Eur. J. Biochem. 1Xh. 597-610 (1989) $, FEBS 1989

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins) Eli7abeth F HOUNSCLL', Alexander M LAWSON*, Mark S STOLL', David P KANE', Geoffrey C CASHMORE', Robert A CARRUTHERS', James FEFNEY and Ten FFIZI'

' Section of Glycoconjugate Rcsearch and Section of Clinical Mdss Spectrometry, MRC Clinicdl Resedrch Ccntre, Harrow, England MRC Biomedical NMR Ccntre, National Institutc for Medical Research, Mill Hill, England

(Received May 10,1989) - EJB 89 0587

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 'H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied aftcr depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J . 256, 397-4011 and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features:

Peripheral Backbone sequences glycosylatlon

GICNACLYI-4 FUCLYI-2

GalpI-4

Galpl-3GlcNAcpl-3GalB1-4 Galpl-3GlcNAcpl-3GaIP1-3

m Gal Galp 1 -4GlcNAcP 1-6

Gal61 -4GlcNAcOl-3

Gal0 1-3

Galpl-3GlcNACBI -3 GICNACO I - ~ ( G ~ c N A c / ~ I - ~ )

Core regions

Gl~NAcpl,~ to GalNAco 1 GlcNAc GlCNACBl/3

GlcNAcPl-3 GalNAco 1

Galp 1-3 GalNAco 1 to Gal GlcNAc,81\6

Gal f i ],3 Ga"co1

Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAcul-4Ga1, repeating-type-1 (Galfil-3ClcNAc) backbones, the backbone branch GlcNAcfil-6(GlcNAcfil- 3)Gal and the backbone sequence GlcNAcfil-6Galfil- in the absence of a substituent at C3 of galactose.

Meconium glycoproteins are representative of the mucins of the human foetal gastrointestinal tract and as such are an abundant source of human mucins that have not been exposed

Correspondence to E. F. Hounsell, Section of Glycoconjugate Research, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, England

Ahhreviation.s. GalNAcol, N-acetylgalactosaminitol; Hex, hex- ose; HexNAc, N-acetylhexosamine; HexNAcol, N-acetylhexos- aminitol; EI-MS, electron-impact mass spectrometry; GC-MS, gas chromatographylmass spectromctry; LSIMS, liquid-secondary-ion mass spectrometry; PMAA, partially methylated alditol acetate.

Preliminary reports or this work have appeared as abstracts: Lawson, A. M., Hounsell, E. F., Wilson, S. B., Cashmore, G., Stoll, M. & Feizi, T. (1988) Proc. 36th A S M S Conference on Muss Spec- trometry uncl Allied Topics, 1211 - 1278; Hounsell, E. F., Lawson, A. M., Feeney, J., Kane, D., Cashmore, G. C., Stoll, M. S. & Feizi, T. (I 988) Proc. 14th Int. Carbohydr. Symp., A92.

to the degradative enzymes of bacteria. In addition, identifi- cation of unusual sequences in these mucins may provide antigenic markers of oncodevelopmental type, i. e. those tran- siently present in the foetus which are diminished during de- velopment and are 're-expressed' in adult neoplasia [I].

In previous reports [2- 41 we have described the character- isation of mono- to tetrasaccharides and a hexasaccharide released from mcconiuin glycoproteins obtained from a blood-group-H active pool depleted of Ii antigenic activities by immunoaffnity chromatography and analysed after mild acid hydrolysis to reduce the chain heterogeneity. We now report the characterisation of the penta- and hexasaccharide chains of these meconium glycopeptides. Complementary studies to these are being carried out by Capon et al. [5, 61 on the oligosaccharides of unfractionated meconium glycopep- tides. Earlier studies [7] have reported the characterisation of the free oligosaccharides found in meconium.

598

MATERIALS AND METHODS

Meconium glycopeptides

Meconium samples were collected from one-day-old hu- man neonates and pooled according to blood group status determined by haemagglutination inhibition assays with saline extracts. The blood-group-H active samples were di- gested with pronase and the glycopeptides purified as de- scribed previously using methods which included immuno- affinity chromatography over an anti-I (Low) antibody column [2]. This yielded an Ii-antigen-depleted fraction which was partially defucosylated by mild acid hydrolysis (0.05 M H2S0, 100 T / l h) and then treated with 0.05 M NaOH/l M NaBH, for 18 h at 50°C [2]. The released oligosaccharides were chromatographed on Bio-Gel P4. Fraction K, corre- sponding to 8 - 10 glucose units/molecule and amounting to 10% by mass of the total oligosaccharides released [2], has been investigated here.

HPLC purification ojfiaction K

Fraction K was further purified by sequential chromatog- raphy by reverse-phase and normal-phase HPLC on columns (250 x 4 mm) of ODs-Hypersil and APS-Hypersil, respective- ly (Shandon Southern Products, Runcorn, UK) using an SP8700 solvent delivery system, an SP8400 variable wave- length detector at 208 nm and an SP4100 computing inte- grator (all from Spectra Physics, Santa Clara, CA95051, USA). Reverse-phase HPLC (Fig. 1 a) was carried out in water. The subfractions K2 ~ K6 (Fig. 1 b) were further purified on the APS-Hypersil column using a gradient of 65% acetonitrile/35% water to 60% acetonitrile/40% water in 10 min (Fig. lc) . One of the subfractions, K2(4), has been described in a previous report [4]. Yields were calculated from the dry mass of the subfractions after reverse-phase HPLC and from the HPLC profiles of the APS-Hypersil subfraction- ation.

Permethylation of'oligosaccharides

Permethylation was carried out using the method de- scribed by Ciucanu and Kerek [S]. Oligosaccharides (approx- imately 10-20 nmol) were dissolved in 75 pl dimethyl- sulphoxide under N2 with sonication for 15 min. A suspension of NaOH (80 mgiml) in 75 pl dimethylsulphoxide was added, followed by 50 p1 iodomethane and the reaction mixture incu- bated under N2 at 22°C for 15 min. This was extracted into chloroform (1 ml) and the chloroform layer washed six times with 4 ml H 2 0 .

