Vol.
177,
June
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
14, 1991
Sophie
FIEVRE, Jean-Michel Jerome LEMOINE, Jean
Laboratoire nO1ll,
Universite
de
Received
April
Chimie des 59655
18,
WIERUSZESKI, MONTREUIL
Biologique, Sciences Villeneuve
Jean-Claude Gerard
and
Unite Mixte de Techniques de Lille d'Ascq Cedex, France
et
720-725
MICHALSKI, STRECKER* Recherche du Flandres-Artois,
CNRS
1991
A novel oligosaccharide has been isolated from human milk and identified aa a trisialylated derivative of lacto-H-hexaose. This compomd was purified by combining ion-exchange chromatography, preparative paper chromatography. structural chromatography and high performanc e liquid analyses by methylation, fast atom bombardmemt-omes spectrometry and proton nuclear magnetic resonance spectroscopy are consistent with the following structure: l&mAc(a2-6)0a1(81-4)01cHAc(01-6), IWuAc(a2-3)Ga1(81-3), Gal(Bl-4)Glc GlcMAc(Hl-3)'
lfeuAc(a2-6)’ 0
1991
Academic
Press,
Inc.
milk
Human
contains
oligosaccharides
the
antigens been
epitopes
structure
[l-9].
characterized.
separation
of
technics
of
Up The
new
numerous
to
fucosylated
of
which
is
related
now,
more
than
70
introduction
isomers
of
that
gel-filtration,
and
could
to
has
be
purified
ion-exchange
cell
surface
oligosaccharides
HPLC
not
sialylated
made
have possible
by
chromatography
the
traditional or
paper
the
first
chromatography. In
the
present
trisialylated
paper,
oligosaccharide
we
describe found
in
the human
isolation
of
milk.
Fractionation of mflk sfalylated olfgosaccharides : Fractionation of milk oligosaccharides leading to the isolation of sialylated oligosaccharides has been previously described [lo]. The sialylated oligosaccharides were eluted from an anion-exchanger (Dowex 1x2; 200-400 mesh; HCOOform) with a discontinuous gradient of pyridine-acetate buffer (pH 5.5) from 5 to 500 mM. * Author
for
Abbreviations: fast atom resonance;
correspondence. HPLC, high bombardment-mass GLC-MS, gas-liqid
CKNX291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
performance
liquid
spectrometry;
chromatography-mass
720
chromatography; NMR, nuclear spectrometry.
FAB-MS, magnetic
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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Analytical procedures : Descending paper chromatography was performed on Whatman no3 paper using the solvent pyridine-ethyl acetate-acetic acid-water (5:5:1:3, by vol.) and sugars were detected with an aniline oxalate reagent [ll]. Preparative reversed phase HPLC of native sialylated oligosaccharides was carried out on two S).zm ODS Zorbax Columns (0.94 cm x 25 cm ; du Pont Instrument, Paris, France). The HPLC apparatus was equipped with a refractometer. The solvent used was a triethylamine-acetic buffer (10 mM, pH 5) 151. Methylation analysis : The oligosaccharide was methylated and then methanolysed (methanol-0.5M HCl, 24 h at SOOC). The partially methylated methyl glycosides were acetylated [12] (1:l pyridine-acetic anhydride, 0.2ml) and the products were analysed by GLC-MS using a capillary column (0.33 mm x 25 m) coated with fused silica CP-SIL 5CB (temperature program : 100 to 240°C, at 4O/min). on a Kratos Concept FAB-MS : FAB-MS was performed spectrometer using xenon as the bombarding atom (operating 7.3 kV, 1.2 ms). The spectra were recorded in positive-ion voltage. permethyled oligosaccharide acceleration The dissolved in 1 ul of methanol, was added to the thioglycerol previous coating with sodium acetate.
II HH conditions mode at (5-10 matrix
mass : 8 kV pg), with
were pfrfqyed on a NMRspectrocopy : The 400 MHz H1-NMK experiments Bruker AM-400 WE spectrometer equipped with a 5 mm H/ C mixed probe-head, operating in the pulse Fourier transform mode and controlled py an aspect 3000 computer. Each oligo?accharide was dissolzed in 0.4 ml exchanges with H20 (99.96% atom H, Aldrich, H 0 after three lyophilisations. The products were ML.f waukee, WI, USA) and intermediate analysed at 300 K with a spectral width of 3000 Hz for 16 K frequency and time domain data points giving a final digital domain points The 20 homonuclear COSY 45 experiments resolution of 0.365 Hz/ point. were performed using the standard Bruker pulse programme COSY. RESULTS AND DISCUSSION The
acidic
oligosaccharides
pyridine-acetate
buffer
chromatography tetraose the
of
which
point during
relative 1)
from to
in
days
order
to
the major further
was
state, Sugar
was (Fig.
analysis
3 Gal,
of the
sialyloligosaccharide and
confirmed
by the
FAB-MS
analysis
a pseudo-molecular
ion
m/z
C108H18g055N5Na
primary
fragments
GlcNAc)
are
mono
and
signifiant
disialylated
at
paper
5
at
chromatography rate
of
as an homogeneous
into
mM
paper
remaining
peaks
1 were
by
isolated
1 indicated
3 NeuAc
furnished formula
by
200
disialyllacto-N-
a migration
2 mg of compound
of
the
the
The material
having
fractionated
by
preparative
0.14 spot HPLC in
a
10 1 of human milk.
