zyxwvutsrqpo
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Eur. .I.Biochem. 203,257-268 (1992)
@) FEBS 1992
Neutral oligosaccharides of bovine submaxillary mucin
A combined mass spectrometry and 'H-NMR study
Wengang CHAI , Elizabeth F. HOUNSELL' , Geoffrey C. CASHMORE' , Jerzy R. ROSANKIEWICZ ', Christopher J. BAUER ',
James FEENEY Ten FEIZT and Alexander M. LAWSON
',
Section of Clinical Mass Spectrometry and ' Section of Glycoconjugates, MRC Clinical Research Centre, Harrow, Middlesex, England
MRC Biomedical NMR Centre, NIMR, Mill Hill, England
(Received June 12/September 17,1991) - EJB 91 0769
Twenty-two neutral 0-linked oligosaccharides ranging from monosaccharides to octasaccharides
were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 'H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other
sources of mucin. The structures include short linear sequences 3-linked to N-acetylgalactosaminitol
(GalNAcol) and branched structures based on either a GlcNAc(p1- 6)[Gal(p1- 3)IGalNAcol or
GlcNAc(B1 - 6)[GlcNAc(pl- 3)IGalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAcal - 6GalNAcol (GalNAc, N-acetylgalactosamine
and the hexasaccharide GlcNAc(p1- 6){GalNAc(al- 3)[Fuc(al-2)]Gal(~1-4)GlcNAc(fil3))GalNAcol (Fuc, L-fucose). Oligosaccharides of the blood-group-A type have not been detected
previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric-mucosal
glycoproteins has been established by classical immunochemical studies.
Bovine submaxillary-gland mucin (BSM) was among the
first mucin glycoproteins to be purified and studied [l - 51.
Early structural investigations of its 0-linked carbohydrate
chains (E 70% of the mass) indicated a predominance of the
acidic disaccharide NeuAc(a2 - 6)GalNAc [6 - 81. Later, the
presence of galactose, L-fucose (Fuc) and/or N-acetylglucosamine [9] was detected in addition to N-acetylneuraminic acid and N-acetylgalactosaminitol, consistent with the occurrence of longer chains and more complex oligosaccharides
in BSM. This has been confirmed by studies on acidic oligosaccharides [lo- 131.
Neutral oligosaccharides comprise approximately one
fifth of the total oligosaccharides and five structures have been
characterised previously [lo]. In the present study a more
comprehensive analysis of neutral oligosaccharides released
from BSM by Carlson degradation [14] has been made by a
combination of liquid secondary-ion mass spectrometry
(LSIMS), GC-MS and 'H-NMR, to establish the range of
structures present and allow comparison with other mucins.
The results show greater diversity of chains than previously
reported, among which are a series of oligosaccharides with
blood-group-A-rela ted sequences.
MATERIALS AND METHODS
Preparation and isolation
of BSM neutral oligosaccharide alditols
BSM, prepared by a procedure essentially as described
by Tettamanti and Pigman [3],was purchased from Sigma
Chemical Co., Dorset, England (Type 1-S).
Oligosaccharides were released from BSM by the digestion
procedure of Carlson [14]. In brief, BSM (450 mg) was incubated with 0.05 M NaOH/1.0 M NaBH, (20 ml) for 16 h at
45°C. After acidification to pH 5 by addition of acetic acid/
H 2 0 (1 : 1, by vol.), the reaction mixture was passed through
a column of ion-exchange resin (Bio-Rad) with a 15 ml lower
bed containing AG 50W-X2 (H' form) and a 20 ml upper
bed containing AG 50W-X8 (H' form), and washed with four
column volumes of HzO. The combined effluent and washes
were lyophilized and the boric acid removed by co-evaporation with several additions of methanol.
The residue was dissolved in 2 mM pyridine/acetate buffer
(pH 5.0, 2 ml) and applied to a Bio-Rad AG 1-X2 (acetate
form, 2 0 ml bed volume) anion-exchange column equilibrated
in the same buffer. The column was eluted with 50 ml buffer
and the effluent lyophilized to give neutral oligosaccharide
alditols (designated as N fraction; 23 mg dry mass). Further
elution with aqueous 0.3 M formic acid gave acidic sialylated
oligosaccharide alditols which were not analysed in the present
study.
The neutral oligosaccharides were chromatographed on a
Bio-Gel P-4 (200-400 mesh, 1.6 x 100 cm) column at 55°C
with elution by water and fractionated to give: N I , 8.8 mg;
N2,4.3 mg; N3, 3.2 mg; N4, 2.6 mg; N5,0.4 mg; N6, 0.2 mg
(Fig. 1).
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Correspondem. to A. M. Lawson, Section of Clinical Mass Spectrometry, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, England
Ahhreviutions. GalNAcol, N-acetylgalactosaminitol; GalNAc,
N-acelylgalactosaminc; dHex, deoxyhexose; Hex, hexose; HexNAc,
N-acetylhexosamine; HexNAcol, N-acetylhexosaminitol; Fuc, Lfucosc; LSIMS, liquid secondary-ion mass spectrometry; BSM, bovine submaxillary-gland mucin.
zyxwvutsrq
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258
HPLC purification
tor operated at 206 nm. Elution was performed with a linear
gradient of CH3CN/H20, 85:15 (by vol.) (solvent A) to
CH,CN/H20, 50:50 (by vol.) (solvent B) at a flow rate of
1 ml/min (for N2: 10% B to 60% B in 35 min; for N3: 20% B
to 70% B in 50 min; for N4 and N5: 30% B to 80% B in
50 min; for N6: 40% B to 90% B in 50 min) (Fig. 2).
The unresolved subfractions, N2-2/N2-3 and N3-3/N3-4, were
further fractionated by the same system using shallower gradients.