Preparation of partially methyluted alditol acetates ( P M A A s )

Permethylated oligosaccharides were hydrolysed for 1 h at 1lO'C in 100 pl 2 M trifluoroacetic acid. The mixture was cooled, evaporated under N2 and residual trifluoroacetic acid removed by evaporation with methanol (three 100-pl aliquots) under N2. The residue was dissolved in 100 ~1 0.1 M sodium borateiNaOH pH 11.3 containing 50 mM NaB2H, and incu- bated at 4°C for 16 h. A drop of glacial acetic acid was added and the mixture evaporated under N2, followed by three evaporations with 200 p1 methanol. The residue was dissolved in 150 pl pyridine/acetic anhydride (1 : 1, by vol.) under N2 with sonication and incubated for 1.5 h a t 100°C. The reaction was evaporated to dryness under N2, extracted with chloro- form (1 ml) and washed six times with 4 ml HzO. The PMAAs

I 23141 5 16171 8 I

b ) C )

I K3

I I I 1 I r l r l . l * 2 4 Q Q 10 t2 2 4 8 8 10 12 14

Mlnulna

Fig. 1. Subfi-uctionurion of oligosaccharidefuaclion K by HPLC. Frac- tion K was the pcnta- to hexasaccharide fraction of oligosaccharides released from meconium which chromatographed on Rio-Gel P4 at 8- 10 glucose units [2]. (a) Reverse-phase ITPLC of fraction K giving subfractions K2 to K6; (b) analytical reverse-phase HPLC of K2 to K6; (c) the subscqucnt normal-phase HPLC of these subfractions. Oligosaccharides in the resulting fractions K2(3) and (4), K3(2) and (3), K4(2) and ( 3 ) , K5(3) and ( 5 ) and K6(2) and (3) have been investi- galed in the present report

were analysed by capillary gas-liquid chromatography/mass spectrometry (GC-MS) as described previously [9].

Liquid-secondary-ion mass spectrometric analysis (LSIMS)

Samples of native oligosaccharide fractions (approx- imately 1 nmol) and permethylated derivatives (200 -

599

500 pmol) dissolved in methanol where mixed with a thin film of glycerol/thioglycerol (1 : 1, by vol.) matrix coated on the stainless steel target. LSIMS analysis was carried out on a VG analytical ZAB2-E mass spectrometer employing a caesium ion gun operated at 35 keV with an emission current of0.5 PA. Mass spectra were acquired at 8 keV accelerating voltage and 1500 resolving power and data were processed by the 11 ~

250 J data system. Mass scans were made at 30 s/decade in the multichannel acquisition mode. Spectra of native carbo- hydrates were acquired in negative mode and of permeth- ylated derivatives in positive mode. Similar fragmentation patterns were seen to those described previously [lo - 131.

N M R spec t voscopj~

High-resolution 500-MHz ‘H-NMR and 2D ‘H-lH COSY experiments were carried out using a Bruker AM500 spectrometer operating in the Fourier-transform mode and equipped with an Aspect 3000 computer as described pre- viously [2-41. The probe temperature was 295 or 285 K as indicated. Chemical shifts were measured using acetone as internal standard which resonates at 2.225 ppm (295 K) with respect to the reference sodium 4,4-dimethyl-4-silapentane-l- sulphonate.

RESULTS

Fraction K was a penta- to hexaaccharide fraction obtained by Bio-Gel P4 chromatography [2] amounting in total to 10% by mass of the oligosaccharides released from the meconium glycopeptides studied. Oligosaccharides were further separated by HPLC (Fig. 1) giving fractions designated K2(3).K2(4),K3(2),K3(4),K4(2),K4(3),K5(3),K5(5),K6(2) and K6(3). The major oligosaccharides in these fractions are summarised in Tables 1 - 3 with their approximate abundance percentage (by mass) of fraction K. The oligosaccharides are categorised into three groups; those having a trihexosamine core (Table l), those having a branched GlcNAcfl1-6(Galfll- 3)GalNAcol core (Table 2) and those with an unbranched GlcNAcpl-3GalNAcol core (Table 3).

Table 1. Oligosaccharzdes having a branched trihexosaminr core with and without residual fucose remaining after mild acid hydrolysis

Oligosacchurides bused on the tri-hexosamine core ClcNAcpl-6fGlcNAcp1-3jG‘alNAcol (K5(3 j and K5[5) J

The major oligosaccharide fraction from reverse-phase HPLC of fraction K was a broad peak K5, eluting at 7 min in 100% water (Fig. 1 a, b). Further chromatography by HPLC on an APS-Hypersil column (Fig. 1 c) gave a major subfraction K5(3) amounting to approximately 30% (by mass) of fraction K and a minor subfraction K5(5) amounting to approximately 2% (by mass) of fraction K .

Oligosarcharide fraction K5(3j . Negative-ion LSIMS of underivatised fraction K5(3) gave a major [M-HI- ion at m/z 952 (Table 4) consistent with a pentasaccharide of com- position Hex2,HexNAc2,HexNAco1. This was supported by the positive-ion LSIMS permethylated spectrum showing an [M+H]+ ion at m/z 1206.6 (Fig.2d). The major PMAA derivatives from methylation analysis (Table 5) gave the com- position and linkages of Gal(l-)/(-6/3)GalNAcol/(-4)GlcNAc- (1-)/(-3)GlcNAc(i -) in the approximate ratios of 2: 1 : 1.4: 0.6. This, together with the molecular mass data, and the intensities of the doublet m/z 464, 432 and the ions m/z 725 and 970 in the permethylated spectrum, was consistent with the presence of a mixture of isomeric structures:

70 432 c 464

Gal( ‘ -4/ 3 )GlcNAc( 1 ~ ~ ~ ) G a l N A c o l

Gal( 1-4/3)GlcNAc( 1 /

The LSIMS spectra of free and permethylated fraction K5(3) indicated the presence of several minor components (Table 4). One of these is suggested as having the sequence Gal-GlcNAc-Gal-CilcNAc as found in fraction K6(2) which would give rise to the ion m/z 91 3. The presence of an internal galactose was confirmed by a signal in the NMR spectrum at 4.1 6 ppm (Fig. 3).

Comparison of the major NMR chemical shift data (Table 6) for fraction K5(3) with those reported for oligo- saccharides of human bronchial mucins [14] showed that it consisted of an approximate 0.7: 1 ratio of the following iso- mers a and h, respectively

GalBl-4GlcNAcB1,

Gal6 1 - 4GlcNAc B 1 2 ‘ ~ a i ~ ~ c o i a

Oligo- Structure sacchdridc

Approx. abundance of fraction K

Gal~1-4GlcNAcpl\

Gal p 1 - 3GlcNAc B 1’3 ‘GalNAcol b

Yo (by mass) K5(3) GalB1-4GlcNAcp1,

Galp1-3GlcNAcfil’

?GalNAcol 6 17

‘6GalNAcol 13 Galpl-4Gl~NAcgl~

:GalNAcol 2 Fucal-2Gal~1-3GlcNAc~1°

The ’H-NMR and 2D-COSY spectra of oligosaccharide fraction K 5(3) are shown in Fig. 3 from which it was possible to obtain additional assignments to those reported in the literature [14-171. Comparison with the data for the trisaccharide Galfll-4GlcNAcjl1-6GalNAcol [3] showed that for the HI -H4 of Gal in this sequence and the H.5 and H6 of GlcNAc the largest difference in chemical shifts is 0.004 ppm. The differences in chemical shift for the HI, H2 and H6’ of GlcNAc and the H2, H6, €16’ of GalNAcol are within 0.04 ppm. On the other hand, differences in the other resonances assigned for GalNAcol were greater than 0.1 ppm, suggesting that the solution conformation is such that the Gal/ll-3/4GlcNAcJ1-3 arm of the two isomers a and b interact significantly with GalNAcol, that the two arms of these iso- mers are spatially removed from each other and that the 1 -

