Glycobiology vol. 11 no. 3 pp. 209–216, 2001
Human α3-fucosyltransferases convert chitin oligosaccharides to products containing a
GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4R determinant at the nonreducing terminus
Jari Natunen, Olli Aitio, Jari Helin, Hannu Maaheimo1,
Ritva Niemelä, Sami Heikkinen, and Ossi Renkonen2
Institute of Biotechnology, P.O. Box 56, 00014 University of Helsinki,
Finland
thesis of J. Natunen [1999] at the University of Helsinki.) The
products of these novel reactions may prove to be useful intermediates for manmade plant hormones and elicitors of defense
functions in plants.
Received on August 8, 2000; revised on October 6, 2000; accepted on
October 14, 2000
Human α3-fucosyltransferases (Fuc-Ts) are known to
convert N-acetyllactosamine to Galβ1-4(Fucα1-3)GlcNAc
(Lewis x antigen); some of them transfer fucose also to
GalNAcβ1-4GlcNAc, generating GalNAcβ1-4(Fucα1-3)GlcNAc
determinants. Here, we report that recombinant forms of
Fuc-TV and Fuc-TVI as well as Fuc-Ts of human milk
converted chitin oligosaccharides of 2–4 GlcNAc units
efficiently to products containing a GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4R determinant at the nonreducing terminus. The
product structures were identified by mass spectrometry
and nuclear magnetic resonance experiments; rotating
frame nuclear Overhauser spectroscopy data suggested
that the fucose and the distal N-acetylglucosamine are
stacked in the same way as the fucose and the distal galactose
of the Lewis x determinant. The products closely resembled
a nodulation factor of Mesorhizobium loti but were distinct
from nodulation signals generated by NodZ-enzyme.
Key words: chitin oligosaccharides/human α3-fucosyltransferases/site-specific, peridistal α3-fucosylation
Introduction
Human α3-fucosyltransferases (Fuc-Ts) are an important
group of glycosylation enzymes, participating, e.g., in
immunological defense (Maly et al., 1996). They transfer
fucose to N-acetyllactosamine, generating the bioactive
epitope Galβ1-4(Fucα1-3)GlcNAc, known as Lewis x antigen
(Edbrooke et al., 1997). Some Fuc-Ts work also with
GalNAcβ1-4GlcNAc (Bergwerff et al., 1993) and 4′-deoxy-Nacetyllactosamine (de Vries et al., 1995). The present experiments revealed that also chitin oligosaccharides, which contain
GlcNAcβ1-4GlcNAc determinants, are acceptors for recombinant
Fuc-Ts V and VI, as well as for mixed Fuc-Ts from human milk.
The acceptors were fucosylated by these enzymes in a sitespecific manner at the N-acetylglucosamine unit adjacent to the
nonreducing end, generating the GlcNAcβ1-4(Fucα1-3)GlcNAcOR determinant. (The data were incorporated in the Ph.D.
1Present
2To
address: VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland.
whom correspondence should be addressed
© 2001 Oxford University Press
Results
α3-Fucosylation of N,N′-diacetylchitobiose (Glycan 2) by
human Fuc-Ts
Incubation of Glycan 2 (4 µmol) with GDP-Fuc (3 µmol) and
purified recombinant human Fuc-TV (50 mU) gave a mixture
of oligosaccharides, from which repeated gel filtration runs on
a Bio-Gel P-2 column, and subsequent runs on a Superdex
Peptide HR 10/30 column gave 880 nmol of a purified
trisaccharide product (Glycan 2′). Its matrix-associated laser
desorption/ionization time of flight (MALDI-TOF) mass
spectrometry (MS) revealed a major signal at m/z 593.3 that
was assigned to [M+Na]+ of Fuc1GlcNAc2 (calc. m/z 593.2).
Glycan 2′ was reduced, permethylated, and subjected to ESI-MS.
