlcNAc
molecules and oligosaccharides shortened by two GlcNAc re-
sidues for all of the fucosylated chitooligosaccharides, whereas
the nonfucosylated substrates were completely digested to free
GlcNAc. It was most likely that the residual components that
were resistant to further digestion by β-N-acetylhexosamini-
dase contain a fucose residue at the nonreducing terminal but
not at the internal residue because the β-N-acetylhexosamini-
dase used in this study is an exo-type glycosidase and is
unable to hydrolyze the substituted GlcNAc at the nonreduc-
ing terminal, for example, it is known that galactosyl GlcNAc
inhibits the action of this enzyme (Li and Li 1970). In fact,
treatment with a combination of β-N-acetylhexosaminidase
and α-fucosidase led to complete degradation to monosaccha-
ride components. This finding suggests that the third GlcNAc
from the nonreducing terminal is fucosylated by FUT8 in chit-
ooligosaccharides. In the case of GN6F, however, fucosylated
chitotriose was observed as a minor component, along with to
the major fucosylated chitotetraose, when digested by β-N-
acetylhexosaminidase.
A
Fucα1
GlcNAcβ1-2Manα1
6
6
Asn
Manβ1-4GlcNAcβ1-4GlcNAcβ-
3
GlcNAcβ1-2Manα1
B
Fucα1
6
GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4[GlcNAc]0-3
C
Fucα1
6
[GlcNAc]0-4 β1-4GlcNAcβ1-4GlcNAc
Fig. 9. Proposed structural requirement for an oligosaccharide substrate for
fucosylation by FUT8. Structures of fucosylated forms of N-glycan and
chitooligosaccharide are shown for FUT8 (A and B) and NodZ (C). Dashed
boxes are the putative essential structures for the reaction of FUT8.
In order to further verify the fucosylation pattern obtained by
glycosidase digestion and normal phase HPLC, nuclear magnet-
ic resonance (NMR) and mass spectrometry (MS) experiments
were performed. As shown in Figure 5, GN5F was prepared by
the reaction of GN5 with FUT8 and purified by normal phase
HPLC for NMR and MS analyses, and product obtained by β-
N-acetylhexosaminidase digestion was also prepared and exam-
ined. Consistent with the chromatographic data (Figure 5), the
MS analysis clearly showed that the difference in mass between
these sugar chains is equivalent to two molecules of HexNAc
(Figure 6), indicating that the β-N-acetylhexosaminidase treat-
ment removed two GlcNAc residues from GN5F. The anomeric
configuration and the position of the fucose residue in GN5F
were determined by NMR. As summarized in Tables 1–4, the
1H and 13C-signals of GN5F and its β-N-acetylhexosaminidase
digest as well as two unfucosylated sugars, GN5 and GN3, were
assigned on the basis of 1D 1H-NMR, 2D 1H-1H COSY (corre-
lation spectroscopy), 2D H-13C HSQC (hetero-nuclear single
quantum coherence) and 2D HSQC-TOCSY (total correlation
spectroscopy) spectra. In the H-NMR spectra of GN5F and
1
1
the digest (Figures 7A and 8A), the observed 3JH1–H2 coupling
constant of the anomeric proton of fucose was 3.6 Hz, indicating
that the fucose is attached to the chitooligosaccharides via an α-
linkage. In the 1H-13C HSQC spectrum of GN5F, the 13C-chem-
ical shift of the C-6 for one GlcNAc residue was 69.7 ppm, while
either 62.8 or 63.3 ppm for the other residues (Figure 7E, Table
1). On the other hand, in the spectrum of the unfucosylated form,
GN5, all of the C-6 signals of GlcNAc were observed at either
62.8 or 63.4 ppm (Figure 7F, Table 2), but no signal was found at
69.7 ppm. The difference in the chemical shifts is consistent
with the view that the fucose is attached to the C-6 position of
a GlcNAc residue, although it was not possible to specify which
GlcNAc residue is fucosylated, due to overlapping signals. In a
comparison of the 1H-13C HSQC spectra of the β-N-acetylhex-
osaminidase-digested product and GN3, however, a similar
difference was observed for the nonreducing terminal residues,
1030

Fucosylation of chitooligosaccharides by FUT8
denoted by “GlcNAc (C)” (70.2 ppm vs 63.3 ppm, Figure 8E
and F, Tables 3 and 4), thus indicating that the fucose residue
is attached to the nonreducing terminal GlcNAc residue in the
β-N-acetylhexosaminidase-digest of GN5F. This indicates that
the fucose is present at the third GlcNAc residue, as indicated by
the chromatographic analyses involving glycosidase digestion.