Fractions N2-N6 were chromatographed on an APSHypersil2 column (NH2, 5 pm, 5 x 250 mm, Shandon Scientific Runcorn, UK) using a Varian 5000 Liquid Chromatograph system with Varian UV-100 variable-wavelength detec-
Preparation of partially methylated alditol acetates
and GC-MS analysis
I
4
Permethylation was carried out essentially by the method
described by Ciucanu and Kerek [15]. The permethylated
oligosaccharide alditols were hydrolysed, reduced and
acetylated as described [16]. GC-MS analysis was performed
on a JEOL JMS-DX303 mass spectrometer using a DB-1
(30 m x 0.244 mm internal diameter x 0.25 pm) capillary column with helium as a carrier gas. The initial column temperature was 50°C programmed to 150°C at 25"C/min, to 250°C
at 5"C/min and to 280°C at 10"C/min. Mass spectra were
recorded at 70 eV electron energy and 300 PA, at a source
temperature of 240°C.
zyxwvutsrqponmlkjihg
zyxwvutsrqponmlkjihgfedcba
zyxwvutsrqponmlkjihgfedcba
5
B
7
6
10
9
11
12
Time (h)
Fig. 1. Bio-Gel P-4 chromatogram of neutral oligosaccharide alditols
released from BSM by alkaline horohydride degradation. The five
fractions N2 - N6 were further fractionated by HPLC. Calibration of
the column was carried out using a hydrolysate of dextran. Glucose
units are indicated by numbers (1 - 11).
Liquid secondary-ion mass spectrometry
LSIMS analysis was carried out on a VG ZAB-2E mass
spectrometer fitted with a caesium ion gun operated at
25 keV (for negative-ion detection) or 35 keV (for positive-
0.20
0.20
1
5
1
-R
0
M
2
N3
0
p5
0.11
e
4
2b
Ib
O''O
I0
30
20
0.15
I
30
0.15
N4
2
1
zyxwvutsrqpo
I
50
40
Time (minl
Time (min)
N6
-zyxwvutsrqponmlkjihg
0.10
(D
0
0
s
%
4
0.05
zyxwvutsrqponmlkjihgfed
J
I
10
20
30
Time (mid
40
,
50
I
10
20
30
Time (mid
1
A0
50
1
I0
20
30
40
50
Time Imtn)
Fig. 2. Elution profiles of oligosaccharide fractions N2 - N6 chromatographed on an APS-Hypersil 2 column. Fractions were collected for
compositional and sequence analysis.
z
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259
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zyx
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Table 1. LSIMS data of native and perrnethylated BSM neutral oligosaccharide alditol fractions.
Oligosaccharide
Native
(M-H)-
Saccharide composition
Permethylated
MH
m1z
+
fragment ions
mlz
N1
222
HexNAcol
N2-1
N2-2
N2-3
N2-4
N2-5
N2-6
42 5
42 5
384
530
587
733
HexNAcHexNAcol
HexN AcHexNAcol
HexHexNAcol
dHexHexHexNAco1
HexHexN AcHexNAcol
dHexHexHexNAcHexNAco1
553
553
512
686
757
93 1
2601228
2601228
29412761262
294/276/262
2601228
2601228
N3-1
N3-2
N3-3
N3-4
N3-5
N3-6
N3-7
587
628
733
587
733
749
89 5
HexHexNAcHexN Acol
HexNAczHexNAcol
dHexHexHexNAcHexNAco1
HexHexNAcHexN Acol
dHexHexHexNAcHexNAco1
Hex,HexNAcHexNAcol
dHexHexZHexNAcHexN Acol
757
798
931
757
931
961
1135
464/432
2601228
260/228,638
2601228
2601228
4641432
464/432
N4-1
N4-6
N4-7
790
(936
936
936
(895
1041
1082
(1041
1244
1187
HexHexNAc2HexNAcol
dHexHexHexN AczHexNAcol
dHexHexHexNAczHexNAcol
dHexHexHexNAczHexN Acol
dHexHexzHexNAcHexNAcol
dHex,Hex2HexNAcHexNAcol
dHexZHexHexNAcZHexNAcol
dHexzHexzHexNAcHexNAcol
dHex2Hex2HexNAczHexNAcol
dHex,Hex2HexNAcHexNAcol
1002
1176
1176
1176
1135
1309
1350
1309
1554
1483
2601228,4641432
883)
2601228
260/228,638/606
4641432)
638/606
260/228, 812
6381606)
260/228,883/638
8 121606
N5-2
N5-3
N5-5
1139
1244
1244
dHexHexHexNAc3HexNAcol
dHex2HexZHexNAczHexNAcol
dHexzHexzHexNAc2HexNAcol
1421
1554
1554
2601228, 883j851
260/228, 8831638
638
N6-2
1447
dHex2HexzHexNAc3HexNAcol
1799
260/228,883/638
N4-2
N4-3
N4-4
N4-5
zyxwvu
zyxw
ion detection) with an emission current of 0.5 pA. Full mass
spectra were acquired at 30 s . decade- using the VG Analytical 11 - 250 J data system in continuum acquisition mode.
Spectra of native oligosaccharides (1 - 3 pg) were recorded in
negative-ion mode and permethylated derivatives ( x 1 pg) in
positive-ion mode. Thioglycerol was used as the liquid matrix.
'H-NMR spectroscopy
500 MHz 'H-NMR spectra were obtained using a Bruker
AM500 spectrometer operating in the Fourier transform
mode and equipped with an Aspect 3000 computer. Chemical
shifts were measured in ppm from the signal for internal
acetone and given with reference sodium 4,4-dimethyl-4silapentane 1-sulphonate taken as 2.225 ppm from acetone at
295 K. The spectra were interpreted with the aid of a computer
program [17] designed to match signals in the spectra with
those reported in the literature. The spectrum of the disaccharide in fraction N2-2 was completely assigned by decoupling
in a one-dimensional difference experiment. The oligosaccharides N2-2, N5-2 and N6-2 were analysed by 600 MHz
'H-NMR spectroscopy on a Varian Unity 600. Spectra were
obtained using a shifted Gaussian weighting function. Phasesensitive dou ble-quantum-filtered correlated spectroscopy
was carried out at 600 MHz on N6-2.
RESULTS
The released neutral oligosaccharide alditols from BSM
were fractionated on a Bio-Gel P-4 column (Fig. 1) and the
peaks N2 - N6 were re-chromatographed separately on an
HPLC column of alkylamine-bonded silica (APS-Hypersil 2).
HPLC subfractions were analysed by LSIMS to establish
composition and purity (Table 1). Fractions N2-1 -N2-6,
N3-1 -N3-7, N4-1 -N4-7, N5-2, N5-3, N5-5 and N6-2
(Fig. 2) contained sufficient amounts of carbohydrate material
for structural elucidation. LSIMS was carried out on native
and permethylated fractions providing evidence of the composition and partial sequence (Table l),with methylation analysis giving linkage information (Table 2). Homogeneous oligosaccharide fractions having a composition not previously reported for BSM carbohydrate chains were analysed by 'HNMR.