2 GlcNAc
1 Glc,
isolate
was obtained
Finally,
from
first
fractionated
further 2).
starting
anion-exchanger a
compound.
The compound
2 weeks.
but
chromatography
of
eluted submitted
to disialyllacto-N-tetraose
(Fig. pure
2
constituted
starting
developped
were
(chemical
the 2459.3 mass
residues.
This
permethylated (Fig. : 2458).
the
3),
presence
result
compound corresponding Moreover,
was which
the
to two
m/z 825 (NeuAc;Gal;GlcNAc) and 1186 (NeuAc 2 ;Gal; of the occurence of two branches , respectively Analysis by GLC-MS of the methylated (Fig. 4).
721
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fig.
1 . Paper chromatography of the sialyloligosaccharides obtained anion exchange chromatography. For experimental conditions, Materials and Methods. 1 : Total fraction; 2 : Fraction containing the trisialyloligosaccharide, obtained by preparative paper chromatography.
Fig.
2 . HPLC of fraction and Methods. R.I. trisialyloligosaccharide.
VI. For experimental : refraction index.
NeuAc(aP-S)G%(P l-3)GJ!NAcl(P
conditions, see Peak 1 corresponds
Materials to the
l-3)
NeuAc(a2-6)’
1186.;
535 825
Fig.
3 . FAB-MS
of
the
permethylated
722
trisialyloligosaccharide.
by see
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ne”ncca*-s,d%p l-4)GIEkMc(PI-6) II GalQ3 l-4dlc NeuAc(42-3)cJaVl(pl-3)Gl~NAc(P1-3) NeuAc(a2-6)’
-3
2la &
0
5.0
Fig.
4 . Zn-COSY
monosaccharides Me3Gal;
of
Me3Glc The
'H-NMN
Gal
and
spectrum
in
2,4
the
ratio
data
are
GlcNAc
with
H-l
of
to the
Glc
of
3,6
given
in
the
upfield
correlated
4,7,8,9
2,3,4
Me4Neu(Me)Ac;
Me2GlcN(Me)Ac;
4 Me
GlcN(Me)Ac
and
respectively. Table
can
314
trisialyloligosaccharide.
3:1:1:1:1:1:1,
downfield
R is
the
presence
Me2Gal;
residues
according
opposition signal
the
Me3Gal;
of
P.P.rn
3.8
4:2
spectrum
showed
2,4,6
2,3,6
4.6
be
1 and easily
with
723
of of
the
Fig.
4.
The
distinguished
resonances resonances
in
H-2
the
the
Gal
GlcNAc
signal
H-l on
H-2 H-2
at
3.320
signals the atoms,
atoms. ppm.
COSY in The
Vol.
177,
No.
BIOCHEMICAL
2, 1991
. 1 H- chemical
Table1 H-l
AND
shifts
H-2
NeuAc(aZ-6)
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(ppm) for trisialyloligosaccharide
H-3
H-4
H-5
H-6
1.714(ax)
3.647
N.D.
N.D.
NAC
H-6’
2.027
2.672(eq) IV' III'
Gal(Rl-4) GlcNAc(Ol-6)
4.443
3.533
3.669
3.926
3.94
4.01
3.55
4.692
3.151
N.D.
N.D.
3.54
3.92
3.79
2.091(a) 2.088(R)
1.783(ax)
NeuAc(aZ-3)
3.678
N.D.
N.D.
-
4.078
3.931
N.D.
N.D.
N.D.
1.684(ax)
3.678
N.D.
N.D.
-
2.037
2.019
2.027
2.758(eq) IV Gal(Rl-3)
4.496
3.529
NeuAc(aZ-6)
2.746(eq) III
GlcNAc(l)l-3)
4.726
3.889
N.D.
N.D.
3.54
3.96
3.82
II Gal(Sl-4)
4.439
3.581
3.717
4.153
N.D.
N.D.
N.D.
I Glc a
5.223
3.608
N.D.
N.D.
N.D.
N.D.
N.D.
8
4.659
3.320
3.640
N.D.
N.D.
N.D.
N.D.