These structural analyses support the conclusion that FUT8 fu-
cosylates chitooligosaccharides via an α1,6-linkage at the third
GlcNAc residue from the nonreducing terminal (Figure 9B).
dated, it is possible that the domain functions to fit the substrate
suitably into the active site to fucosylate the third GlcNAc of the
nonreducing terminal. For example, the domain may recognize
the terminal GlcNAc and then allow the third residue to be po-
sitioned in close proximity to the catalytic center. As shown by
fucosylation of GN6 in Figure 5, however, other GlcNAc posi-
tions can be fucosylated to a much lesser extent in the case of a
sufficiently long chitooligosaccharide, hexaose or probably lon-
ger. Thus, the reducing terminal region consisting of
trisaccharide or more could also contribute slightly to selecting
the position to fucosylate. As demonstrated by the present study
of the fucosylation of chitooligosaccharides, the nonreducing
terminal trisaccharide, but not the reducing terminal saccharide,
is of critical importance for FUT8 to fucosylate the acceptor.
This view is consistent with the inability of FUT8 to fucosylate
chitobiose (Quesada-Vincens et al. 1997; Quinto et al. 1997;
Chazalet et al. 2001). These considerations may explain
how FUT8 acts on N-linked oligosaccharide for core fuco-
sylation: it is probable that the enzyme determines the
position of fucosylation via recognizing the β1,4-linked tri-
saccharide sequence, Manβ1-4GlcNAcβ1-4GlcNAcβ-, in
N-glycans rather than the reducing terminal GlcNAc which
is linked to asparagine.
It is well known that core-fucosylated N-glycans play var-
ious roles in the functions of glycoproteins (Miyoshi et al.
1999; Taniguchi et al. 2006). However, fucosylated chitooli-
gosaccharides produced by FUT8 have not been reported nor
have their properties been examined. The fucose residue that
is added by FUT8 to relatively long chitooligosaccharides
such as a pentaose and hexaose is markedly resistant to di-
gestion by α-fucosidase whereas such resistance was not
observed in the case of the shorter oligosaccharides exam-
ined (Figure 5). This inconsistent result might be due to
the occurrence of a conformational difference between the
molecules that protect the fucose from the glycosidase but
can be formed only in the GlcNAc polymers of five residues
or more. In addition, our analyses suggested that the pres-
ence of a fucose residue at the nonreducing terminal
GlcNAc prevents the digestion of the fucosylated chitooligo-
saccharide by exo-hexosaminidase (Figure 5). It seems likely
that these properties of the fucosylation by FUT8 potentially
contribute to the biological stabilization of the longer chitoo-
ligosaccharides. Therefore, industrial applications of FUT8
might be useful in terms of developing new carbohydrate-
based biomaterials.
Discussion
The fucosylation of chitooligosaccharides has been reported for
α1,3- and α1,6-fucosylation (Sanjuan et al. 1992; Bec-Ferté et
al. 1994; Olsthoorn et al. 1998; Natunen et al. 2001). The α1,3-
fucosylation of chitooligosaccharides has been found in a natu-
ral product from rhizobium (Olsthoorn et al. 1998); however,
the α1,3-fucosyltransferase responsible for the synthesis of
the structure has not been identified in rhizobia. As indicated
by the in vitro chemoenzymatic synthesis of a fucosylated oli-
gosaccharide using a human enzyme, it is likely that the human
α1,3-fucosyltransferase also has the ability to fucosylate chitoo-
ligosaccharides (Natunen et al. 2001). On the other hand, it is
known that only NodZs from rhizobia catalyze the α1,6-fucosy-
lation of chitooligosaccharide (Sanjuan et al. 1992; Bec-Ferté et
al. 1994). This α1,6-fucosylation by NodZ occurs at the reduc-
ing terminal GlcNAc residue of the oligosaccharide, while the
α1,3-fucosylation of GNs by rhizobial and human enzymes acts
on internal GlcNAc residues. The findings herein show that hu-
man α1,6-fucosyltransferase has the ability to catalyze the
synthesis of α1,6-fucosylated chitooligosaccharides as well as
the core fucosylation of N-glycans, and furthermore that the
α1,6-fucosylated chitooligosaccharide formed by the mamma-
lian enzyme is distinct from that produced by the bacterial
enzymes in terms of the position of fucosylation.