Each Bio-Gel P-4 fraction was initially screened by negative-ion LSIMS to determine its homogeneity. As a representative example, the spectrum from fraction N3 (Fig. 3) indicates the presence of at least five oligosaccharide molecular
species ([M-HI- ions: m/z 587, 628, 733, 749 and 895). The
relative abundance of these ions and the ultraviolet detection
response at 206 nm obtained from individual components by
HPLC of N3 (Fig. 2) were in good agreement.
260
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II
3
i n1
zyx
zy
(7)
1:49
895
Fig. 3. The negative-ion LSI mass spectrum of native oligosaccharides
in fraction N3 from Bio-Gel P-4 chromatography. The peaks in the
HPLC chromatogram of N3 (see Fig. 2) containing the assigned [MHI- ions are shown in parenthesis.
Table 3. BSM oligosaccharides with unbranched core GalNAcol. Abundance was based on the dry mass of Bio-Gel P-4 fractions and the
subsequent HPLC profiles determined by ultraviolet absorbance at
206 nm.
Approximate
abundance
of fraction N
(by mass)
Fraction
Structure
N1
N2-1
N2-2
N2-3
N2-4
N3-1
N3-3
GalNAcol
44
GlcNAc(1- 3)GalNAcol
11.5
GalNAc(a1 - 6)GalNAcol
3.0
0.8
Gal(1 - 3)GalNAcol
Fuc(l-2)Ga1(1-3)GalNAcol
5.0
Gal(l-4)GlcNAc(l-3)GalNAcol
0.3
GalNAc(1 -3)[Fuc(l-2)]Ga~(I -3)GalNAcol 1.0
%
0
I
I I i,l
I
I
I
I
I I
I
l + Z l
I
I
I
IS1
I
zyxwv
zyxwvu
I
The major ohgosaccharides identified were categorised
into three groups; those having an unbranched core GalNAcol
(Table 3), those having a trihexosamine core (Table 4) and
those with a branched GlcNAc(1- 6)[Gal(l- 3)IGalNAcol
core (Table 5). The relative abundance of oligosaccharide
components were determined by HPLC.
I
0
I I S 1 I
h
Oligosaccharides having unbranched core regions
(fractions N l , N2-1- N2-4, N3-1 and N3-3)
I
I
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As expected from its elution position on Bio-Gel P-4 and
from its liquid secondary-ion (LSI) mass spectrum, fraction
N1 contained the monosaccharide GalNAcol identified previously amongst the oligosaccharides released from BSM [lo].
Oligosaccharides in fractions N2-1 and N2-3, identified by
mass spectrometry (Tables 1 and 2) as GlcNAc(1-3)GalNAcol and Gal(1- 3)GalNAcol, respectively, were presumed
to be the major oligosaccharides identified earlier [ 101 and
were not analysed further.
The LSI mass spectrum of permethylated fraction N2-2
(Fig. 4) and methylation analysis (Table 2) indicated the pres-
z
zy
261
Table 4. BSM oligosaccharideswith core type: GlcNAc(1- 6)(GlcNAc(l- 3)jCalNAcol.
Fraction
Structure
Approximate abundance
of fraction N (by mass)
~~
Yo
N3-2
GlCNAc(P1- 6)
\ GalNAcol
3.9
GlcNAc(P1- 3) /
Gal(P1- 4)GlcNAc(Pl - 6)
N4- 1
'
Fuc( 1 -2)Gal(1 -4)GlcNAc(l- 6)
N4-3
1.2
\ GalNAcol
2.6
GlcN Ac( 1 - 3) /
F~~(~-~)G~(~-~)[Fuc(I-~)]G~cNAc(~
-6)
N4-5
\ GalNAcol
GlcNAc(1- 3) /
N5-2
0.5
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zyx
ClcNAc(/ll- 6)
GalNAc(al-3)[Fuc(al-2)]Gal(/lI
N5-5
zy
zyxw
GalNAcol
GlcNAc(P1- 3) /
'
GalNAcol
0.5
\ GalNAcol
0.2
/
-4)GlcNAc(/?1-3)
Fuc(a1- 2)Gal(PI -4)GlcNAc(/l1-6)
/
Fuc(al-2)Gal(~1-4)ClcNAc(~1-3)
zyxwvutsr
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zyxwvutsrqponmlk
260
I
MH +
88
2
B
553
68
I
4 48
d
28
8
m z
8
Fig. 4. The positive-ion LSI mass spectrum of permethylatcd fraction N2-2, GalNAc(a1- 6)GalNAcol.
-
HB'GalN
-
H4GalN
-
HlGalN
H2GalN
H201
H5GalN
I
.
.
m
- y i l v - T - ,
< -
4
1
I
d
i
a ,
H6GalN
z
-- H501
H3GalN
Ha01
H6'ol
Hlol
m
-
H401
H601
Hl'Ol
7
1
,
4
"
3 J
3 0
9 7
Chemical Shift (ppm)
Fig. 5.600-MHz 'H-NMR spectrum of oligosaccharide fraction N2-2, GalNAc(a1- 6)GalNAcol.
3 6
3 5
3 4
262
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z
Table 5. BSM oligosaccharideswith core type: GlcNAc(1- 6)[Gal(l- 3)]GalNAcol. Fractions containing the same oligosaccharide are listed together.
