Sulphation causes heterogeneity of gastric mucins

Beurden-Lamers, W M O Van; Spee-Brand, Rita; Dekker, Jan; Strous, Ger J.

doi:10.1016/s0304-4165(89)80039-x

Cited by 19 publications

(8 citation statements)

References 39 publications

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“…It appeared that mucin with a high buoyant density and a corresponding high electrophoretic mobility had an amino acid composition slightly different from that of mucin with both low buoyant density and low electrophoretic mobility. Recently, we have found that these differences in buoyant density and electrophoretic mobility of rat gastric mucin can be explained by differential sulphation (Van Beurden-Lamers et al, 1989). We now present evidence that rat gastric mucin consists of only one glycoprotein.…”

Section: Introductionmentioning

confidence: 50%

“…The oligosaccharides and sulphate residues on the mucus glycoprotein were labelled by incubating stomach segments for 2 h in the presence of [3H]galactose and [35S]sulphate as described by Van Beurden-Lamers et al (1989). The stomach segments were washed with ice-cold phosphate-buffered saline and the [3H,35S]mucin was homogenized in 6 M-guanidinium chloride-containing buffer, carboxymethylated, and isolated as described above.…”

Section: Metabolic Radiolabelling Of Gastric Mucinmentioning

confidence: 99%

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Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone

Dekker

Beurden-Lamers

Oprins

et al. 1989

Biochemical Journal

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We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.

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Section: Introductionmentioning

confidence: 50%

Section: Metabolic Radiolabelling Of Gastric Mucinmentioning

confidence: 99%

Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone

Dekker

Beurden-Lamers

Oprins

et al. 1989

Biochemical Journal

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“…The rat mucin is probably more negatively charged than the human mucin. The glycopeptide of the rat mucin carries an intrinsic highly negative charge, due to oligosaccharide sulphation [10,11,24]. As sialic acid content is very low in both mucins, sulphate is the major charged residue on the carbohydrate moieties ( Table 1).…”

Section: Vol 277mentioning

confidence: 99%

The oligomeric structure of rat and human gastric mucins

Dekker

Aelmans

Strous

1991

Biochemical Journal

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Intact oligomeric gastric mucins were isolated from the fundic part of rat and human stomach. Physicochemical properties of the oligomeric mucins from both species, such as buoyant density, molecular mass, proteinase-resistance, amino acid composition and monosaccharide composition were similar. Biochemical analysis showed that the oligomeric mucins from both species consist of disulphide-linked mucin monomers exclusively: no other covalently attached proteins were detected in purified monomeric mucin. Four major differences were found between the monomeric mucins of these species: (1) the human monomer is larger, (2) the proteolytic-digest peptides derived from proteinase-sensitive portions of the polypeptide backbone displayed no sequence similarity, (3) the human mucin was less sulphated compared with rat mucin, and (4) the proteinase-sensitive part of the human mucin was relatively larger. However, analyses of [3H]galactose-labelled mucin from both species on gel filtration revealed that both gastric mucins were exclusively synthesized as oligomers. The results indicate that the oligomeric structures of human and rat gastric mucin are similar and their biosyntheses are not affected by the differences in the subunits.

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“…Nevertheless the distribution of the labelled material was broader than that of partially purified rat gastric mucous glycoprotein and the position of the main peaks of labelled and unlabelled material did not exactly coincide. The profiles of mucous glycoproteins present in and secreted by rat fundic mucosal segments pre-incubated with [35S]sulphate and [3H]galactose and material scraped from the surface of rat stomach have been compared by using caesium chloride density gradient centrifugation (van Beurden-Lamers, Spee-Brand, Dekker & Strous, 1989). Some of the labelled material ran at a higher density than the unlabelled material isolated from mucosal scrapings.…”

Section: Nature Of High Molecular Weight Materialsmentioning

confidence: 99%

Regulation of mucus secretion by cells isolated from the rat gastric mucosa.

Keates

Hanson

1990

The Journal of Physiology

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SUMMARY1. A suspension of cells, containing about 30 % mucous cells, was isolated from the rat fundic mucosa, and was pre-incubated with D-[6-311]glucosamine. 3H-labelled material subsequently released into the medium was separated by Fast Protein Liquid Chromatography on a Superose 6 column.2. A sharp peak of labelled high molecular weight material eluted from the column close to the void volume. This material was identified as mucous glycoprotein by its similar chromatographic behaviour to partially purified rat gastric mucous glycoprotein, by its resistance to complete degradation by papain and by its behaviour on treatment with dithiothreitol. On a caesium chloride density gradient the labelled material was virtually all located between densities of 1t35 and 1-53 g/ml, with the main peak at 1-40 g/ml.3. A broad peak of lower molecular weight material was also eluted from the column. The release of this unidentified material did not seem to be closely associated with the release of mucous glycoprotein from the cells.4. Release of mucous glycoprotein was stimulated by secretin (half-maximally effective concentration 2-a nM, 84% stimulation above basal release at 100 nM), and by isoprenaline (half-maximally effective concentration 34 nm, 33 % stimulation at 1 tM). Carbachol (0-5 mM) produced a significant (18-29%) stimulation of mucus secretion, but gastrin (100 nM), histamine (0-5 mM) and epidermal growth factor (200 nM) were without effect.5. The preparation should prove useful in the identification of the agents which regulate gastric mucus secretion.

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Sulphation causes heterogeneity of gastric mucins

Cited by 19 publications

References 39 publications

Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone

Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone

The oligomeric structure of rat and human gastric mucins

Regulation of mucus secretion by cells isolated from the rat gastric mucosa.

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