Interestingly, while NodZ fucosylates the reducing terminal
GlcNAc (Figure 9), our findings indicated that FUT8 transfers
a fucosyl unit to the third GlcNAc position from the nonre-
ducing terminal of chitooligosaccharides, suggesting that the
mechanism(s) by which the residue in the acceptor oligosac-
charides is selected for fucosylation is different between the
mammalian and bacterial enzymes. Because the two enzymes
are very similar in terms of both overall structure and the for-
mation of an α1,6-linked fucose, it is reasonable to assume
that their catalytic mechanisms are also similar with the ex-
ception that the mammalian and bacterial enzymes natively
act on N-linked oligosaccharides and chitooligosaccharides,
respectively. Although it may have been thought that these
two enzymes catalyze the fucosylation of the reducing termi-
nal GlcNAc of any oligosaccharide as the acceptor, our results
indicate that FUT8 does not necessarily recognize the reduc-
ing terminal of substrates.
The activities of FUT8 toward the chitooligosaccharide se-
ries are comparable to those of NodZ (Quesada-Vincens et
al. 1997; Quinto et al. 1997; Chazalet et al. 2001), suggest-
ing that the apparently unusual fucosylation can occur if an
appropriate, native acceptor substrate is present in the organ-
ism which expresses FUT8 or its ortholog. Such a possible
involvement of FUT8 in the metabolism and function of
chitooligosaccharides or related types of oligosaccharides re-
mains to be explored in addition to elucidation of its roles in
the biosyntheses of N-glycans.
The difference in the fucosylation of chitooligosaccharides
may be attributed to how the enzymes recognize and capture
the acceptors, the process of which involves the interaction of
the enzyme with the substrates. Such an interaction must be con-
ferred by the structure of the enzyme, and it seems likely that the
additional domain(s) of FUT8, which is lacking in NodZ, plays a
role in this distinct fucosylation. Although the exact function of
the domain of FUT8, e.g. the SH3 domain, remains to be eluci-
Materials and methods
Materials
GDP-β-L-fucose was purchased from Wako Pure Chemicals
(Osaka, Japan). Free and pNP-labeled chitooligosaccharides
1031

H Ihara et al.
were purchased from Seikagaku Biobusiness Corporation (To-
kyo, Japan). 4-Methylumbelliferyl triacetylchitotriose and 4-
methylumbelliferyl N-acetylglucosamine were purchased from
Toronto Research Chemicals Inc. (Toronto, Canada). β-N-
Acetylhexosaminidase (β-N-acetylglucosaminidase) from jack
bean and α-fucosidase from bovine kidney were purchased
from Sigma (St. Louis, MO). Other common chemicals were
obtained from Wako Pure Chemicals, Nacalai Tesque (Kyoto,
Japan) and Sigma.
using a TSK-gel Amide-80 column (4.6 × 250 mm) (Tosoh),
and elution was performed isocratically with 76% acetoni-
trile at a flow rate of 1.0 mL/min and 30°C. The structures
of the digested products were assigned by comparison with
standard chitooligosaccharides (Seikagaku Biobusiness
Corporation).
NMR experiments
NMR experiments were performed using a Bruker AVANCE-
1
600 spectrometer (600 MHz for H resonance frequency), and
Preparation of recombinant protein
probe temperature was set at 25°C. The oligosaccharide sam-
ples were dissolved in 100% D2O solution to be 2.5 mM
(GN5F and its β-N-acetylhexosaminidase-digest) or 5.0 mM
(GN5 and GN3). Chemical shifts were adjusted with the sin-
glet signal of sodium 3-trimethylsilyl-1-propanesulfonate
(DSS) at 0.00 ppm. To confirm the structures of GN5F and
The recombinant FUT8 was expressed in soluble form, with a
C-terminal polyhistidine tag, using a baculovirus/insect cell ex-
pression system and purified by Ni2 chelating affinity
chromatography as described previously (Ihara et al. 2006).
1
β-N-acetylhexosaminidase-digest of GN5F, H-NMR, 2D
Protein assay
1H-1H COSY, 2D 1H-13C HSQC and 2D HSQC-TOCSY spec-
tra were collected. For the use of NMR experiments,
chitopentaose (GN5) and chitotriose (GN3) were purchased
from Seikagaku Biobusiness Corporation.