~
~~
Approximate abundance
of fraction N (by mass)
Structure
Fraction
Yo
GlcNAc(P1- 6)
N2-5/N3-4
\ GaiNAcol
1.0, 3.2
\ GalNAcol
0.2, 5.9
Gal(B1- 3) /
GlcNAc(P1-6)
N2-6/N3-5
Fuc(al-2)Gal(Pl-
zyxw
3) /
N3-6
G ~ I ( / ~ ~ - ~ ) G I C N A 6)
C(~~I\ GalNAcol
Gal(jl1- 3) /
N3-7
Gal(P1- 4)GlcNAc(P1-6)
1.O
\ GalNAcol
0.7
\ GalNAcol
/
1.5
\ GalNAcol
6.6
\ GalNAcol
0.5
Fuc(a1-2)Gal(B1 - 3) /
GlcNAc(P1- 6)
N4-2
GalNAc(al-3)[Fuc(al -2)]Gal(P1-3)
Fuc(a1- 2)Gal(/?1-4)GlcNAc(fi1-6)
N4-4
/
Fuc(al-2)Gal(Bl-3)
Fuc(a1-2)Gal(jl1 -4)[Fuc(al-3)]GlcNAc(P1-6)
N4-7
Fuc(al-2)Gal(~1 - 3) /
GalNAc(a1- 3)[Fuc(al- 2)]Gal(B1-4)GlcNAc(jll- 6)
N4-6/N5-3
Fuc(al-2)Gal(BlGaINAc(a1- 3)[Fuc(al-2)]Gal(/?1-4)GlcNAc(P1-
N6-2
6)
zy
zyx
\ GalNAcol
3) /
'
GalNAc(a1- 3)[Fuc(d- 2)]Gal(al- 3) /
0.1,0.7
0.3
GalNAcol
Table 6. Comparison of the 'H-NMR chemical shifts and coupling constants for disaccharide a-wGalNAc(1- 3)-~-GalNAcol1 (data from [HI)
and a-D-GalNAc(l-6)-D-GalNAcol 2. Coupling constants given to one decimal place are k0.3 Hz and those given to two decimal places are
kO.01 Hz.
Disaccharide
Residue
Chemical shift
zy
H1
H1'
H2
H3
H4
H5
H6
H6'
NAc
2
2
GalNAc
GalNAcol
GalNAc
GalNAcol
5.103
3.810
4.916
3.739
3.718
3.676
4.235
4.395
4.186
4.256
3.921
3.888
4.075
3.872
4.043
3.680
3.992
3.397
4.073
3.749
4.084
3.990
3.791
3.647
3.750
3.565
3.768
3.647
3.779
3.797
2.049
2.060
2.046
2.055
Disaccharide
Residue
Coupling constant
J1,z
Ji,,z
J2.3
J3.4
4.07
5.60
3.9
6.10
8.22
1.94
11.32
2.32
10.99
10.99
3.11
8.00
2.95
1.83
1
1
Jt.1.
zyxwvut
J4.5
J5.6
j5,6'
J6,6'
1.20
2.08
1.2
9.77
5.36
7.00
11.74
4.88
4.88
1.30
7.37
11.9
10.37
Hz
~~
1
1
2
2
GalNAc
GalNAcol
GalNAc
GalNAcol
11.45
11.3
z
zy
zyxwv
263
Table 7. 'H-NMR chemical shifts of oligosaccharide fractions having the GlcNAc(l-6)[GlcNAc(l-3)~GalNAcol core. D-GalNAcol, (0);
p-Dgalactose, (0);
j-D-N-acetylglucosamine,(0);u-L-fucose,( A ) ; a-D-N-acetylgalactOSamine,(H). The superscripts at the name of the sugar indicate
the linkage positions of the subsequent monosaccharides in the sequence.
Residue
GalNAcol
Gal(l43)
,2)4.3
Signal
H2
H3
H4
H5
NAc
HI
H2
H4
HI
H5
Chemical shifts of fraction
N3-2
N4- 1
N5-2
4.285
3.989
4.238
2.045
4.283
3.987
3.518
4.242
2.045
4.282
3.985
3.514
2.044
N5-5
zyxw
CH3
GlcNAc
(1-6)
GlcNAc
(1+3)
Gal
(1 -+4)
Fuc
(I -21496
Fuc(1-3)
Fuc( 1 -2)
Blood group A
GalNAca
HI
H6
NAc
HI
H6
NAc
H1
H2
H4
HI
H5
CH3
HI
145
CH3
HI
H4
H5
CH3
HI
NAc
4.538
2.064
4.599
-
2.082
ence of a novel disaccharide GalNAc(1- 6)GalNAcol. Small
amounts of the partially methylated alditol acetates for
GlcNAc(1 -) and (- 3)GalNAcol were due to carry over from
the previous HPLC fraction N2-1. The 'H-NMR spectrum
(Fig. 5) showed a downfield a-anomeric signal o f J l . z = 4 Hz
and chemical shift 4.916 ppm, characteristic of a GalNAccl
residue. Comparison of the signals of GalNAcol with the
related oligosaccharides GalNAc(a1- 3)GalNAcol (Table 6)
and Ga1(~1-4)GlcNAc(~1-6)GalNAcol[IS, 191 gave confirmatory evidence for the assignment GalNAc(al-6)GalN Acol.
LSIMS analysis of the native fraction N2-4 ([M-HI-,
m/z 530, Table 1) and of the permethylated fraction (MH',
m/z 686) together with methylation analysis (Table 2) showed
that the sequence Fuc(1- 2)Ga1(1- 3)GalNAcol commonly
found in mucins is also present in BSM.
4.562
3.998
2.060
4.597
3.952
2.082
4.412
4.542
2.066
4.597
3.949
2.080
5.598
4.280
3.510
4.225
2.043
5.31 1
4.249
1.234
4.540
2.066
4.602
2.082
4,54014,526
3.89213392
5.305
3.925
1.234
5.348
3.837
4.320
1.251
5.177
2.039
zyz
zyxwvu
N3-1 contained a trisaccharide with a Hex/HexNAc/
HexNAcol (Hex, hexose; HexNAc, N-acetylhexosamiae;
HexNAcol, N-acetylhexosaminitol) composition, [M-HI- at
rnjz 587 (Table 1). Permethylation gave a MH' at m/z 757
with a strong fragment doublet at m/z 464/432 indicative of
the Hex(1- 4)HexNAc sequence. Methylation analysis was
consistent with the sequence ga1(1 - 4)GlcNAc(l- 3)GalNAcol.
Fraction N3-3 was of limited amount but could be partially
characterised. LSIMS of the free and permethylated oligosaccharide indicated the dHex/Hex/HexNAc/HexNAcol (dHex,
deoxyhexose) composition (Table 1). The approximate
1 : 1 : 1 :1 ratio of Fuc(1 -)/GalNAc(l -)/( -2,3)Gal(l-)/
( - 3)GalNAcol linkages by methylation analysis was consistent with two possible structures but most likely to represent
the scquence GalNAc(1- 3)[Fuc(l- 2)]Ga1(1- 3)GalNAcol.
