Mebius F. Kramer

Isolation and partial characterization of rat gastric mucous glycoprotein

Mucus glycoproteins from the rat stomach were characterized after their isolation from homogenates of the superficial gastric mucosa by equilibrium centrifugation in CsCl density gradients. Water-soluble as well as water-insoluble glycoproteins were studied. The latter were solubilized by 2-mercaptoethanol reduction of the homogenate. From both homogenate fractions the sames two glycoproteins 1 and 2 were purified, glycoprotein 1 being present in considerably higher amount than glycoprotein 2. Their respective buoyant densities in a CsCl gradient were 1.47--1.50 g/ml and 1.56--1.58 g/ml. The two glycoproteins expressed slight differences in gel electrophoresis and gel filtration. The results from column chromatographic comparisons between reduced and unreduced glycoproteins indicated strongly that both glycoproteins 1 and 2 were built from subunits kept together by S-S bonds. The s20,w values of the reduced glycoproteins 1 and 2 were 15.7 S and 11.6 S. Glycoprotein 1 contained 5% protein, 70% carbohydrate and 1--2% sulphate, whereas these percentages for glycoprotein 2 were 10% protein, 65% carbohydrate and 10% sulphate. The molar proportions of the main sugar components galactose, fucose, glucosamine and galactosamine were 4 :2 : 4 : 1 (glycoprotein 1) and 3 : 2 : 3 : 1 (glycoprotein 2). Blood-group activity A was expressed by glycoprotein 1, whereas glycoprotein 2 showed mainly blood-group activity Leb, some B activity and also some A activity, but to a lesser extent than glycoprotein 1.

Terminal α(1→4)-linked N-acetylglucosamine: A characteristic constituent of duodenal-gland mucous glycoproteins in rat and pig. A high-resolution 1H-NMR study

Abstract The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract was examined for species- and tissue-specificity. To this purpose, oligosaccharides were released from purified glycoprotein preparations of rat and pig gastric, duodenal-gland and small-intestinal mucus, by alkaline borohydride reductive cleavage. Based on the results of 500-MHz 1 H-NMR spectroscopy and of sugar analysis of the total oligosaccharide fractions, terminal GlcNAc, α(1→4)-linked to galactose, appears to be a characteristic constituent of duodenal-gland oligosaccharides. Similarly, NeuAc in α(2→3)-linkage to galactose turns out to be a typical constituent of small-intestinal mucous glycoproteins. In general, glycoproteins from gastric mucus possess larger and more-branched carbohydrate chains than those from duodenal-gland and small-intestinal mucus. Comparing rat and pig, oligosaccharide structures for corresponding tissues are less complex for the former. After fractionation, the rat duodenal-gland oligosaccharides could be characterized by application of 1 H-NMR spectroscopy as being branched tetra- up to hexa-saccharide chains, all sharing the italicized trisaccharide element. The chains exhibit microheterogeneity as to the termination by fucose in α(1→2)- or by GlcNAc in α(1→4)-linkage to galactose. The following structures can be proposed for the most abundant rat duodenal-gland oligosaccharides:

Effect of carbenoxolone on the synthesis of glycoproteins and DNA in rat gastric epithelial cells.

The influence of carbenoxolone on the synthesis of glycoproteins in the surface mucous cells and the production of new cells in the rat gastric mucosa was studied by means of a vascular perfusion system. The rate of incorporation of tritiated galactose, glucosamine, serine, and sulphate in surface mucous cells, studied by autoradiography, was not affected by the addition of carbenoxolone to the drinking water. The sugar composition (determined by gas-liquid chromatography) of the gastric glycoproteins (isolated by centrifugation in CsCl), was not changed in carbenoxolone-treated rats. Compared with untreated animals, the number of [3H]-thymidine labelled nuclei per fundic pit increased by 38% to 76% in carbenoxolone-treated rats, implying a higher number of mitotically active cells. This results in an increased supply of young mucous cells; if this also proves to be true in human gastric mucosa, it may be relevant to the therapeutic effect of carbenoxolone.

Glycoprotein synthesis in gastric epithelial cells of the rat: Properties of microsomal glycoprotein glycosyltransferases

1. Optimal assay conditions were determined for a microsomal glycoprotein galactosyl- and fucosyltransferase derived from gastric epithelial scrapings with both exogenous and endogenous acceptor glycoprotein. 2. Subcellular fractionation of the homogenate yielded microsomal fractions enriched in glycosyltransferases. 3. The effect of feeding on galactosyltransferase activity per cell was examined. 4. Endogenous acceptor molecules were identified as glycoproteins after labeling by means of UDP-[3H]galactose in the cell-free system.