Protein concentrations were determined using a bicinchoninic
acid kit (Pierce, Rockford, IL), with bovine serum albumin as
the standard.
FUT8 activity assay
α1,6-Fucosyltransferase activity was assayed using free and la-
beled chitooligosaccharides under modified conditions using
N-glycans as reported previously (Uozumi, Yanagidani et al.
1996; Yanagidani et al. 1997; Ihara et al. 2006). Recombinant
FUT8 was incubated at 37°C with 1 mM of acceptor substrate
and 2 mM GDP-β-L-fucose as a donor in 0.1 M MES-NaOH,
pH 7.0. The reactions were terminated by boiling after an ap-
propriate reaction time, and the reaction mixtures were
centrifuged at 15,000 × g in a microcentrifuge for 10 min.
The resulting supernatants were injected to a normal phase
HPLC (2695 Separation Module, Waters, Milford, MA)
equipped with TSK-gel, Amide-80 (4.6 × 250 mm) (Tosoh, To-
kyo, Japan), as reported about chitinase experiment previously
(Abdel-Banat et al. 2002; Kabir et al. 2006). The product and
substrate were separated isocratically with 70% acetonitrile as
the irrigant at a flow rate of 1.0 mL/min and 30°C. UV absor-
bance of the column elute was monitored with a UV monitor
(2487 Multi λ Absorbance Detector, Waters) for detection of
free and pNP-labeled chitooligosaccharides, at 210 and
300 nm, respectively (Abdel-Banat et al. 2002; Murata et al.
2005; Kabir et al. 2006). The fluorescence of the column elute
for 4MU-labeled sugars was detected with a fluorescence de-
tector (2475 Multi λ Fluorescence Detector, Waters), at
excitation and emission wavelengths of 315 nm and 380 nm,
respectively (Tazawa et al. 1998).
MS spectrometry
Matrix-assisted laser desorption/ionization time of flight
mass spectrometry (MALDI-TOF MS) spectra were mea-
sured using a Shimadzu AXIMA-CFR spectrometer
(Kyoto, Japan) with reflectron mode, and 2,5-dihydroxyben-
zoic acid was employed as the matrix. Angiotensin II was
used for the calibration.
Funding
This work was supported by Core Research for Evolutional
Science and Technology (CREST) of Japan Science and
Technology Agency (JST).
Conflict of interest statement
None declared.
Abbreviations
4MU 4-methylumbelliferone; COSY correlation spectroscopy;
DSS sodium 3-trimethylsilyl-1-propanesulfonate; Fuc fucose;
FUT8 α1,6-fucosyltransferase; GDP guanine nucleotide di-
phosphate; GN1 GlcNAc or N-acetylglucosamine; GN2 N,
N′-diacetyl chitobiose; GN3 N,N′,N″-triacetyl chitotriose; GN4
N,N′,N″,N‴-tetraacetyl chitotetraose; GN5 N,N′,N″,N‴,N‴′-
pentaacetyl chitopentaose; GN6 N,N′,N″,N‴,N‴′,N‴″-hexaace-
tyl chitohexaose; GNF fucosylated chitooligosaccharide; GNs
chitooligosaccharides; HPLC High performance liquid chroma-
tography; HSQC hetero-nuclear single quantum coherence;
MALDI matrix-assisted laser desorption/ionization; MS
mass spectrometry; NMR nuclear magnetic resonance; pNP
p-nitrophenol; TOCSY total correlation spectroscopy; TOF
time of flight; UV ultraviolet.
Structural analysis of fucosylated chitooligosaccharide by exo-
glycosidase digestion
Fucosylated chitooligosaccharides were digested by α-fucosi-
dase (bovine kidney, Sigma), β-N-acetylhexosaminidase (jack
bean, Sigma) and their combination. Samples were incubated
at 37°C for 24 h in 50 mM citrate buffer, pH 5.0. The reac-
tions were terminated by boiling, and the reaction mixtures
were centrifuged at 15,000 × g in a microcentrifuge for
10 min, after the addition of an equal amount of acetonitrile.
The supernatants were then analyzed by normal phase HPLC
1032

Fucosylation of chitooligosaccharides by FUT8
products containing a GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4R determinant
at the nonreducing terminus. Glycobiology. 11(3): 209–216.
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