264
zyxwvutsrq
zyxwvutsrqponmlkjihg
- -- -
-
HlGaN
H 1Fuc
/
5.4
I
,
I
H2ol
HlGlN
HlGal
, l , , , , , l , j1 /
,
,
,
,> ,
5.3
5.2
ppm
-
H501
n
HlGlN
H301
-
HSFuc H2GaN
m
H5GaN
(
4.5
,
,
,
,
1
4.4
,
,
,
,
,
4.3
/
1
,
1
(
,
,
,
4.2
,
4.1
/
,
,
~
(
1
4.0
1
-
H401
H4FuC
rl
H6GlN
I
l
l
,
3.9
,
I
I
l
l
I
I
7
8 1 ,
3.7
3.8
I
7
I
)
3.6
'
,
I
'
,
3.5
"
'
I
/
3.4
Chemical Shift (ppm)
Fig. 6. 600-MHz 'H-NMR spectrum of oligosaccharide fraction N5-2, GlcNAc(B1- 6)(GalNAc(ul- 3)[Fuc(al- Z)]Gal(Bl- 4)GlcNAc(B1- 3))
GalNAcol.
zyxwvutsrqp
zyxwvutsrqponm
zyxwvutsrqponm
xi0 '0
'XIO'O
P
2
3
0
2
zyxwvutsrqponm
zyxwvutsr
883
68.
MH+
1421
5 48- ,228
2
B
851
MNa'
1443
Fig. 7. The positive-ion LSI mass spectrum of permethylated fraction N5-2, GlcNAc(B1- 6)(GalNAc(ul- 3)Fuc(al- Z)]GaI(B1-4)GlcNAc(/?l- 3))
GalNAcol.
Oligosaccharides based on the trihexosamine core
GlcNAc(p1- 6)[GlcNAc(jl- 3)]GalNAcol
(fractions N3-2, N4-1, N4-3, N4-5, N5-2 and N5-5)
The minimum structure of this series, the trisaccharide
core, was present in fraction N3-2 as indicated by its LSI mass
spectrum ([M-HI-, m/z 628) and 'H-NMR analysis (Table 7).
The latter gave a spectrum having chemical shifts typical of
GalNAcol linked at C6 and C3 by GlcNAcP [16,17] and two
additional N-acetamido methyl signals and two p-anomeric
doublets of coupling constant J1, = 8.3 Hz, indicative of
the two GlcNAc residues. LSIMS and methylation analysis
agreed fully with this interpretation (Tables 1 and 2, respectively).
The saccharide composition ([M-HI-, m/z 790) and 'HNMR analysis (Table 7) of fraction N4-1 were consistent with
its major component being a tetrasaccharide having GalPl -4
linked to the GlcNAc on the 6 branch of the core (Table 4),
the 'H-NMR data for which has been described previously
for an oligosaccharide of bronchial mucins [20]. The LSI mass
spectrum of the permethylated fraction (MH', m/z 1002) had
fragment ions, mjz 260/228 and 4641432 corresponding to
GlcNAc and Gal-GlcNAc sequences, respectively and were in
accord with this assignment. Additional ions in the spectrum
at nz/z 1176 (MH') and mlz 883 (fragment ion) indicated the
presence of a less-abundant component which from methylation analysis (Table 2) showed minor amounts of linkages
Fuc(1 -), GalNAc(1 -), (-2,3)Ga1(1-) and (-3)GalNAcol
and suggested the structure GalNAc(1- 3)[Fuc(l-2)]Gal(1-4)GlcNAc(l- 3)GalNAcol.
LSIMS of the native fraction N4-3 ([M-HI-, m/z 936 and
895) was consistent with the presence of two components with
compositions dHex/Hex/HexNAc2/HexNAcol and dHex/
Hex2/HexNAc/HexNAcol.The ratio of partially methylated
alditol acetates produced from fraction N4-3 representing the
linkages Fuc(1 -)/Gal(l -)/GlcNAc(l -)I( - 4)GlcNAc(l-)/
(- 2)Gal(1-)/( - 3,6)GalNAcol in the approximate ratio of
1 :0.3 :0.8 : 1 : 1 : 1 (Table 2), were interpreted as indicating an
80: 20 mixture of the blood-group-H-containing structure
Fuc(1 - 2)Ga1(1 - 4)GlcNAc(l - 6 / 3)[GlcNAc(l - 6 / 3)]GalNAcol and Gal(l-4)GlcNAc(l- 6)[Fuc(l-2)Ga1(1- 3)]GalNAcol (Tables 4 and 5). Fragment ions in the LSIMS
permethylated spectrum, mlz 2601228 (HexNAc), mlz 4641432
(Hex/HexNAc) and rn/z 6381606 (dHexlHexlHexNAc) were
in agreement with these assignments. Subsequent microscale
sequence analysis based on TLC-MS of neoglycolipid derivatives [21] confirmed the presence of the major component with
Fuc-Hex-HexNAc on the 6 arm of GalNAcol and HexNAc
on the 3 arm (data not shown).
Fraction N4-5 was a mixture of several components with
carry over from fraction N4-4 and consequently could not be
completely characterised. The LSI mass spectrum of permethylated fraction N4-5 indicated its major component to
have a composition dHexz/Hex/HexNAc,/HexNAcol
(MH'
at m/z 1350, Table 1) and fragment ions m/z 260 (terminal
GlcNAc) and mlz 812 (dHexz/Hex/HexNAc).Together with
the methylation analysis (Table 2) N4-5 was deduced to contain an analogue of N4-3 (Table 4) having an additional fucose residue linked 1-3 to GlcNAc giving Fuc(lh2)Gal(l-t4)[Fuc(1 +3)]GlcNAc sequence on the 1-6 arm.
zyxwvutsrq
z
zyxwvut
zyxwvuts
zy
zyxwvutsr
26 5
Confirmation of this was obtained by microsequencing analysis [21].
The [M-HI- at m/z 1139 obtained from LSIMS of native
fraction N5-2 implied a hexasaccharide composition of dHex/
Hex/HexNAc,/HexNAcol. Results of H-NMR analysis
(Table 7 and Fig. 6) indicated N5-2 to be a novel structure
and were consistent with the trihexosamine core extended on
the 3-linked GlcNAc by the blood-group-A sequence GalNAc(a1- 3)[Fuc(ctl-2)]Gal(/31-4). The composition was
confirmed from the permethylated LSI mass spectrum which
indicated the GlcNAc and GalNAc-(Fuc-)Gal-GlcNAc
branching sequences from fragment ions at m/z 260 and 883,
respectively (Fig 7). Linkage information from the partially
inethylated alditol acetate derivatives was also consistent with
the hexasaccharide structure showing Fuc(1- )/GlcNAc(l -)/
GalNAc(1 -) /(-4)GlcNAc(l-)/(-2,3)Ga1(1-)/(-3,6)GalNAcol in the approximate ratio 1 : 1 :1: 1: 1: 1 (Table 2).