600

Table 2. Oligosaccharides having a branched Glc~~Acpl-6(GalBI-3)Ga6~Acol core, ( A ) with peripherul region Glchr.4ca1-4 substitution or ( B ! with unsubstituted backbones

Oligosaccharide Structure Approx. abundance of fraction K

not further characterised

6 3GalNAcol GlcNAcul-4Gal~l’

GlcNAcal-4Galp1-4GlcNAc~1,

Pucul-2Galpl/

3GalNAcol 6

GalB1-4GlcNAcpl\

Gal p 1 - 3 / 4GlcNAc B 1 - 3Galp 1’ 6 3Ga lNAcol

% (by mass)

20

16

2

2

10

12

4.5

Table 3. Unbranched digosaccharides

Oligosaccharide Structure Approx. abundance of fraction K

YO (by mass) Gal~1-3GlcNAc~l-3Galpl-4GlcNAcpl-3GalNAcol 1

Gal~1-3GlcNAc~1-3Galpl-3GlcNAcpl-3GalNAcol 0.5

6 arm adopts a similar conformation in both the tri- and pentasaccharides.

Oligosaccharide fraction K5j.5). L S I M S analysis of frac- tion K5(S) provided molecular mass data consistent with hexasaccharide components of composition Fuc, Hex,, HexNAc2,HexNAcol [native: m/z 1098 (M - H)-; permeth- ylated: m/z 1380.7 (M +H)+]. Methylation analysis confirmed the presence of the linkages Fuc(1-), Gal(1-), (-2)Gal(l-), (-6/3)GalNAcol, (-4)GlcNAc(l-) and (-3)GlcNAc(l-) (Table 5). The formation of mjz 638 in the permethylated spectrum of KS(5) unaccompanied by m/z 606 indicated a Fuc-Gal- GlcNAc glycosidic cleavage fragment in which the 3 position of the GlcNAc was substituted (Fig.2e). The absence of the PMAA derivative for (-4/3)GlcNAc(l-) pointed to the fucose being I-2-linked to Gal.

Gal(l-4)GlcNAc(l,

638 1 6/3)GalNAcol Fuc(l-2)Gal(l-3)GlcNAc(l

The ‘H-NMR chemical shift data of fraction K5(5) were compatible with thc major component being a fucosylated hexasaccharide based on the major pentasaccharide of frac- tion KS(3), isomer b, as a backbone and having fucose linked a1-2 to Gal on the type 1 backbone linked 1-3 to GalNAcol. The signals include fucose H1 and CH3 at 5.212ppm and 1.232 ppm, respectively, as previously described for this oligosaccharide isolated from human bronchial mucins [ 3 4, 171. The presence of the other isomeric sequence Fucal- 2Gal~l-4GlcNAc~I -, linked to either C3 or C6 of GalNAcol, could be excluded by a comparison of the chemical shift data with those in the literature [14, 171.

Additional fucose HI and CH3 signals in the ‘H-NMR spcctrum at 5.193 and 1.223 ppm respectively, together with 8-anomeric signals between 4.43 ~ 4.57 ppm, possible H2 and H3 signals of GalNAcol between 4.18-4.30 ppm and NAc signals at 2.089, 2.068 and 2.048 ppm showed the presence of additional minor fucosylated components. The linear isomer Fucal - 2Galpl- 3/4GlcNAcpl- 3Galp1- 3/4GlcNAc/31-3Gal- NAcol could be ruled out duc to absence of appropriate

60 1

Table 4. Summary ojproposed structures and MS data ojoligosaccharide fractions K2j3) to K6(3). Fractional mass valucs have been included for ions abovc 1000 Da.

Oligosaccharide Assigned Structure Fract ion

m/z of [N-HI- m/z of [ M + H ] ‘ Other d i a g n o s t i c ( n a t i v e ) (permothylated) ions

(permethylat ed)

6

3 GalNAcol

mipi GlcNAc( 1,

,6/3)GalNAcol tial(l-3/4)ClcNAc(l~3)G~l(l

G a l pI-4GlcNAcBlt 6 GalNAcol

,3 G a B l l

m a j o r 952

minor 952

minor 749

1206.6

1206.6

961

709,260,228

464,432,260,218

464.437

6

3 GalNAcol m a l o r 1114.4 ,

G~lp1-4GlcNAc~l-M;alBl GLcNAc-Gal -(Fuc)GlcNAc,

6/3GalNncol minor 1098.4 Gal’

KZ( 3 ) ? minor 952

141@.7

1380.7

1206.6

175,668,464,432

883

109,26@.2LU

K3(4) GalB1-4GlcNAcp1, 6

3 GalNAcnl major 1114.4

I Gal~1-4/3GlcNAcpl-3Cal~l Gal-GlcNAc-Gal-GlcNAc,

6/3GalNAcol minor 1114.4 Gi 1’

minor 952 iiiinor 149

141U.7

1410.1

1206.6 961

J25.668.164.432

913

6 GalNAcol rnajnr 1155.4

t3 GlcNAcal-Gale1 FUC(F?.~-G~CNAC) ~Gnl NAcol minor 1098.4

1451.7

1380.7

709,260,228

1087.6

6 GalNAcol major 1114.4 3 ,

Gall31 K412) ? minor 1155.4

Gal-GslNAcol. K2(4), K3(4) 7 minor 1114.4 Gal -i;lcNAc,

G ~ I - G ~ ~ N A C ’ K2(3) 7 m i n o r 952

1410.7

1451.7

1410.7

1206.6

913,681,464,432

709,260,228

125,660,464,132

709,260,228

Gal81-4GlcNAcBl 6 GalNAcol major 952

G~lg1-4/3GlcNAcP~ minor 190

K6(2) m i nor 152 minor 587

1206.7

1002.6 1706 .1 757

970,125,464,432

913,464,432

KS(5) Galp1-4tilcNAcB 1, 6

3 I

G a l N k o l major 1098.4

Fucal-2Gal B1-3GkNArPI FIN (Gal -GlcNAc)2GalNncol minor 1090.4

1380.7

1380.7

630,464,432

1087.6.638

K6(2) Gal~l-3GlcNAc~1-3Gal~l-4/3GlcNAc~l-3tialNAcol major 952 1LOb.7 413,464,432

K 6 l 3 ) G l ~ N A c a 1 - 4 G o l g l - 4 t i l c N A c B 1 , 6

3 I

Gal Nncol majov 1098.4

Fucnl-2Gal61 K6(2) m i n o r 957

1380.7

1206.7

709,260,228

913,464,432

chemical shifts predicted from previous data [I 8, 191 and from fraction K6(2) in this study (see below). A possible alternative sequence consistent with the data is Galfil-3/4GlcNAcfil- 3(Fucal-2)Gal/l1-3/4GlcNAc/l1-3GalNAcol. Evidence for the presence of this oligosaccharides as a minor component came from permethylation analysis (Table 5 ) showing (-3/2)Gal( 1-) and (-3)GalNAcol linkages and the intense peak at m/z 1087.6 for a Hex,-HexNAc,-Fuc- fragment on LSIMS analysis of the methylated fraction (Fig. 2e). The out- standing PMAAs (Table 5 ) arising from (-4)Gal(1-) and GlcNAc(1-) were presumed to originate from a contaminating GlcNAcctl-4Gal sequence as in K2(3) described below.