The [M+Na]+ ion (m/z 749.6) was selected for tandem mass
spectrometry (MS/MS). The majority of the fragments
produced could be assigned to the trisaccharide alditol
GlcNAcβ1-(Fucα1-)GlcNAcred (Figure 1A). The Y1a fragment
(according to the nomenclature of Domon and Costello [1988])
at m/z 490.4 is produced by the loss of the nonreducing end
GlcNAc. In addition, the Y1a/Y1b fragment at m/z 302.2 (as
well as its dehydration product, m/z 284.2) will only arise from
a doubly substituted GlcNAcred. However, small but clear ions
(marked with an asterisk in Figure 1A) reveal also the presence
of another isomer with fucose bonded to the nonreducing end
GlcNAc. The Y1* ion (m/z 316.2) represents a singly substituted
GlcNAcred, and the B2* ion at m/z 456.2 carries a disaccharide
fragment representing the Fucα1-GlcNAc unit from the the
nonreducing end of the small side product. In conclusion, the
MS/MS data established that Fuc-TV transferred mostly to the
N-acetylglucosamine of the reducing end of N,N′-diacetylchitobiose.
The data of the 1H-NMR spectrum of Glycan 2′ (Table I)
show that the fucose is α1,3-linked and confirm that it is
bonded to the reducing end GlcNAc. (i) The coupling constant
of the fucose H1 resonance (J1,2 = 3.6 Hz) establishes the
presence of an α-linkage. (ii) The fucose H1, H5 and H6 resonances resemble those of the GlcNAcβ1-4(Fucα1-3)GlcNAc
determinant at the reducing end of unconjugated plant Nglycans reported by Lhernould et al. (1992). By contrast, the
fucose resonances of Glycan 2′ are distinct from those
observed for the trisaccharide GlcNAcβ1-4(Fucα1-6)GlcNAc
(Glycan 2″; Table I). (iii) The H1 signals of the reducing end
209
J. Natunen et al.
Fig. 1. ESI-MS/MS spectra from reduced and permethylated derivatives of fucosylated chitin oligosaccharides. The fragment ion nomenclature of Domon and
Costello (1988) is used. (A) collision-induced dissociation (CID)-MS of the [M+Na]+ ion (m/z 749.6) from Glycan 2′ that had been generated by Fuc-TV. The ions
marked with an asterisk originate from a small side product that represents an isomer of Glycan 2′. (B) CID-MS of the [M+2Na]2+ ion (m/z 508.8) from Glycan 3′
that had been generated by Fuc-TV. (C) CID-MS of the [M+2Na]2+ ion (m/z 631.6) from Glycan 4′ that had been generated by Fuc-Ts of human milk.
GlcNAc unit C of Glycan 2′ are at 5.077/4.684 p.p.m., whereas
in the fucose-free chitin oligosaccharides they are at 5.185/
210
4.689 p.p.m.; analogous differences have been reported by
Niemelä et al. (1999) between the reducing end GlcNAc H1
Chitin oligosaccharide products of human α3-fucosyltransferases
Table I. 1H Chemical shifts (ppm) of structural reporter groups of selected Glycans at 23 °C.
Residuea
Proton
H1
Glycans
2′ b
2″ c
3′ b
4
4′ dn.d.
A
—
—
—
5.185(α)/4.689(β)
5.184(α)/4.689(β)
B
—
—
5.185(α)/4.688(β)
4.58
4.579/4.570e)
C
5.077(α)/4.684(β)
5.181(α)/4.693(β)
4.580/4.571e)
4.58
4.570
D
4.544/4.527e
4.640/4.644
4.522
4.58
4.519
fucose
5.113
4.893/4.901
5.124
—
5.119
H4
D
3.240
ND
3.238
ND
3.237
H5
fucose
4.767
4.101/4.137
4.764
—
4.762
CH3
fucose
1.268
1.209/1.221
1.268
—
1.267
aThe
residue notation is as follows:
D
C
B
A
GlcNAcβ1-4(Fucα1-3/6)GlcNAcβ(1-4)GlcNAcβ1-4GlcNAc
bThe data refer to the Fuc-TV-generated product.
cGlycan 2″ is GlcNAcβ1-4(Fucα1-6)GlcNAc.
dThe data refer to Glycan 4′ that was generated by human milk Fuc-Ts.
eThe two chemical shift values given arise from signals representing the α- and β-pyranosic forms of the oligosaccharides, respectively.