N5-5 had a monosaccharide composition of dHex2Hex2HexNAc2HexNAcol ([M-H]-, m / z 1244) and an NMR spectrum consistent with that of a heptasaccharide, previously
identified in bronchial mucins [20], having two blood-groupH-containing chains, Fuc(a1- 2)Gal(p1- 4)GlcNAc(/31-),
on the core GalNAcol. The LSI mass spectrum of
permethylated N5-5 showed an M H t at mjz 1554 and an
intense fragment ion at m/z 638 indicative of the dHex/Hex/
HexNAc. Methylation analysis confirmed the linkages
Fuc(1 -)/( - 2)Ga1(1 -)I( -4)GlcNAc(l-)/( - 3,6)GalNAcolin
an approximate ratio 2: 2: 2: 1 (Table 2).
-
3.5-
'
1
zyxwvutsrqpon
H5GaN
g;
H4G
HBGaNIol
H2GaN
H201,-
so
.;f
H3Gal
. ..
'
-e
fg
-:0004:
HGGaN
-HGGaN
;..
H3GaN
H5Fuc
HlFuc
H 1 GaN
HlGal
HlGlN
zyxwvuts
Oligosaccharides having a GlcNAc(b1- 6)[Gal(bl- 3)]GalNAcol core (fractions N2-5/N3-4, N2-6/N3-5, N3-6, N3-7,
N4-2, N4-4, N4-6/N5-3, N4-7 and N6-2)
The trisaccharide core structure GlcNAc(/YI - 6)[Gal(/313)]GalNAcol (fractions N2-5/N3-4) and its analogue fucosylated at C2 of galactose on the (2-3 branch (fractions
N2-6jN3-5) were identified by Tsuji and Osawa [lo] and have
been characterised previously from other sources by 'H-NMR
[18, 20, 22-25]. Their structures were fully established in the
present study (Tables 1, 2 and 8).
Oligosaccharides representing a Gal(1- 4) extension of
the 6-linked GlcNAc of the above structures were found in
fractions N3-6 and N3-7. The tetrasaccharide N3-6 gave an
[M-HI- at mjz 749 in negative LSIMS indicating the Hex2/
HexNAc/HexNAcol composition and an 'H-NMR spectrum
(Table 8) in accordance with this commonly found mucin
oligosaccharide [18, 20, 22, 24-26]. LSIMS of the native
([M-HI-, mjz 895) and permethylated (MH', m/z 1135) fraction N3-7 indicated a pentasaccharide structure (Table 5) and
'H-NMR data were consistent with previously reported data
[23-271 (Table 8). Fragment ions, m/z 464/432, in the permethylated spectrum indicated the Gal-GlcNAc- branch while
a small ion at mjz 961 may have arisen from fucose loss from
the MH' ion or from the tetrasaccharide in N3-6 due to some
overlap in fraction collection.
The composition of N4-2 was established as dHex/Hex/
HexNAc2/HexNAcol from LSIMS of the native oligosaccharide ([M-HI-, m/z 936). The NMR spectrum (Table 8) was
consistent with the previously characterised blood-group-Acontaining pentasaccharide [28]. LSIMS of the permethylation product MH' m/z 1176 and the partially methylated
alditol acetates (Table 2) were consistent with this interpretation, the latter clearly showing the GalNAc(1 -) and
(-2,3)Gal(l -) features.
4.5
Fig. 8. The double-quantum-filtered correlated spectrum at 600 MHz
of oligosaccharidefraction N6-2 GalNAc(a1- 3)(Fuc(al- Z)1Gal(/31- 4)GlcNAc(B1- 6){GalNAc(~l-3)Fuc(al- 2)1Gal(!l- 3)]GalNAcol.
The monosaccharide composition of fraction N4-4 was
m/z 1041). The
'H-NMR spectrum (Table 8) matched previously recorded
data [23,24,26] for a hexasaccharide having a terminal bloodgroup-H determinant on both arms. Its permethylated LSIMS
MH' ion at m/z 1309 and intense fragment ions m / z 638/606,
together with methylation analysis (Table 2), were in accord
with this assignment.
An analogue of N4-4 having an additional fucose residue was identified in fraction N4-7 (dHex,Hex,HexNAcHexNAcol; [M-HI-, m/z 1187). The NMR spectrum
(Table 8) showed the presence of a heptasaccharide previously
characterised in bronchial mucin [24]. The LSI mass spectrum
of the permethylated fraction yielded mjz 1483 (MH') and
fragment ions mjz 812, produced from cleavage of the
GlcNAc(1-6) glycosidic bond, and m/z 606 from subsequent
elimination of the 3-linked fucose from mlz 812 [29]. Methylation analysis confirmed this linkage with the presence of the
partially methylated alditol acetate from (- 3,4)GlcNAc(l-)
as well as substantiating all other linkages (Table 2).
The NMR spectra of N4-6/N5-3 and N6-2 (Table 8)
showed the presence of blood-group-A-containing heptasaccharides and octasaccharides previously characterised in
blood-group-A-active human ovarian mucins [28]. Chemical
shifts were assigned by reference to the literature [28] and
the two-dimensional correlated spectrum shown in Fig. 8 for
fraction N6-2. Their structures were confirmed by mass spectrometry as follows: fractions N4-6/N5-3 contained a heptasaccharide of composition dHex2Hex2HexNAc2HexNAcol
([M-HI-, m/z 1244). The permethylated LSI mass spectrum
was in agreement with this composition ( M H + ,m/z 1554) and
showed the 6-linked oligosaccharide chain from the fragment
ion mjz 883, HexNAc(dHex-)HexHexNAc, and m/z 2601228
for the terminal HexNAc. Methylation analysis gave an approximate 2: 1 : 1: 1 : 1 : 1 ratio of partially methylated alditol
acetates representing Fuc(1 -)/GalNAc(l -)/( - 2)Ga1(1-)/
dHex2/Hexz/HexNAc/HexNAcol
([M-H]-,
zyxwvutzz
N
Table 8. 'H-NMR chemical shifts of oligosaccharide fractions having the GlcNAc(l-+6)1Gal(l- 3)1GalNAcol core structure. Abbreviations and superscripts as used in the legend to Table 7.