Oligosuccharides having u GlcNAcfiI-6(Gul~l-3) GulNAcol core and a peripheral GlcNAccc I -4Gal sequence [fructions K2(3) , K3(2), K4(2) and K6(3)]

Characteristic signals in the ‘H-NMR spectra for a GlcNAcd -4 chain-terminating substituent and from methy- lation analysis for GlcNAc(1-) and (-4)Gal(l-) were found for oligosaccharide subfractions K2(3), K3(2), K4(2) and K6(3) making the sequence GlcNAccrl-4Gal a major structural mo- tif among the penta- to hexasaccharides of the meconium fraction (Table 2). As reported for oligosaccharides of pig stomach mucins [16], the characteristic signals include an H1

h

v

*.

0

?.

c h v w ". h

v

0-

3 P

Re

lati

ve

In

ten

sit

y (%

I4

8 x" 3 ln

Re

lati

ve

In

ten

sity

(%

I

Q).

O

Fi

0

h

7 OI

X

w

P

v

P)

m

-0

N

603

*

1206.6

* 709

Fig. 2

1380.7 *

Table 5. Linkages and saccharide composition assigned from methylation analysis of oligosaccharide fractions Symhots used: (*) PMAAs arising from minor components; (+) inadequate concentration of PMAAs for quantification; (-) not detected; (#) low response hut no other GalNAcol detected

Linkage Oligosaccharide fractions

K2(3) K2(4) K3(2) K3(4) K4(2) K4(3) KS(3) K5(5) K6(2) K6(3)

Fuc(l-) Gal( 1 -) (-4)Gal(l-) (-3)Cral( 1 -) (-2)GaI(l-) (-6)Gd(l-) (-3/2)GaI( I-)

(-3)GalNAcol (-6/3)GalNAcol

(-6/3)Gal( 1 -)

ClcNAc(l-) (-4)GlcNAc(l-) (-3)GlcNAc( 1 -) (-4/3)CrlcNA~(l-)

-

1 0.7 0.3 - *

- * 1 1 1 *

* 2 *

-

I

2 *

- *

- -

0.7 2 0.2 * 1 1 - -

- * - -

0.2 0.2 -

3 1 1 .s 1 2 0.1

-

-

*

+ *

-

+ + + -

-

2 - * *

- * 1

1.4 0.6

-

1 1 0.7

1

0.4

0.6 1 0.7 1 1

-

-

- - - 0.2# *

1 1

0.6 1 1.4 -

-

-

resonance with coupling constant of 3.7 Hz detected at 4.8 - 4.9 ppm for experiments carried out at 285 K and an H5 signal with a distinctive multiplet at 4.17-4.20ppm (295 K) as shown in Fig. 4 for the oligosaccharide fractions K6(3) and K2(3). (The former signal is obscured by the water signal for NMR experiments carried out at 295 K, but is identified at lower temperatures, e. g. 280 or 285 K, where the water signal is shifted downfield.)

The oligosaccharide fractions containing the GlcNAccl substituent amounted to approximately 40% (by mass) of fraction K (Table 2A). Fraction K3(2) was very heterogenous and was not analysed further in this study. Fractions K2(3), K4(2) and K6(3) were relatively homogeneous and structural assignments could be made for their major components as follows.

Oligosaccharide fraction K2(3). Molecular mass data from LSIMS (Table 4) and methylation analysis (Table 5) were con- sistent with a saccharide composition of Galz.GlcNAc2, GalNAcol as the major constituent of K2(3). One of the

branches on the GalNAcol core was deduced as a GlcNAc- Gal-GlcNAc sequence from the ion at mjz 709 together with thc m/z 260, 228 doublet from the terminal GlcNAc. This implied that a single galactose formed the other branch, and the overall pentasaccharide structure of the major oligosac- charide was deduced to be

228 + 2l GlcNAc(1- )Gal(l-4)GlcNAc( 1 Gal 701 ( 1’ 6/3)GalNAcol

The loss of methanol from the glycosidic cleavage ion m/z 709 to give miz 677 (Fig.2a) was only a minor process despite the proposed substitution pattern of the (-4)GlcNAc(l-) as compared, for example, with the abundant rnjz 432 fragment formed from mjz 464 for the Gal(l-4)(ilcNAc terminal se- quence.

604

G a l , 9 - 4 G l c N A c E 1

G a l S 1 - 3 N G l c N A c B l '

':GalNAcol

H I H I HI GN ON Gal

3.5 2.1 2.0 4.7 4.5 43 4 1 3.9 3.7 nnm

Fig.3. 500-MHz ' H - N M R spectrum of oligosaccharide subfraction K 5 ( 3 ) and the 2D 'H- 'H COSY spectrum at 295 K

A comparison of the 'H-NMR chemical shift data for the major oligosaccharide in fraction K2(3), given in Table 7, together with those for mucin oligosaccharides previously reported in the literature, reveals that the H2, H3 and H5 chemical shifts observed for GalNAcol are characteristic of such an alditol substituted at C6 by GlcNAcP and at C3 by Gal#?. That the Gal#?l-3GalNAcol branch is not further substituted is evident from comparison of the chemical shift data for the two monosaccharides in this sequence with the extensive data on related oligosaccharides [14 - 17, 20 - 251. In particular, the shift of the H4 of GalNAcol and H1 of Gal are characteristic of this sequence in the absence of a substituent at Gal. The remainder of the assigned chemical shifts are similar to those given by van Halbeek et al. 1161 for a GlcNAcal-4Galfl1-4GlcNAc#?1-6 branch in a hexasaccharide from pig stomach mucins having two terminal GlcNAcctl-4 rcsidues as in oligosaccharide K4(2) (Table 7).