ND, not determined.
—, not appropriate.
signals of Lewis x and LacNAc determinants of polylactosamines. (iv) The double doublet of Glycan 2′ at 4.144 p.p.m. is
identical with the αH2 resonance of the α3-fucosylated,
reducing-end GlcNAc of the unconjugated plant N-glycans
(Lhernould et al., 1992). No nuclear magnetic resonance
(NMR) evidence was obtained for the presence in Glycan 2′ of
the minor side product detected in the MS experiments.
All 1H- and 13C-signals of Fuc-TV-generated Glycan 2′ were
assigned (Table II). All resonances of the fucose unit and most
resonances of the reducing end GlcNAc unit of Glycan 2′
resembled closely those reported by Lhernould et al. (1992) for
the unconjugated N-glycan of plant type, Rβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc. Comparison with the 13C-signals of Glycan 2
reported by Bock et al. (1984) reveals large shifts in Glycan 2′
Table II. 1H and 13C chemical shifts of Glycan 2′ and (distal) parts of Glycan 4′
Glycan 2′a
Glycan 4′a
GlcNAc
Db
GlcNAc
Cα
GlcNAc
Cβ
Fuc
GlcNAc
D
Fuc
H1
4.544/4.527
5.077
4.684
5.113
4.519
5.119
H2
3.737
4.144
3.881
3.705
3.735
3.707
H3
3.546/3.539
3.980
3.832
3.947
3.535
3.938
H4
3.240
3.904
3.893
3.806
3.237
3.803
H5
3.435
3.912
3.514
4.767
3.428
4.762
H6
3.607
3.774
3.737
—
3.607
—
H6′
3.964
3.851
3.908
—
3.963
—
CH3
—
—
—
1.268
—
1.267
C1
101.73
92.20
96.03
99.95
ND
ND
C2
57.14
55.31
58.11
69.02
ND
ND
C3
74.96
74.09
76.17
70.52
ND
ND
C4
72.04
74.96
74.96
73.36
ND
ND
C5
77.31
72.74
76.72
67.97
ND
ND
C6
62.93
61.09
61.27
16.81
ND
ND
aResidue
notation is shown in Table I.
two chemical shift values given arise from signals representing the α- and β-pyranosic forms of the oligosaccharides, respectively.
ND, not determined.
—, not appropriate.
bThe
211
J. Natunen et al.
Table III. Assigned interresidue ROEs in Glycan 2′
Proton Paira
Proton Pair
Fuc H1 ←→GlcNAc Cα H2
Fuc H4 ←→GlcNAc D H4
Fuc H1 ←→GlcNAc Cα H3
Fuc H5 ←→GlcNAc D H2b
Fuc H1 ←→GlcNAc Cα H4
Fuc H5 ←→GlcNAc D H4
Fuc H1 ←→GlcNAcCβ H3
Fuc H6 ←→GlcNAc D H2b
Fuc H1 ←→GlcNAc C NAc
aFor
residue notation, see Table I.
ROE contact was observed also in Glycan 4′.
bThis
of the acceptor and 400 nmol of the donor in 100 µl of the
reaction mixture, afforded the purified product in an apparent
yield of 180 nmol. The yield of the product was determined by
its UV absorption at 214 nm, which was compared to the
absorption of a GlcNAc reference. This method appears to
overestimate the yield of the fucosylated products by about
20%. The 1D 1H NMR spectrum of the product was identical
with that of the Fuc-TV-generated Glycan 2′ (not shown).
