Primary structure of a trisialylated oligosaccharide from human milk

Vol.

177,

June

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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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

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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

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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

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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

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