The major component of oligosaccharide fraction K2(3) is therefore as shown in Tables 2 and 7. Two minor components in subfraction K2(3) are shown to be present from analysis of signals in the 'H-NMR spectrum of lower intensity than those assigned to the major oligosaccharides

(Fig.4) including additional signals in the HI anomcric region (4.7-4.4 ppm) and the presence of a signal at 4.13 ppm characteristic of an internal galactose linked at C3 such as that present within the sequences Gal#?l-3/4GlcNAc~l-3Gal#?l- or GlcNAc~l-3Gal~1 -3/4GlcNAc. In addition, there were sig- nals in the spectrum consistent with the presence of the tetrasaccharide Gal~1-4GlcNAc~l-6(Gal#?l-3)GalNAcol pre- viously characterised [2]. The presence of this last tetrasaccha- ride was confirmed by the MS results which gave a minor molecular ion in the spectrum of miz 749 for the underivatised oligosaccharidc (Table 4) and a relatively intense doublet miz 464,432 in the spectrum of the permethylated oligosaccharide (Fig. 2a) indicating a Galfll-4GlcNAc sequence. The absence of additional molecular ions suggested that the second minor component was isomeric with the K2(3) major penta- saccharide and from these data, together with the identifica- tion of a (-3)Gal(l-) linkage by methylation analysis, this minor component was assigned as

605

Table 6 . ‘H-NMR chemical shqt data for oligosaccharide having a lrihexosamine core region sequence Values are measured from sodium 4,4-dimethyI-4-silapentane-l- sulphonate at 295 K . Symbols used: (0) GalNAcol; (0) GlcNAcB; (3) Gala. Superscripts refer to the position of linkages

Residue Protori K 5 ( 3 )

GalNAc6

Gal4, ti

GlcNAc3

GalNAcol H1 HI’ H2 H3 H4 H5 H6 H6‘ NAc

HI H2 H5 H6 H6’ N Ac

H1 H2 H3 H4

Hi H2 H3 H6 H6‘ NAc

H1 H2 H3 H4

3.624 3.6-3.7 4.283 3.998 3.520

4.233 4.245 3.905 3.701 2.043

4.561 4.564 3.790 3.811

3.602 3.998 3.838 2.062

4.473 3.539 3.669 3.925

4.649 4.624 3.923 3.908

3.955 4.022

2.070 2.079

4.455 4.456 3.531

3.917 3.925

3.6 -3.7

3.7-3.8 3.8-3.9

3.6-3.7

Tdblc 7. ‘ H - N M K chemical shiji data ,for oligosaccharides having a terminal GlcNAcal-4Gal .sequence Values are measured from sodium 4,4-diinethyl-4-siIapentane-l- sulphonate at 295 or *285 K. Symbols used: (0) GalNAcol; (0) GlcNAcB: (U) GalB; (@) GlcNAcct; ( A ) Fuca. Superscripts refer to the position of linkage

Residue Proton K Z ( 3 ) K 4 ( 4 K 6 (3)

~

GalNAcol HI, HI’ 3.75 - - H2 H3 H4 H5 I16 H6’ NAc

~ ~ 1 3 HI H2 H3 H4

GlcNAd‘ H1 FI 2 H6 H6‘ NAc

Ga14.6 HI H2 H4

GlcNAca4. ‘ HI H5 NAc

Fuc’,~ or I T 1 GlcNAccr4, H5

H6

4.401 4.065 3.460 4.291 3 .?4 3.24 2.068 4.466 3.562 3.668 -

4.564 3.15 4.017 3.84 2.068 4.523 3.75 3.982

4.852 * 4.194 2.068

4.41 2 4.086 3.517 4.268 -

-

2.054 4.521 -

- 3.975

4.575

4.021

2.067

4.525

3.982

4.853 * 4.194 2.070 4.863 * 4.177 2.108

-

-

-

4.408 4.086 3.500 4.274 - - 2.055

4.563 -

-

-

4.562

4.020

2.059

4.530

3.983

4.85 * 4.195 2.067 5.222 4.281 1.244

~

-

-

Oligosaccharicle fractioii K4(2 ) . The major component in this fraction was established by LSIMS (Table 4) and methyla- tion analysis (Table 5) as a branched hexasaccharide of com- position Gala.GlcNAc,,GalNAcol containing mainly (-4)Gal(l-), GlcNAc( 1 -), (-6/3)GalNAcol and (-4)GlcNAc( 1 -). The low concentration of oligosaccharide in this fraction made quantification of the PMAA derivatives impractical. The se- quence ion m/z 709 in thc permethylated LSIMS spectrum (not shown) indicated the trisaccharide GlcNAc-Gal-GlcN Ac as one branch while the other was deduced as GlcNAc-Gal from the presence of the m/z 260. 228 doublet for a non- reducing terminal GlcNAc and the absence of an intense mjz 464 ion. The major component of fraction K4(2) could there- fore he assigned the following sequence

228 + 26d 7 0 9 1

GlcNAcl 4Gal( 1’

228 t 260 1 Comparison of the NMR chemical shift data with those

in the literature identified this oligosaccharide as the hexasac-

charide having two GlcNAcd -4Gal sequences linked PI-4 to GlcNAcfil -6GalNAcol and fil-3 to GalNAcol as described for an oligosaccharide of pig stomach mucins [16].

Evidence for a minor component in K4(2) containing a fucosylated Gal-GlcNAc-Gal-GlcNAc sequence was indi- cated by the mjz 1087.6 ion in the LSIMS spectrum of the permethylated fraction. This was confirmed by the ‘H-NMK spectrum which showed signals at 1.22 and 5.23 ppm consis- tent with fucose CH3 and H1, respectively.

Oligosuccharide,fraction K 6 ( 3 ) . The major component of this fraction was idcntified as a hexasaccharide with the com- position Fuc, Hex2, HexNAc,, HexNAcol from the observed molecular ions at m/z 1098 [M-HI- and m/z 1380.7 [M+ HI’, of native and permethylated material in negative and positive ion LSIMS respectively (Table 4). The presence of ions m;z 709, 260 and 228 in the latter spectrum (Fig.2g) suggested a HexNAc-Hex-HexNAc non-reducing terminal se- quence and together with GC-MS analysis of the PMAAs (Table 5) was in agreement with K6(3) having the following sequence

GlcNAc(1 4)Gal(l-4)GlcNAc(l 70g1 6/3)GalNAcol

228 + 260

~ u c ( 1 - 2 ) ~ a i ( 1’

1

606

A

B

J VJl i GlcNAcul-4Galj31-4C LcNAcSl

‘tGalNAcol HI

Ga 1~ 1/ GNB H l

-A __Id- .... i- I I I I I - . L - - - . L I - . A

4 . 4 4 .3 d.2 4 . 1 4 . 0 3.9 3.8 3.7 3.6 3.5 b . i - _ L - I

PPY 5 . 1 5 . 0 1.9 4.8 4 .7 d . 6 4.5

PPM

Fig.4. 500-MHz ’ H - N M R specirum of~iligosacchnride fraction K6(3) ( A ) nnd K2/3) [ B ) ni 259 K. The inset shows the anomcric signals for K6(3) at 185 K. Asterisks denote signals arising from oligosaccharide K6(2) in fraction K6(3) and minor componcnts in fraction K2(3)

Comparison of the NMR chemical shifts in Table 7 shows that those given for oligosaccharides K6(3) are similar to those for oligosaccharides K2(3) and K4(2), the most diver- gent being for the H4 resonance of GalNAcol and the H1 of Gal linked PI-3 to GalNAcol. As discussed above for oligosaccharide K2(3), the H4 rcsonance of GalNAcol is par- ticularly susceptible to changes in shielding depending on the substituent on Gal linked pl-3 to it. This, together with the identification of chemical shifts characteristic of a GlcNAcal- 4GlcNAcfll-6 branch (discussed above) and those for fucose linked a1-2 to Gal in p1-3 linkage to GalNAcol [14, 22-24] gave the assignment shown in Tablcs 2 and 7 for the major component of fraction K6(3).