Human milk α3-fucosyltransferases, too, converted Glycan 2
to Glycan 2′ that was isolated in pure form and identified by
the 1D 1H NMR spectrum (not shown).
Fig. 2. Expansion of the ROESY spectrum of Glycan 2′. The assigned
interresidual ROE contacts between proton pairs of the fucose (F in the
spectrum) and the N-acetylglucosamine C at the reducing end (C in the
spectrum), and those between the fucose and N-acetylglucosamine D at the
nonreducing end (D in the spectrum) are boxed. The correlation between Fuc
H1 and acetyl-CH3 of N-acetylglucosamine C is not shown. The data are
presented as a list of assigned interresidue ROEs in Table III.
only at C-3 and C-4 of the reducing end GlcNAc. These
changes resemble closely the differences at C-3 and C-4 in the
GlcNAc units of (conjugated) Lewis x and (free) LacNAc
(Bock et al., 1984).
The rotatine frame nuclear Overhauser spectroscopy
(ROESY) spectrum of Glycan 2′ (Figure 2) revealed several
contacts between the fucose H1 and the protons H2, H3, and
H4 of GlcNAc unit C at the reducing end (for residue notation
in chitin oligosaccharides, see footnotes of Table I), but not
between the fucose H1 and the protons at position 6 of GlcNAc
C (cf. Table III). No cross peaks were observed between Fuc
H1 and the protons of GlcNAc D in Glycan 2′. Analogous data
have been reported for Galβ1-4(Fucα1-3)GlcNAcβ1-3′Lactose
by Wormald et al. (1991). Taken together, the NMR data
confirm and extend the MS observations on the primary
structure, showing that Glycan 2′ represents the trisaccharide
GlcNAcβ1-4(Fucα1-3)GlcNAc.
The ROESY spectrum also showed correlations between
several interresidual proton pairs of the fucose and the GlcNAc
D at the non-reducing end of of Glycan 2′ (Table III),
suggesting that the fucose and the distal GlcNAc D are
stacked.
Also recombinant human Fuc-TVI converted Glycan 2 to
Glycan 2′. This reaction, which was performed with 200 nmol
212
α3-Fucosylation of N,N’,N′′-triacetylchitotriose (Glycan 3) by
human Fuc-Ts
Incubation of Glycan 3 (1.0 µmol) with GDP-Fuc (1.0 µmol)
and purified recombinant human Fuc-TV (25 mU) in 200 µl of
the reaction buffer gave a mixture of oligosaccharides, from
which 190 nmol of a purified tetrasaccharide product, Glycan
3′, was isolated. The tetrasaccharide revealed in MALDI-TOF
MS a major signal at m/z 796.5 that was assigned to [M+Na]+
of Fuc1GlcNAc3 (calc. m/z 796.3) (not shown).
Glycan 3′ was reduced, permethylated, and subjected to
electrospry ionization mass spectrometry (ESI-MS). The
doubly charged [M+2Na]2+ ion (m/z 508.8) was selected for
MS/MS. All fragments obtained could be assigned to the tetrasaccharide alditol GlcNAcβ1-(Fucα1-)GlcNAcβ1-GlcNAcred
(Figure 1B). A loss of terminal, nonsubstituted GlcNAc unit is
evident from the B1 ions at m/z 282.2 (sodiated) and m/z 260.0
(protonated). Loss of methanol from the m/z 260.0 ion
accounts for the m/z 228.2 and m/z 196.2 ions. The Y1 ions at
m/z 316.2 and m/z 338.2 (carrying one and two sodiums,
respectively) indicate that the N-acetylglucosaminitol unit of
the reduced Glycan 3′ carried only one monosaccharide
substituent. Furthermore, theY2a /B2 ion at m/z 442.4 can only
arise by loss of a distal, unsubstituted GlcNAc accompanied by
loss of the GlcNAcred unit. No fragments were observed, even
in close inspection, which would represent a fucosylated
reduced end (i.e., Fucα1-GlcNAcred at m/z 490.4; cf. fragments
of Glycan 2′ in Figure 1A) or a fucosylated nonreducing end
GlcNAc (at m/z 456.2). It is also noteworthy that fragmentation of
the fucose unit produced mainly Z ions. This behavior is
reportedly characteristic to 3-substitution (Viseux et al., 1997),
implying that the fucose was 1,3-linked to GlcNAc. The origin
of the fairly intense fragment at m/z 455.2 is complex. Its
nature was revealed by producing the B2, Y2a, and Z2b fragments
with a high orifice voltage and by collecting MS/MS/MS data
Chitin oligosaccharide products of human α3-fucosyltransferases
with these skimmer fragments (not shown). Only the Y and Z
ions generated the m/z 455 ion, so it must contain the reduced
end. We suggest that this is an 0,4X1 ion, arising by a cross-ring
cleavage of the middle GlcNAc ring. Taken together, the MS/MS
data established that Fuc-TV transferred exclusively to the
middle GlcNAc of N,N’,N′′-triacetylchitotriose.