Human duodenal gland (Brunner's Gland) mucus glycoprotein analysis

A mucus glycoprotein of the duodenal gland is characterized. The glycoprotein was isolated from a water-soluble homogenate fraction of the submucosal tissue of the most proximal part of the small intestine, containing the duodenal gland, and was purified from contaminating protein by two sequential equilibrium-centrifugation steps in CsCl density gradients. Structural analysis of the purified glycoprotein showed two regions in the protein core: one part characterized by the presence of essentially all of the cysteine residues and another by the presence of most of the serine and threonine. Carbohydrate was found linked to the latter part. Rat (H. L. Smits, P. J. M. van Kerkhof, and M. F. Kramer (1982) Biochem. J. 203, 779-785.) and human duodenal gland mucus glycoprotein show homology in chemical composition. Both glycoproteins have a relatively high protein content and contain little sulfate and no neuraminic acid. In man the mucus glycoprotein, however, has a higher content of serine plus threonine, a lower content of N-acetylglucosamine, a slightly higher content of fucose, and a lower molar ratio of N-acetylgalactosamine relative to serine plus threonine.

Role of galactosyl-transferases in rat gastric epithelial glycoprotein synthesis

Two galactosyl-transferases have been found in the Golgi-enriched subcellular fractions derived from rat gastric mucosa. One incorporates galactose into ovomucoid at optimal pH 6.8. The reaction can be completely inhibited by acetylglucosamine. The apparent Km for UDPgalactose is 0.024 mM. The other galactosyl-transferase incorporates galactose into desialated ovine submaxillary mucin at optimal pH 7.5 and the transfer cannot be inhibited by acetylglucosamine. The apparent Km for UDPgalactose is 0.191 mM. Both enzymes require Mn2+ and Triton X-100 for optimal galactose incorporation. The enzymes could be separated by polyacrylamide gel electrophoresis. Incorporation into endogenous glycoprotein was studied in conditions optimal for the two galactosyl-transferases: (1) at pH 6.8, using Mes as buffer system, and (2) at pH 7.5, using Tris-HCl in the presence of an inhibitory excess of acetylglucosamine. In both cases, most radioactive galactose is incorporated into macromolecules, which could be identified as epithelial glycoprotein. Endogenous incorporation in the presence of excess acetylglucosamine results in the formation of a substantial amount of a disaccharide (probably galactose-beta-(1-3)acetylgalactosamine), whereas upon incorporation at pH 6.8 almost no disaccharide is formed. Quantitative immunoprecipitation experiments with specific antibodies to the endogenous product, labelled by [3H]galactose in the presence of varying amounts of desialated ovine submaxillary mucin and/or acetylglucosamine, indicated that other galactosyl-transferases are involved in the biosynthesis of epithelial glycoprotein.

Synthesis of a mucous glycoprotein in the human uterus

Scrapings of endometrium and uterine contents of 10 women were analysed. In the uterine lumina of two women that used synthetic progestagens, a considerable amount of mucus was present. We fractionated the mucus by CsCl density equilibrium centrifugation into glycoprotein and protein fractions. With sugar and amino acid analysis the glycoprotein could be classified as a typical epithelial glycoprotein, resembling the cervical glycoprotein. It contains neuraminic acid (6.2%) and sulfate (8.4%). From the uteri of the other 8 women, who did not use hormones, a small amount of a similar glycoprotein could be isolated.

COMPOSITION AND STRUCTURE OF RAT BRUNNER'S GLANDS AND GASTRIC MUCOUS GLYCOPROTEIN

Previously, the isolation and partial characterization of rat gastric mucous glycoprotein (MG) has been described (Spee-Brand et al., 1980). We now have isolated a MG from Brunner’s glands of the rat duodenum. The composition and size of the O-glycosidically linked oligosaccharide chains of both gastric and Brunner’s glands MG are described.

Intracellular Site of Glycosyl- and Sulfate-Transferases in the Surface Mucous-Cells of the Rat Stomach

Publisher Summary The surface mucous cells of the rat stomach produce mainly glycoproteins that consist of ca. 20% of amino acid, 80% of sugar residues, and 3% of sulfate groups. Most sugars are attached to the glycoprotein in the Golgi apparatus. The levels of glycosyltransferases involved in glycoprotein synthesis could be determined in the homogenates of mucosal scrapings, as well as in sub cellular fractions, by means of endogenous and exogenous acceptor molecules. When radioactive sugars are transferred from nucleotide sugars to endogenous macromolecules, these molecules could be identified as glycoproteins with the same density and electrophoretic behavior as the gastric epithelial glycoprotein. The sulfate-transferase assay is linear with enzyme–protein concentration and with time for 60 min.

Carbohydrate Chains of Human Pre- and Postovulatory Cervical Mucous Glycoprotein

Mucous glycoproteins (M.G.) of cervical mucus are characterized by their great number of O-glycosidically linked carbohydrate chains. For cervical mucus of Macaca radiata differences in these chains have been reported between the various phases of the menstrual cycle, apart from variations in bound neuraminic acid (AcNeu) content (Nasir ud Din et al, 1979).Our study of human cervical M.G. (Van Kooij et al., 1980) and that of Wolf et al. (1980) did not reveal systematical variations with cycle phase, in AcNeu content nor in the relative amount of the other sugars. In the present study we report data on size and nature of the oligosaccharide chains of human cervical M.G.