Comparison of the ‘H-NMR chemical shift data for the major oligosaccharides in fractions K2(3) and K6(3) suggests that the presence of the fucose residue perturbs the chemical shift for the acetamido group of GlcNAcpl-6, this resonancc having a significantly different shift in the two oligosac- charides (Table 7). Although thc ovcrall chemical shifts are consistent with the structural interpretations given for the oligosaccharides, this difference suggests that, in the solution conformation of the major oligosaccharide of fraction K6(3), the fucose residue is probably in close proximity to the acetamido group of the GlcNAcjl-6 residue.

The NMR spectrum for fraction K6(3) givcn in Fig.4 also shows signals arising from contaminant K6(2) (see below) as predicted from the HPLC trace (Fig. 1 c) the GC-MS results (Table 5) and the LSIMS which showed quasi-molecular ions nz/z 952 in the negative ion spectrum of the native

oligosaccharide (Table 4) and m/z 1206.7 in the positive ion spcctrum of the permethylated fraction (Fig. 2g).

Oligosucchurides having a GlcNAcpl-6 (Gulpl-3) GulNAcol core and unsubstituted backbone Gal-GlcNAc-Gal sequences [fractions K214), K3j4) and K4(3)]

Analysis of the NMR spectrum of oligosaccharide fractions K2(4), K3(4) and K4(3) together with a comparison of the chemical shift assignments given in Table 4 and those reported previously [4] with those in the literature showed that the major oligosaccharides in these fractions had a GlcNAcpl- 6(Galfl1-3)GalNAcol core in the absence of chain-terminal GlcNAcx or significant amounts of fucose. These fractions together amounted to approximately 26% (by mass) of the penta- and hexasaccharide fraction K. The characterisation of the major oligosaccharide of fraction K2(4) has been discussed previously [4]; however, the MS data are included in the present report (Tables 4 and 5) with the probable assignments of minor components. The oligosaccharide fractions K3(4) and K4(3) were shown to contain isomeric hexasaccharides with unsubstituted backbone sequences having one or two Galfll-3/4GlcNAc~1-3 repeats as described below.

Oligosacchuride ,fraction K3(4). Negative ion LSIMS (Table 4) showed that fraction K3(4) contained one major component with a composition of Hex3,HexNAc2, HexNAcol (m,iz 11 14.4) and two minor components Hex,,HexNAc,,- HexNAcol (m/z 952) and Hex2,HexNAc,HexNAcol (m/z 749). The approximate ratios of 2:1:1:2 of Gal(1-)/

607

_.<-

A G a l 6 1 -4G lcNAc6 1

Ga181-3/4GlcNAcQ1-3Ga 1P1 I l l HI HlGal G N ~ -

H5 , I !:I -01

Gal# 1-3GlcNAcPl-3Ca1~1-4Gl~NAc#l \:GalNAcol NAc

G a 101'

c Gal~l-3GlcNAcpl-3Gal~l-3/4GlcNAc~l -3GalNAcol * * HlGal H4

I __I_ I I I - U L L . I - - - _ L 1 I I I \ I -1

, I 2 . 1 2 . 0 4 . 7 4 .6 4 . 5 4 . 4 4 . 3 4 2 1.1 4 0 3.5 3 . 0 3.7 3 6 PPH

Fig. 5. 5(10-hfHz ' H - N M R spectrum qf' oligosaccharidefruction K 3 ( 4 ) ( A ) , oligosaccharide fraction K4(3) ( B ) and oligosaccharide fruction Kh(2J (C). The structures givcn are for the major component in each fraction. Asterisks denote lion-carbohydrate impurities

(-3)Ga1(1-)/(-6/3)GalNAcol/(-4)GlcNAc(l-) obtained from the major PMAAs by methylation analysis (Table 5), together with the results of negative LSIMS of neoglycolipids (unpub- lished observations) obtained from fraction K3(4) and positive LSIMS of permethylated K3(4) (Fig. 2b) gave the major struc- ture as Gal(l-&)GlcNAc(l, , 668J6/3)GalNAcal

The ion doublet at m/z 464,432 indicated the Gal(1-4)GlcNAc non-reducing end sequences while mjz 668 confirmed the Gal-GlcNAc-Gal sequence. Small amounts of (-4)Gal(l-), (-6)Ga1(1-) and (-3)GlcNAc(l-) were evident from methylation analysis and must arise from minor components as did thc miz 913 ion in the LSIMS spectrum (Fig.2d).

The 'H-NMR chemical shifts for fraction K3(4) shown in Table 8 are similar to those given for an oligosaccharide of human bronchial mucins having the GalPl-4GlcNAcfll-6 and Galfll-4GlcNAcfll-3Galfll-3 sequences [I71 and human ovarian cyst mucins having the Galpl-4GlcNAcfll-6 and Galpl-3GlcNAcfll-3Galfll-3 sequences [25]. There are some differences in chemical shift which are outside the normal variation between laboratories (approximately 0.005 ppm). However, the NMR data combined with those obtained from LSlMS and GC-MS analysis, are most compatible with the oligosaccharide having two non-reducing Galpl-4 sequences as the major component with the possibility of a minor amount of the alternative isomer having a Galfl1-3GlcNAcjl- 3Gal sequence. The assignments of the signals of Galfi1-3/ 4GlcNAc were made by comparison with those reported [19]

G a l ( 1-4 ) G l c N A c ( 1 3 )Gal( 1

432 + 464

for the linear tetrasaccharides of milk having the two isomeric sequences linked to glucose.