The 1D 1H-NMR spectrum of Glycan 3′ (Table I) lacks the
4.144 p.p.m. H2-signal of the reducing end GlcNAc (i.e., unit
C) of the α-form of Glycan 2′, confirming that the reducing
end of Glycan 3′ is different from that of Glycan 2′. By
contrast, the H4 resonance of GlcNAc D in Glycan 3′ is
identical to that of Glycan 2′, implying that the nonreducing
ends in Glycans 2′ and 3′ are similar. Also the H1, H5, and H6
signals of fucose in Glycan 3′ resemble their counterparts in
Glycan 2′. Taken together, the NMR data confirm and extend
the MS data, establishing that Fuc-TV-generated Glycan 3′
represents GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc. Human
milk Fuc-Ts, too, converted Glycan 3 to Glycan 3′, which was
isolated in purified form and was identified by 1D 1H-NMR
and MS/MS data (not shown).
α3-Fucosylation of N,N′,N″,N′′′-tetraacetylchitotetraose
(Glycan 4) by human Fuc-Ts
Glycan 4 (6 µmol) was incubated with GDP-Fuc (3 µmol) and
partially purified Fuc-Ts of human milk (4.3 mU) in 1.2 ml of
the reaction buffer for 2 days and with additional 4.3 mU of the
enzyme for further 2 days. A mixture of oligosaccharides was
obtained from which an isocratic high pH anion exchange run
on a CarboPac PA-1 column, using 40 mM NaOH, gave
740 nmol of a pentasaccharide, Glycan 4′. It revealed in
MALDI-TOF MS a major signal at m/z 999.7 that was
assigned to [M+Na]+ of Fuc1GlcNAc4 (calc. m/z 999.4) (not
shown).
Glycan 4′ was reduced, permethylated, and subjected to ESI-MS.
The doubly charged [M+2Na]2+ ion (m/z 631.6) was selected
for MS/MS. The low-mass region of the MS/MS spectrum
(Figure 1C) resembles that of Glycan 3′, showing the same B1
fragments from the unsubstituted terminal GlcNAc unit (m/z
282.2, m/z 260.2), and Y1 ions from the GlcNAcred (m/z 316.2,
m/z 338.2). In addition, theY2a /B2 ion (m/z 442.4), the B3 ion
(m/z 946.8), and the Y3a ion (m/z 980.6) establish that the
fucose unit is linked to either of the two midchain GlcNAcs.
The Y2 ion at m/z 561.4 indicates that the GlcNAcβ1-4GlcNAcredfragment originally carried only one saccharide substituent,
implying that the fucose must reside at the penultimate
GlcNAc residue C, close to the nonreducing end. This notion is
directly confirmed by the B2 ion at m/z 701.6, carrying the
methylated fragment GlcNAcβ1-(Fucα1-)GlcNAc derived
from the nonreducing terminus of Glycan 4′. As above, fragmentation of the fucose unit produced Z ions, implying the
presence of a Fucα1,3-linkage. The MS/MS data did not reveal
any specific fragments for other pentasaccharide isomers. We
conclude that Glycan 4 was fucosylated solely to the penultimate GlcNAc residue next to the nonreducing end.