~~~~~~
Residue
Signal
GalNAcol
Gal( 1--t 3)
GlcNAc
~~
Chemical shifts of fraction
N3-4
N3-5
N3-6
N3-7
N4-2
N4-4
N4-6/N5-3
N4-7
N6-2
4.395
4.061
3.466
4.282
2.067
4.464
3.560
3.899
4.403
4.084
3.503
4.257
2.058
4.573
3.538
3.92
5.222
4.277
1.244
4.550
3.94
2.054
4.398
4.063
3.460
4.287
2.067
4.465
3.560
3.897
4.30
3.579
4.161
2.036
3.529
4.209
4.406
4.084
3.499
4.263
2.054
4.514
3.922
5.221
4.278
1.243
4.551
3.997
2.060
4.404
4.080
3.498
4.271
2.053
4.573
3.923
5.221
4.227
1.240
4.549
3.985
2.060
4.405
4.082
3.489
4.255
2.055
4.573
3.584
-
4.558
3.999
2.065
4.404
4.084
3.498
4.255
2.055
4.579
3.538
3.925
5.223
4.277
1.245
4.566
4.001
2.055
4.081
3.584
2.036
4.696
3.898
4.225
5.184
4.319
1.228
4.567
4.410
3.537
3.924
4.471
3.538
3.926
4.539
3.922
5.331
4.229
1.233
4.598
-
4.051
zyxwvutsrqponm
Fuc( 1-+2)
(1 -4
H2
H3
H4
H5
N Ac
HI
H2
H4
H1
H5
CH3
HI
H6
NAc
H1
H6
NAc
H1
H2
H4
H1
H5
CH 3
HI
H5
CH3
HI
H2
H3
H4
H5
HI
H2
H5
CH3
o\
o\
GlcNAc
(1-3)
Gal
(1 +4)
Fuc
(1 +2)4,6
Fuc(1+3)
GalNAcx
Fuc(l+2)
Blood group A
4.535
3.932
2.067
4.554
3.935
2.055
-
2.057
4.596
3.906
4.216
5.283
1.236
5.104
1.272
5.178
5.176
3.999
2.047
5.372
3.996
2.039
5.319
4.316
1.232
4.319
1.252
-
5.219
4.276
1.243
4.556
2.050
-
5.174/5.184
4.244
3.9
-
2.04512.045
5.349
3.8
4.318
1.247
z
zyxwvutsrqp
zyxwvut
zyxwvutsr
267
+ xi0 '0
I88
XiO'O
260
883
MH+
zyx
638
I
P
4 48
2
zi?
228
I
1799
28
E
288
Fig. 9. The positive-ion LSI mass spectrum of permethylated fraction N6-2, GalNAc(a1- 3))IFuc(al-2)jGal(/?1-4)GlcNAc(~1- 6)(GalNAc(a1- 3)[Fuc(al- 2)jGal(B1- 3))GalNAcol.
zyxwvutsrqp
zyxwvutsrqp
(-4)GlcNAc(l-)/( -2,3)Gal(l-)/( - 3,6)GalNAcol, supported the assigned linkages (Table 2). For fraction N6-2 a
[M-HI- ion at m/z 1447 of the native oligosaccharide indicated
a composition of dHex2/Hex2/HexNAc3/HexNAcol.
The permethylated LSI mass spectrum (MH+, m/z 1799) gave rise to
fragment ions from the 6-linked and 3-linked sequences that
provided evidence for the blood-group-A-containing sequences at n ? / 883,
~ GalNAc(Fuc-)Gal-GlcNAc- and m/z 638,
GalNAc(Fuc-)Gal-, the latter arising from a less common
glycosidic cleavage at galactose (Fig. 9). Methylation analysis
substantiated the composition and monosaccharide linkages
of N6-2 (Table 2) and its assigned structure (Table 5).
DISCUSSION
This study highlights the complexity of neutral 0-linked
oligosaccharides present in BSM in contrast to the previously
perceived simplicity of chains from this source. Of the 22
oligosaccharides identified, ranging from monosaccharides to
octasaccharides, only five have been detected previously in
BSM [lo].
A combination of LSIMS of native and permethylated
oligosaccharides, 'H-NMR and methylation analysis was
used as appropriate to define the oligosaccharide alditols
released from BSM under the conditions employed by Carlson
[14]. Alternative conditions of alkali/borohydride hydrolysis
used to liberate oligosaccharides from the glycoprotein [ 10,
111 were found by LSIMS screening of released oligosaccharides, to yield lower amounts of the larger oligosaccharides
and give rise to small amounts of unidentified but degraded
oligosaccharides (results not shown). The source of BSM and
preparation method used might also be expected to affect the
final proportions of individual oligosaccharides determined.
The chromatographic isolation strategy to obtain relatively pure fractions of oligosaccharides was based on gelfiltration chromatography and subsequent HPLC as this provided the best means of optimizing separation of components
differing in molecular mass and concentration. Negative-ion
LSIMS screening of Bio-Gel P-4 and HPLC fractions proved
of particular value for estimating the approximate distribution
of molecular species present. The relative intensity of [M-HIions representing each component in a mixture agreed closely
with their HPLC profile from ultraviolet detection at
206 nm. This is likely to be dependent upon the dHex, Hex
and HexNAc composition of the oligosaccharides analysed
and the balance of HexNAc to Hex or dHex in relation to
greater surface activity in the LSIMS matrix and ultraviolet
absorbance of HexNAc. The commonly observed suppression
of LSIMS signals of one component by another in a mixture
(e. g. peptides) is not as apparent for native oligosaccharides
due to structural similarity.