Signals of low intensity shown in Fig.5A at 4.062 ppm and 3.456 ppm for H3 and H4 of GalNAcol, respectively, suggest the presencc of additional oligosaccharides in fraction K3(4) having a Gal~l-3(GlcNAc/31-6)GalNAcol core where the Gal is not further substituted (Tables 7 and 8). The second signal at 4.158 ppm for H4 of an internal Gal residue (Fig. 5A), together with an ion m/z 913 in the mass spectrum (Table 4). suggests that one of the minor components with this core is related to the major component of fraction K4(3) described below, but probably having a repeating type-2 back- bone sequence linked at C6 of GalNAcol to account for the PMAA data and the lack of an HI GlcNAc resonance at 4.719 ppm (Fig. 5B, H1 GN). Additional H1 Gala signals to those given in Table 8 at 4.45-4.47 ppm are consistent with the presence of the tetrasaccharide Gal/ll-4GlcNAc/?l- 6(Gal#H -3)GalNAcol which would give rise to the molecular ions 749,961 (Tdbk 4). An assignment could not be made for the preposed pentasaccharide having mjz 952 and 1206.6.

Oligosarrharide pactinn K4(3) . As indicated from the mass spectral data given in Table 4 this fraction was princi- pally a hexasaccharide of composition Ga13,GlcNAc2, GalNAcol containing Gal(l-)/(-3)Gal(l-)/(-6/3)GalNAcol/ (-4)GlcNAc(l-)/(-3)~ilcNAc(l-) in the ratio 2: 1 : 1 : 1 : 1. The most probable structural assignment

I 6/3)GalNAcol 881 + 913

Gal( 1-3)GlcNAc( 1-3)Gal( 1-4)GlcNA~( 1

Gal(1'

60 8

Table 8. H - N M R chemical shift data for ohgosaccharides having backbone Gal-GlcNAc-Gal sequencvs Valucs are measured from sodium 4.4-dimethyl-4-silapentane-I-sulphonate at 295 K. Symbols used: (0) GalNAcol; (0) GlcNAcP; (U) Ga1,O. Superscripts rcfcr to positions of linkage. Square brackets enclose proposcd minor isomers

Kesidue Proton K3(4) K4(3) W 2 ) 3 3 4 3

GalNAcol

~ ~ 1 3

GlcNAc6

GlcNAc'

GlcNAc3

Gal". '

(to GalNAcol)

(to Gal)

H 2 H3 H 4 H5 NAc

Hi H 2 H 4

HI H6 NAc

HI NAc

H1 NAc

HI H 2 H 4

H1 H4

TI1 H2 H4

4.405 4.052 3.442 4.276 2.068

-

4.131

4.552 3.997 2.059

4.695 [4.675] 2.032 [2.037]

4.468

3.921 -

4.482 [4.449] 3.536 [3.52] 3.921

4.400 4.062 3.456 4.288 2.067

4.463 3.559 3.911

4.553 3.996 2.063

4.719 2.028

4.468 3.647 4.156

4.442 3.521 3.91 I

4.292 4.002 3.538 4.153 2.037

4.629 [4.650] 2.081 [2.076]

4.720 2.027

4.442 [4.457] 4.157

4.442 3.524 3.910

was supported by the spectrum of the permethylated fraction which showed glycosidic cleavage of the tetrasaccharidc branch producing an intense m/z 913 ion for a Hex2-HexNAc, branch (Fig.2~). Methanol elimination from this ion was pre- sumed to give m/z 881 and to indicate a (-4)GlcNAc(l-j sub- stituent linked to GalNAcol. The low intensity of m/z 432 was considered to point to the terminal Gal being linked predominantly 1-3 to GlcNAc.

Permethylation analysis of K4(3) indicated the presence of minor components which includcd the linkages (-4)GaI-( 1 -), (-6)G al( 1 -), (-3)GalNAcol, GlcNAc(1-) and (-6/3jGal( 1 -j and were assumed to arise from oligosaccharides designated K2(3), K2(4) and K4(2) and including one having the sequence

G~CNAC(~, 6/3)Gal(l-

GlcNAc( 1'

The chemical shift assignments given in Table 8 for the major component of K4(3) supported the hexasaccharide as- signment given above. Signals for the H2, H5 and NAc of GalNAcol, NAc of GlcNAc and H1 of Gal linked to GalNAcol are similar to those reported in the literature for a hexasaccharide of ovarian cyst mucins having the sequences

Gal~l-3GlcNAc~l-3Gal/31-4GlcNAc/31-6 and Galpl- linked at C6 and C3 of GalNAcol respectively [21]. The chemical shifts for the tetrasaccharide sequence are also similar to those given for a pentasaccharide lacking the galactose linked at C3 of GalNAcol [25] thus further confirming the assigned structure. In addition, the chemical shifts for H1, H2 and H4 of Gal at 4.442, 3.521 and 3.911 ppm respectively are characteristic of oligosaccharides having a terminal GalPl - 3GlcNAc sequence [ 191. The alternative isomeric sequence Gal~l-4GlcNAc on a linear chain [19] has an H1 signal for Gal at 4.480 ppm and a H2 signal at 3.539 ppm and these were not detected in the 'H-NMR spectrum of subfraction K4(3). The chemical shifts at 4.468, 3.647 and 4.156 ppm were compatible with signals of the H1, H2 and H4 of an internal Galpl-4 residue, but the presence of the alternative isomeric sequence Gal~l-3GlcNAc/3l-3Gal~l-3GlcNAc linked at C6 could not be ruled out.

The presence of a minor component is confirmed by the presence of H1 Galp signal at 4.458 ppm and a GalNAcol H2 signal at 4.391 ppm. Additional significant signals at 4.607 ppm (p anomeric signal of GlcNAc), 2.054 and 2.045 ppm (NAc) in the NMR spectrum are consistent with the presence of an oligosaccharide having an internally branched galactose residue [16]. 'l'hese data together with the results of negative LSIMS analysis of oligosaccharide fraction