1D 1H-NMR spectrum of purified Glycan 4′ (Table I) was
analogous to that of Glycan 3′. The proton signals of the fucose
and the GlcNAc D (Table II) were assigned as described by
Maaheimo et al. (1997). The data reveal a great similarity in the
spin systems of the fucose and the GlcNAc D in Glycans 2′ and
4′. In addition, the ROESY spectrum of Glycan 4′ (not shown)
revealed cross peaks between fucose H5 and GlcNAc D H2 as
well as between fucose H6 and GlcNAc D H2 (compare
Table III), suggesting that the fucose and the GlcNAc D are
stacked also in Glycan 4′. Taken together, the NMR data confirm
and extend the MS/MS results, establishing that Glycan 4′ represented GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAcβ1-4GlcNAc.
Even Fuc-TV converted Glycan 4 to Glycan 4′, which was
isolated in purified form in a yield of 22% and was identified
by its 1D 1H-NMR spectrum (not shown).
Discussion
The present data show that recombinant human Fuc-TV, in
truncated soluble form, catalyzed site-specific transfer of α3-linked
fucosyl groups to completely N-acetylated forms of chitobiose
(Glycan 2), chitotriose (Glycan 3), and chitotetraose (Glycan
4) as shown in Equation 1.
Eq. 1. GlcNAcβ1-4GlcNAcβ1-OR + GDP-Fuc →
GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-OR + GDP
The fucose unit was transferred selectively to the N-acetylglucosamine unit adjacent to the nonreducing end in all three
chitin oligosaccharide acceptors. No other isomers were
observed in Glycans 3′ and 4′, the fucosylated products from
N,N′N″-triacetylchitotriose and N,N′,N″,N′′′-tetraacetylchitotetraose, respectively. However, Glycan 2′, the fucosylated
N,N′-diacetylchitobiose, GlcNAcβ1-4(Fucα1-3)GlcNAc, was
contaminated by a small amount of an isomeric side product
where the fucose was linked at the nonreducing end GlcNAc of
the acceptor.
Even a soluble form of recombinant human Fuc-TVI, and
partially purified human milk Fuc-Ts catalyzed the reactions of
Equation 1. It is not known at present whether these Fuc-Ts
generate also the distally fucosylated side product when
working with N,N′-diacetylchitobiose.
The products of Fuc-T reactions were characterized by
MALDI-TOF MS, ESI-MS/MS, and 1D as well as 2D-NMR
experiments. The NMR data confirmed that the Fuc-Ts, working
with the chitin oligosaccharides, generated Fucα1-3GlcNAc
linkages rather than Fucα1-6GlcNAc bonds. The novel
reactions of Equation 1 are analogous to the reactions of
neutral i-type polylactosamine chains catalyzed by several
human Fuc-Ts. Even these acceptors are α3-fucosylated at the
peridistal N-acetylglucosamine unit by several human Fuc-Ts
(Niemelä et al., 1998; Nishihara et al., 1999). However, many
human Fuc-Ts transfer efficiently even to inner N-acetyllactosamine units of i-type polylactosamine chains, e.g., to the
middle GlcNAc of the hexasaccharide Galβ1-4GlcNAcβ1-3Gal−
β1-4GlcNAcβ1-3Galβ1-4GlcNAc (de Vries et al., 1995;
Niemelä et al., 1998; Nishihara et al., 1999). Remarkably, the
present experiments with Glycan 4 did not lead to transfer at
sites other than the peridistal GlcNAc, implying that a difference exists in the acceptor activities of the nondistal parts of
chitin saccharides and i-type polylactosamines.