Of the oligosaccharides characterised with short linear
sequences linked to GalNAcol, the disaccharide GalNAc(a16)GalNAcol has not been previously identified in BSM or, to
our knowledge, on any other mucin glycoprotein. In 0-linked
oligosaccharides of glycoproteins, N-acetylgalactosamine,
when found as a chain-terminating monosaccharide, is usually
linked a1 - 3 to galactose or N-acetylgalactosamine. This
new disaccharide suggests the existence of an N-acetylgalactosaminyl transferase which facilitates cll - 6 linkage of
GalNAc to GalNAcal-protein. The majority of structures
elucidated contained the branched (- 3,6)GalNAcol core,
based either on GlcNAc(j1- 6)[Gal(P1- 3)IGalNAcol or
GlcNAc(~1-6)[GlcNAc(PI - 3)IGalNAcol. In the latter series a novel hexasaccharide, GlcNAc(P1- 6)(GalNAc(al- 3)Fuc(clI-2)]Gal(PI -4)GlcNAc(PI - 3))GalNAcol, was identified. The blood-group-A structure has not been found previously in BSM although it has been detected by immunochemical methods in bovine gastric-mucosal glycoproteins by
Kabat and associates [30]. In the present study an additional
three oligosaccharides were identified with the blood-groupA sequence in structures with a GlcNAc(P1- 6)[Gal(Pl- 3)]GalNAcol core region. The anomeric configurations were not
assigned in an unbranched oligosaccharide, GaINAc(1- 3)[Fuc(l - 2)]Ga1(1- 3)GalNAcol, but it may also have the
blood-group determinant.
In its structural diversity of 0-linked oligosaccharides,
BSM resembles other mucin glycoproteins and indeed,
together with the acidic structures present, which represent
some 80% of total oligosaccharides in BSM, it is a readily
available source of oligosaccharides for structural and functional studies.
The authors gratefully acknowledge the assistance of C.-T. Yuen
in gel-filtration aspects of the work.
REFERENCES
1. Gottschalk, A. (1960) Nature 186, 949-951.
2. Tsuiki, S., Hashimoto Y. & Pigman, W. (1961) J. Bid. Chem.
236, 2172-2178.
3. Tettamanti, G. & Pigman, W. (1968) Arch. Biochem. Biophys.
124,41- 50.
zyxwvutsrq
zyxwvutsrq
zyxwvutsrq
4. Tanaka, K., Bertolini, M. & Pigman, W. (1964) Biochem. Biophys.
Res. Commun. 16,404-409.
5. Tanaka, K. & Pigman, W. (1965) J . Biol. Chem. 240, PC14871488.
6. Gottschalk, A. & Graham, E. R. B. (1959) Biochem. Biophys.
Acts 34, 380 - 391.
7. Bhavanandan, V. P., Buddecke, E., Carubelli, R. & Gottschalk,
A. (1964) Biochem. Biophys. Res. Commun. 16, 353-357.
8. Ozeki, T. & Yosizawa, Z. (1971) Arch. Biochem. Biophys. 142,
177 - 183.
9. Bertolini, M. & Pigman, W. (1970) Curhohydr. Res. 14, 53-63.
10. Tsuji, T. & Osawa, T. (1986) Carbohydr. Res. 151, 391 -402.
11. D’Arcy, S. M., Donoghue, C. M., Koeleman, C. A. M., van den
Eijnden, D. H. & Savage, A. V. (1989) Biochem. J . 260,389393.
12. Savage, A. V., Donoghue, C. M., D’Arcy, S. M., Koeleman, C.
A. M. & van den Eijnden, D. H. (1990) Eur. J . Biochem. 192,
427 - 432.
13. Savage, A. V., Donohue, J. J., Koeleman, C. A. M. & van den
Eijnden, D. H. (1990) Eur. J. Biochem. 193, 837-843.
14. lyer, R. N. & Carlson, D. M. (1971) Arch. Biochem. Biophys. 142,
101- 105.
15. Ciucanu, 1. & Kerek, F. (1984) Curbohydr. Res. 131,209-217.
16. Hounsell, E. F., Lawson, A. M., Stoll, M. S., Kane, D. P.,
Cashmore, G. C., Carruthers, R. A., Feeney, J. & Feizi, T.
(1989) Eur. J . Biochem. 186, 597-610.
17. Hounsell, E. F. &Wright, D. J. (1990) Curbohydr. Res. 205,1929.
18. Hounsell, E. F., Lawson, A. M., Feeney, J., Gooi, H. C.,
Pickering, N. J., Stoll, M. S., Lui, S. C. & Feizi, T. (1985) Eur.
J . Biochem. 148. 367 - 377.
19. Feeney, J., Frenkiel, T. A. & Hounsell, E. F. (1986) Curbohydr.
Res. 152, 63 - 72.
20. Breg, J., van Halbeek, H., Vliegenthart, J. F. G., Klein, A.,
Lamblin, G. & Roussel, P. (1988) Eur. J . Biochem. 171, 643654.
21. Stoll, M. S., Hounsell, E. F., Lawson, A. M., Chai, W. C. & Feizi,
T. (1990) Eur. J . Biochem. 189, 499-507.
22. van Halbeek, H., Dorland, L., Vliegenthart, J. F. G., Kochetkov,
N. K., Arbatsky, N. P. & Derevitskaya, V. A. (1982) Eur. J .
Biochem. 127, 21 - 29.
23. Rao, B. N. N., Dua, K. K. & Bush, C. A. (1985) Biopolymers 24,
2207 - 2229.
24. Klein, A,, Lamblin, G., Lhermitte, M., Roussel, P., Breg, J., van
Halbeek, H. & Vliegenthart, J. F. G. (1988) Eur. J. Biochem.
171, 631 -642.
25. Lamblin, G., Boersma, A,, Lhermitte, M., Roussel, P., Mutsaers,
J. H. G. M., van Halbeek, H. & Vliegenthart, J . F. G. (1984)
Eur. J . Biochem. 143,227-236.
26. Capon, C., Leroy, Y., Wieruszeski, J.-M., Ricart, G., Strecker,
G., Montreuil, J. & Fournet, B. (1989) Eur. J. Biochem. 182,
139- 152.
27. Lawson, A. M., Hounsell, E. F., Stoll, M. S., Chai, W., Feeney,
J., Rosankiewicz, J. R. & Feizi, T. (1991) Curbohydr. Res. 221,
191-208.
28. Dua, V. K., Rao, B. N., Wu, S.-S., Dube, V. E. & Bush, C. A.
(1986) J . Biol. Chem. 261, 1599-1608.
29. Egge, H. & Peter-Katalinic, J . (1987) Muss Spectrom. Rev. 6 ,
331 -393.
30. Beiser, S. M . & Kabat, E. A. (1952) J . Immunol. 68, 19-40.
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