609

K4(3) as neoglycolipids (unpublished observations) gave the structure

Galpl-3/4GlcNAci31,

6Galp1 -3GalNAcol / 3

Galp1-3/4GlcNAcpl

Periphery Backbone Core

GalNAco 1

Oligosaccharides having unbranched core region and backbone sequences

0ligosarcharide.fraction Kti(2). The oligosaccharide frac- tion K6(2) amounted to 1 - 2% of fraction K (Fig. 1). The major LSIMS [M -HI- ion of this underivatised fraction was at m/z 952 indicating a composition of Hex2,HexNAc2,- HexNAcol. This was confirmed in the permethylated spec- trum by the [M + HI + ion at m/z 1206.6. Significant ions at m/z 913 and 464 in the spectrum pointed strongly towards an unbranched structure containing a Hex-HexNAc-Hex- HcxNAc sequence (Fig. 20. Linkage analysis was consistent with this assignment showing (-3)Ga1(1-) with (-3)GalNAcol and no (-6/3)GalNAcol, while the presence of PMAAs for (-4)GlcNAc(l-) and (-3)GlcNAc(l-) indicated both type 1 and 2 sequences being present. The m/z 464 ion without a mjz 432 permethylated spectrum suggested a Gal(1-3)GlcNAc non- reducing terminal giving the overall structure Gal(1-3) GlcN Ac( 1 -3)Gal(l-3/4)GlcNAc( 1 -3)GalNAcol. Comparison of the chemical shifts for the GalNAcol residue of subfraction K6(2) with those in the literaturc [2, 14-17] confirmed the linear GlcNAcj?l-3GalNAcol core (Table 8, Fig. 5). The chemical shifts assigned for the HI, H6 and NAc of the inter- nal GlcNAc residue linked to GalNAcol compared with those for the two trisaccharides, Gal~l-4GlcNAc~l-3GalNAcol and Gal~l-3GlcNAc~l-3GalNAcol obtained from bronchial mucins 114, 16, 171 and meconium glycopeptides [2] shows that the major component in K6(2) has the internal GalP1- 4GlcNAc~l-3GalNAcol sequence. A terminal Galal-3Glc- NAc sequence for both of the isomers is assigned from compa- rison of the chemical shifts for the non-reducing terminal galactose residue with those previously described [19]: as dis- cussed above, the absence of the chemical shifts 4.480 and 3.539 ppm in the spectrum of fraction K6(2) suggests that non-reducing terminal Gal/?l-4GlcNAc sequences are absent as confirmed by the MS results.

DTSCUSSION

Tn the present study penta- and hexasaccharide structures of human roetal gastrointestinal mucins (meconium) were characterised by the combined use of mass spectrometry and ‘H NMR. LSIMS and GC-MS provided molecular mashes and information on sequence, pattern of branching and link- age as has been shown by our studies and those of others [I0 ~ 131 on structurally related oligosaccharides. Together with NMR studies, the complete structural assignment for the major oligosaccharides was made. In addition, information was given on minor oligosaccharides which, even after exten- sive chromatographic fractionation, wcrc not separatcd from the major components. LSIMS, and particularly GC-MS of PMAAs were invaluable in identifying these minor com- ponents.

The structural features of the oligosaccharides charactcr- ised are shown as a composite diagram in Fig. 6 together with those from other oligosaccharides (mono- to tetrasaccharide [2] and one hexasaccharide [4]) previously identified in the meconium fraction studied. Seven different types of core re-

i GlcNAcO I-~(GIcNAcO 1-31 GalgIJGalNAcol

GalNAcnl-3GalNAcol

Galpl-4-----GlcNAc~1-6GalNAco I

Galgl-4----GlcNAcgl ‘6

GalNAco I

Gaipl-4

F ~ c ~ 1 - z ................................... -Gal(j.3

G~~NAC~~.~...-- ............................ ~alg1.4

Gal0 I -3GlcNAcgI -3Galpl-3 Galg I-3GlcNAc(3I -3GaloI -4

GalNAco I

GlcNAcnl -4 .............................................. or Fuca.1-2

‘6 Galgl-4----GlcNAcPI

GalNAcoI Gal0 I-4GIcNAcOI-6

Galg 1-4 Gal0 1-3

Gal 01 -3GlcNAcf31-3Gal~I -4 ----GlcNAcB I -3GalNAco 1 Gal~l-3GlcNAcpl-3G;11~1-3 I

F i g . 6. Composite diugrum of the proposed peripheral, hackhune und core domains of the mono- to hexusuccharides characferisedjrom human meconium glycopeptides using N M R ond M S

gions can be discerned: GalNAcol, Calfll-3GalNAco1, GalNAcctl-3CalNAco1, GlcNAc~1-3GalNAco1, GlcNAcPl- 6CalNAco1, GlcNAc~l-6(GlcNAc~l-3)GalNAcol and Glc- NAcfll-6(GalP1-3)GalNAcol.

Extended glycosylation occurs on both branches at C6 and C3 of CalNAcol and both repeating Galfl1-3GlcNAc and alternating Gal~1-3GlcNAc/Gal~1-4GlcNAc sequences are found giving rise to seven different linear backbones : Galfll -4, Galpl-3, Gala1 -3GlcNAcfll-3Galal-4, Gala1-3Glc- N Acfll-3Galal-3, GalPl-4GlcNAcflI-6, Galfil-3GlcNAcflI- 3 and GalPl-4GlcNAcfil-3. Evidence was also found for a branchcd backbone in one of the fractions [fraction K4(3)], GlcNAcfl1-6(GlcNAc~1-3)Gal.

Identification of oligosaccharides having the possible com- binations of GlcNAc linked at C6 and/or C3 of both the core GalNAc and backbone Gal suggests that biosynthesis of branches can occur either by initial addition of GlcNAc to C6 or C3. However, whilc degradation by bacterial enzymes seems unlikely, cleavage at branch points by endogenous glycosidase giving rise to unbranched GlcNAcPl-6Gal/ GalNAc sequences cannot be ruled out. Overall therc is a paucity of terminal GlcNAcBl-residues suggesting that ad- dition of this residue is the rate-limiting step and that sub- sequent galactose addition occurs rapidly as has been pro- posed earlier [26]. Several of the chains are terminated with a GlcNAcrl -4 substituent linked to galactose. Fucose is present as a peripheral substituent showing that not all of this residue was removed by the mild acid hydrolysis conditions used.

Comparison of the NMR data for this comprehensive series of oligosaccharides has provided scveral insights into their conformation which it is planned to explore more fully by nuclear Overhauser enhancement experiments. Confor- mational details concerning the relative positions of branches on either Gal or GalN-Acol, discussed abovc for thc major

610

isomers of fraction K5(3) and previously [4, 271 for the major oligosaccharides of fractions K2(4) and K3(4), predict that therc is no through-space interaction between sequences linked 1-6 and 1-3 at a branch point but that a 1-6-linked sequence can interact with sequences linked 1-6 to other resi- dues in the oligosaccharide. However, this may not be true for branches linked to GalNAcr-SerlThr rather than GalNAcol. The proposed spatial orientation of the periphcral substi- tutions, GlcNAcml-4 linked to Galpl-4GlcNAcfil-6 in frac- tions K2(3), K4(2), and K6(3) and Fucal-2 linked to Galpl- 3GalNAcol in fraction K6(3), suggest that these form rela- tively globular conformational domains as has been shown for the blood group and related antigens, H, A, B, Lea, Leh, SSEA-1 and their sialylated analogues [lX, 19, 22, 28-31]. This, together with the possible restricted distribution of thc C?lcNAcnl-4Gal sequence and its relative abundance in hu- man foetal gastrointestinal mucins, suggests its potential as an epithelial marker for differentiation stage and neoplastic transformation.

The authors wish to thank Mrs J . Gilbert and Miss E. White for typing the manuscript.

REFERENCES 1. Hounsell, E. F. & Feizi, T. (1982) Med. Bid. 60, 227-2236, 2. Hounsell, E. F., Lawson, A. M., Feeney, J.. Gooi, H. C.,

Pickering, N. J.: Stoll, M. S.. Lui, S. C. & Feizi. T. (1985) Bur. J . Biochern. 148, 367 - 377.

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