There are some predecessors of the present findings. N,N′diacetylchitobiose was fucosylated by a human lymphocyte
enzyme (Hoflack et al., 1978), that was probably an α3-fucosyltransferase; the known α6-fucosyltransferases do not react
with N,N′-diacetylchitobiose (Voynow et al., 1991). Fucosylation of some chito-type saccharides by human Fuc-TVI
213
J. Natunen et al.
(Nimtz et al., 1998) and fucosylation of N,N′-diacetylchitobiose-6-sulfate (Tran et al., 1998) have also been reported.
The site-specific and efficient transfer of α3-linked fucose to
chitin oligosaccharides of the present experiments generated
products that resemble a Nod factor of Mesorhizobium loti
(Olsthoorn et al., 1998) but are distinct from the α6-fucosylated
nodulation signals synthesized from chitin oligosaccharides by
the NodZ enzyme (Quinto et al., 1997). Indeed, in the history
of life, chitin oligosaccharides may have been the first acceptors
for α3-fucosyltransferases, which are highly conserved from
bacteria to humans (Oriol et al., 1999; Leiter et al., 1999).
Consequently, the present-day glycoconjugates with N-acetyllactosamine units may be relatively late “clients” of these
enzymes. Additional acceptors for these enzymes may be
available in saccharide sequences like cello-oligosaccharides,
laminaribiose, Manβ1-4GlcNAc, and GlcUAβ1-3GlcNAc.
The present ROESY data (summarized in Table III) suggest
that the fucose and the distal GlcNAc residue in the α3-fucosylated chitin saccharides are stacked in a way reminiscent of the
stacking of fucose and galactose in the Lewis x determinant
(Wormald et al., 1991; Miller et al., 1992). Analogous interactions have been reported also in the GalNAcβ1-4(Fucα13)GlcNAc epitope (Bergwerff et al., 1993), in the α3-fucosylated core of plant N-glycans (Bouwstra et al., 1990), and in
the Nod-factor lipo-oligosaccharide from M. loti (Olsthoorn et
al., 1998). All these examples of uniquely rigid trisaccharide
determinants are probably recognized by several proteins and
perhaps also by other saccharides; the details are best understood for
the Lewis x binding. For instance, the rigidity of the GlcNAcβ14(Fucα1-3)GlcNAc determinant in the core of N-glycans in
plants may contribute importantly to the allergenicity characteristic for many plant glycoproteins (Garcia-Casado et al.,
1996; van Ree et al., 2000). On the other hand, the α3-fucosylation of chitin oligosaccharides is likely to restrict the action
of chitinases, β4GlcNAc transferases, acyl transferases, and
lectins recognizing chitin-type chains; analogous effects of α3fucosylation on polylactosamine binding are well known
(reviewed in Renkonen, 2000).
The in vitro Fuc-T reactions with chitin oligosaccharides and
their derivatives may prove useful for synthesis of man-made
plant growth regulators (Röhrig et al., 1995; Staehelin et al.,
1994) and elicitors of defense functions (Yamaguchi et al.,
2000). Developmentally important derivatives of N-acetylchito-oligosaccharides are believed to occur even in vertebrates, but their exact structures are not known (Semino et al.,
1996), and no functional relations between these saccharides
and the α3-fucosyltransferases are known at present.
Materials and methods
(Uppsala, Sweden). Dowex AG 1-X8 (AcO–, 200–400 mesh),
Dowex AG 50W-X8 (H+, 200–400 mesh) and Bio-Gel P-2
were from Bio-Rad (Richmond, CA). D2O was from
Cambridge Isotope Laboratories (Woburn, MA). Partially
purified human milk Fuc-Ts (EC 2.4.1.152 and EC 2.4.1.65)
were prepared according to Natunen et al. (1994) and assayed
as described in Eppenberger-Castori et al. (1989).
