Characterisation by mass spectrometry and 500‐MHz proton nuclear magnetic resonance spectroscopy of penta‐ and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)

Abstract

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-2GalpI-4 Galpl-3GlcNAcpl-3GalB1-4Galpl-3GlcNAcpl-3GaIP1-3 m GalGalp 1 -4GlcNAcP 1-6 Gal61 -4GlcNAcOl-3Gal0 1-3Galpl-3GlcNACBI -3 GICNACO I -~( G~c N A c /~I -~) Core regionsGl~NAcpl,~ to GalNAco 1 GlcNAc GlCNACBl/3GlcNAcPl-3 GalNAco 1Galp 1-3 GalNAco 1 to Gal GlcNAc,81\6Gal fi ] , 3Ga"co1Sequences 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 to the degradative enzymes of bacteria. In addition, identification of unusual sequences in these mucins may provide antigenic markers of oncodevelopmental type, i. e. those transiently present in the foetus which are diminished during development and are 're-expressed' in adult neoplasia [I].In previous reports [2-41 we have described the characterisation 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 glycopeptides. Earlier studies [7] have reported the characterisation of the free oligosaccharides found in meconium.

Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids

“…Despite the three chromatographic steps used in the fractionation of the penta-and hexasaccharides (Bio-Gel P4, reverse-and normal-phase HPLC), multiple components were present in several of the fractions as deduced from MS and NMR analysis [3]. TLC of the free oligosaccharides ( Fig.…”

“…4A) and identified as G a l~l -4 G l c N A c~1 -6-OY (Table 2) and the slower, at position 10, gave an LSIMS spectrum (Table 2) with molecular ion [M-HI-m/z 1362.8 and fragment ions m/z 1200, 997 and 835 consistent with the sequence GalPl-4GlcNAcfil-3Galfi1-3-OX. The identification of these products was important in assigning the structure of the major oligosaccharide in this fraction (Table 1) in conjunction with MS and NMR data on the free alditols [3]. The minor neoglycolipid bands at positions 1 and 12 of Fig.…”

“…Reductive amination of the resulting aldehyde groups gives derivatives characteristic of each branch which can be analysed with high sensitivity by thin-layer chromatography/liquid-secondary-ion mass spectrometry. Studies of a series of oligosaccharide fractions derived from human meconium glycopeptides [3,5,6] show that this approach has considerable potential for deriving sequence 3GlcNAcPl -3Gal and Gal,% -4GlcNAcPl-3Gal (designated 0 3 and 0 7 , respectively) were gifts of Dr A. Veyrieres.…”

Neutral oligosaccharides of bovine submaxillary mucin

“…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 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.…”