The is
structural
feature
characterized
ppm and
6= 4.726
ppm,
H-3
at
6=4.078
ppm,
It
shifts
to GlcNAc-III
H-l
resonances
by comparison
[7]. chemical
a-2,6-linked
Gal
as infered
disialyllacto-N-tetraose characteristic
NeuAc(a2-3)Gal(Sl-3)[NeuAc(a2-6)]GlcNAc(l3l-3)
by significant
can
of the
[7],
and the
correlated
with
with
be
H-3ax
also and
lowfield
the
observed
H-l
at
reference deduced
H-3eq
of
shifted and H-2
6=4.499 compound
from NeuAc
the
residue
value
of GalIV
resonances
on the
confirmed
by the
COSY spectrum. The structure relevant
of the
B-1,6-linked
NMR parameters
of the
described
in compounds
previously
branch
can be also
NeuAc(a2-6)Ga1(01-4)GlcNAc(R1-6) S2:10:6
and S2:11:4
by Grijnberg
sequence et
al.
151.
The
combination
spectroscopy
allows
of
sugar
to
propose
analysis the
methylation, FAH-MS following structure for
oligosaccharide: NeuAc(a2-6)Ga1(01-4)GlcNAc(Rl-6) NeuAc(a2-3)Gal(Rl-3) NeuAc(a2-6)
\ GlcNAc(Bl-3) I 724
\ /
Gal(Ol-4)Glc
and
NMH
the
new
Vol.
177,
No.
2, 1991
In spite
BIOCHEMICAL
of
in
human
milk,
of
this
new
the it
relative of
core
of
tetraose
which
Gal is
The Gal(Bl-4)GlcNAc II
had not
yet
and the
it
predominates
(type
major
I)
is
to bear
the it
among the sialylated
at
as
in
oligosaccharide
a-2,6-sialylated, an a-2,3-linked
COMMUNICATIONS
trisialyloligosaccharide Indeed,
respectively,
disialylated is
RESEARCH
represents
which
GlcNAc,
found
that
of this
material.
branch
branch been
amount
BIOPHYSICAL
sialylated
[13],
The Gal(Rl-3)GlcNAc position
low
may be assumed
series
lacto-N-hexaose
AND
and this sialic
major
compound
possesses hexaose the
the
series.
C-3
and C-6
disialyllacto-Nof human milk. branch acid
of
type
residue.
Acknowledpents : This research was supported in part by the Centre National de la Recherche Scientifique (Unite Mixte nOll1: Relations structure-fonction des constituants membranaires; Director: Professor Andre Verbert), by the Universite des Sciences et Techniques de Lille Flandres-Artois and by the MinistBre de 1'Education Nationale. The authors are grateful to the Conseil Regional du Nord-Pas-de-Calais, the Centre National de la Recherche Scientifique, the Ministere de la Recherche et de 1'Enseignement Superieur, the Ministere de 1'Education Nationale and the Association pour la Recherche sur le Cancer for their contribution in the acquisition of the 400 MHz NMR apparatus. We are indebted to Miss Catherine Alonso (CNRS technician) for her skillful technical assistance. REFNRENCNS
(11 Kobata,
A. (1977) in "The Glycoconjugates" ( Horowitz, J. and Pigman, W. Eds) I, 423-440, Academic Press New York. P.A., McCrumb, D.K. and Wang, W.C. (1987) J. [21 Smith, D-F., Prieto, Biol. Chem. 262, 12040-12047. Bruntz, R., Dabrowski, U., Dabrowski, J., Ebersold, A., [31 Peter-Katalinic, J. and Egge, H. (1988) Biol. Chem. Hoppe-Seyler 369, 257-273. Strecker, G., Wieruszeski, J.M., Michalski, J.C. and Montreuil, J. [41 (1988) Glycoconjugate J. 5, 385-396. G., Lipniunas, P., Lundgren, T., Erlansson, K., Lindh, F. [51 Gronberg, and Nilsson, B. (1989) Carbohydr. Res. 191, 261-278. H., Nakada, H., Kurosaka, A., Hiraiwa N., Numata, Y., [61 Kitagawa, Fukui, S., Funakoshi, I., Kawasaki, T., Yamashina, I., Shimada, I. 28, 8891-8897. and Inagaki, F. (1989) Biochemistry Strecker, G., Wieruszeski, J.M., Michalski, J.C. and Montreuil, J. [71 (1989) Glycoconjugate J. 6, 67-83. G., Wieruszeski, J.M., Michalski, J.C. and Montreuil, J. [81 Strecker, (1989) Glycoconjugate J. 6, 169-182. G., Lipniunas, P., Lundgren, T., Lindh, F. and Nilsson, [91 Gronberg, B. (1990) Arch. Biochem. Biophys. 278, 297-311. J.M., Chekkor, A., Bouquelet, S., Montreuil, J.. 1101 Wieruszeski, Strecker, G., Peter-Katalinic, J. and Egge, H. (1985) Carbohydr. Res. 137, 127-138. S.M. (1950) Biochem. Sot. Symp. 3, 52-61. 1111 Partridge, B., Strecker, G., Leroy, Y. and Montreuil, J. (1981) Anal. [=I Fournet, Biochem. 116, 489-502. Kobata, A. and Ginsburg, V. (1972) J. Biol. Chem. 247, 1525-1529. r131
725