Fucosyltransferase reactions
Fuc-TV reactions were carried out under conditions similar to
those described by Palcic et al. (1989); the nominal enzyme
concentrations were initially 12.5 mU/100 µl, and the reaction
mixtures were incubated for 5 days at room temperature. In
Fuc-TVI reactions, the nominal enzyme concentration was
initially 10 mU/100 µl, and the reaction mixtures were incubated for 3 days at 37°C. For human milk Fuc-T reactions,
Glycan 2 was incubated with the enzyme at 10 mM and
Glycans 3 and 4 at 5 mM; the initial enzyme concentration was
typically 360 µU/100 µl and fresh enzyme (360 µU/100 µl)
was added after 2 days; the incubations were performed at
37°C for a total of 4 days.
Chromatographic methods
Chromatographic experiments were performed as described by
Maaheimo et al. (1995) and Natunen et al. (1997).
Mass spectrometry
MALDI-TOF MS was performed in the positive ion delayed
extraction mode with a BIFLEX™ mass spectrometer (BrukerFranzen Analytik, Bremen, Germany) using 2,5-dihydroxybenzoic acid as the matrix. ESI-MS of reduced and permethylated (Ciucanu and Kerek, 1984) glycans were collected using
an API365 triple quadrupole mass spectrometer (Perkin-Elmer
Instruments, Thornhill, Ontario). The samples were dissolved
in 50% aqueous methanol containing 0.5 mM sodium
hydroxide and injected into the mass spectrometer with a nanoelectrospray ion source (Protana A/S, Odense, Denmark) at a
flow rate of about 30 nl/min. MS/MS spectra were acquired by
colliding the selected precursor ions to nitrogen collision gas
with acceleration voltages of 35 V (doubly charged precursors)
or 55 V (singly charged precursors).
NMR spetroscopy
The NMR experiments were performed on a Varian Unity
500 spectrometer at 23°C in Shigemi tubes (Shigemi Co.,
Tokyo) essentially as described in Maaheimo et al. (1997). The
assignments are based on the structural reporter group
resonances of Table I and DQFCOSY, TOCSY, 1D selective
TOCSY, HSQC, 2D HMQC-TOCSY and DEPT 135 experiments.
Materials
N,N′-diacetylchitobiose (Glycan 2) and GlcNAcβ1-4(Fucα1-6)GlcNAc (Glycan 2″) were from Sigma (St. Louis, MO),
N,N′,N″-triacetylchitotriose (Glycan 3) and N,N′,N″,N′′′tetraacetylchitotetraose (Glycan 4) were from Seikagaku
(Tokyo). GDP-fucose was a gift from Prof. B. Ernst (Universität
Basel). Fuc-Ts V (EC 2.4.1.65) and VI (EC 2.4.1.152) were
from Calbiochem (La Jolla, CA). GDP-[U-14C]fucose was
from Amersham International (Little Chalfont, UK). Superdex
Peptide HR 10/30 HPLC-column was from Pharmacia
214
Acknowledgments
The work was supported in part by grants from the University
of Helsinki, the Academy of Finland (38042, 40901, 44318
and 41413), the Technology Development Centre, Helsinki
(TEKES 40057/97 and 40368/99), as well as Emil Aaltonen
Foundation, Tampere. The support from the Graduate School
of Bioorganic Chemistry (University of Turku) and from the
Foundation of Jenny and Antti Wihuri to Jari Natunen are also
acknowledged.
Chitin oligosaccharide products of human α3-fucosyltransferases
Abbreviations
CID, collision induced dissociation; ESI, electrospray
ionization; Fuc, L-fucose; Fuc-Ts, human α1,3-fucosyltransferases III–VII and IX; Gal, D-galactose; GalNAc, N-acetylD-galactosamine; GlcNAc, N-acetyl-D-glucosamine; GlcNAcred,
N-acetyl-D-glucosaminitol; LacNAc, Galβ1-4GlcNAc; MALDITOF, matrix-assisted laser desorption/ionization time of flight;
MS, mass spectrometry; MS/MS, tandem mass spectrometry;
ROESY, rotating frame nuclear Overhauser spectroscopy.
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