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G J Strous

Publications and source records attributed to G J Strous.

At least 109 records · Page 6Linked to original sources
PubMed1984

16,16-Dimethyl prostaglandin E2 stimulates galactose and glucosamine but not serine incorporation in rat gastric mucous cells.

It has been suggested that the cytoprotective effect of prostaglandins could be mediated by an increased mucus glycoprotein secretion in the stomach. In the rat we studied the mucus glycoprotein synthesis after an in vivo treatment with 16,16-dimethyl prostaglandin E2 (10 micrograms/kg X day subcutaneously) for 1 h or 7 days. The incorporation rate of [3H]galactose, [3H]glucosamine, and [3H]serine was determined by light microscopic autoradiography in the ex vivo vascularly perfused stomach. Prostaglandin increased the rate of [3H]galactose and [3H]glucosamine incorporation twofold to fourfold in the fundic surface mucous cells; but the total protein synthesis as measured by [3H]serine incorporation was not increased. Analyses of purified mucus glycoprotein did not show an effect on carbohydrate composition, oligosaccharide chain size, nor on buoyant density, after prostaglandin treatment. The present study reveals that 16,16-dimethyl prostaglandin E2 stimulates the mucus glycoprotein synthesis in the fundic mucous cells. This effect may well be one of the mechanisms by which prostaglandin protects the stomach against noxious agents.

16,16-Dimethylprostaglandin E2↗
PubMed1983

Biosynthesis of the major glycoprotein associated with zymogen-granule membranes in the pancreas.

The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr = 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-beta-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides. Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5-6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73000-61000. We conclude that GP-2 contains five or six N-linked carbohydrate chains. From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (greater than 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (less than 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.

Animals↗
PubMed1983

Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis.

In rats infused with asialoglycoprotein for 60 min, receptor-mediated endocytosis of the ligand occurred exclusively in hepatic parenchymal cells. We have used double-label immunoelectron microscopy on ultrathin cryosections of rat liver to identify the site at which the asialoglycoprotein receptor and its ligand dissociate following their common endocytosis. Asialoglycoprotein receptor, ligand and clathrin were identified and quantitated by the use of monospecific antibodies followed by gold-protein A complexes. Both receptor and ligand were found associated with the membrane of clathrin-coated vesicles close to the cell surface. We identified other vesicles that contained ligand accumulated within the lumen. The membranes of these latter vesicles contained little receptor, but receptor was concentrated in tubular extensions that were largely free of ligand. We call this organelle CURL (compartment of uncoupling of receptor and ligand). CURL vesicles appear to transform into secondary lysosomes, wherein the ligand is degraded. The tubular vesicles are, we propose, an intermediate in recycling the receptor to the cell surface.

Animals↗
PubMed1983

Transport and topology of galactosyltransferase in endomembranes of HeLa cells.

HeLa cell membranes were studied for the distribution and orientation of the Golgi marker enzyme uridine diphosphate-galactose:beta-D-N-acetylglucosamine beta, 1-4 transferase (GT). Short pulse labeling in the presence of [35S]methionine resulted in two precursor species (Mr = 44,000 and 47,000), present in a microsomal fraction with a density of 1.18 g/ml in sucrose, presumably derived from the rough endoplasmic reticulum. Processing of the N-linked oligosaccharide(s) occurred only after the precursor molecules migrated to lighter density fractions, presumably derived from the Golgi complex. The mature GT molecules (Mr = 54,000) contain O-linked oligosaccharides as shown by beta-elimination of metabolically incorporated [3H]galactose. The O-glycosylation occurred mainly in the light density fractions. The topology of GT was studied on membrane fractions after labeling with [35S]methionine as well as immunocytochemically on ultrathin cryosections at the electron microscope level. Our results indicate that both the antigenic determinants of GT as well as polypeptide chain are present intramembraneously and at the luminal side of the membranes of the Golgi complex and rough endoplasmic reticulum.

Animals↗
PubMed1983

Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles.

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.

Albumins↗
PubMed1983

The pathway of the asialoglycoprotein-ligand during receptor-mediated endocytosis: a morphological study with colloidal gold/ligand in the human hepatoma cell line, Hep G2.

The human hepatoma cell line, Hep G2, was used as a model system to delineate the morphological aspects of receptor-mediated endocytosis of asialoglycoproteins (ASGP). These hepatoma cells, which have several structural features in common with liver parenchymal cells, contain a well-differentiated endocytotic apparatus which includes coated vesicles and CURL (compartment of uncoupling receptor and ligand) similar to that found in rat liver cells in vivo (Geuze et al., 1983). In order to precisely identify the route of ligand from the cell surface to the CURL sorting vesicles, Hep G2 cells were allowed to endocytose in synchrony ASGP bound to 12 nm colloidal gold for 5 to 60 min. Thereafter the subcellular distribution of the gold particles was examined in ultrathin cryosections. This study demonstrates that, once internalized, ASGP ligand enters a system of CURL tubules prior to delivery to the lumenal contents of attached endocytotic vesicles. These vesicles then in turn transform into multivesicular bodies and secondary lysosomes. Thus, the tubular portions of CURL provide the entry for ASGP ligand into the sorting compartment.

Asialoglycoprotein Receptor↗
PubMed1982

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells.

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.

Animals↗
PubMed1980

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.

Animals↗
PubMed1980

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.

ABO Blood-Group System↗
PubMed1979

Initial glycosylation of proteins with acetylgalactosaminylserine linkages.

Epithelial glycoprotein like that produced by the gastric surface consists of a polypeptide chain rich in serine and threonine; to these amino acid residues oligosaccharide chains of variable length are linked. The linking sugar is acetylgalactosamine. To find out whether the initial glycosylation takes place at the ribosomal level. I treated purified peptidyl-tRNA, derived from rat gastric membrane-bound polysomes, with alkali in the presence of boro[3H]hydride. Alkali specifically splits glycosidic bonds between serine or threonine and oligosaccharide side chains (beta-elimination reaction). The linking sugar is converted to an alditol and simultaneously labeled. GalNAc was identified as the linking sugar by paper chromatography. Furthermore, nascent peptides with lengths between 40 and 60 amino acid residues already contained this linking sugar. Gel filtration on Bio-Gel P-2 of 3H-labeled saccharides revealed that nascent chains contained mainly monosaccharides, but some di- or trisaccharides were found with GalNAc as the linkage sugar. These findings demonstrate that initial glycosylation of epithelial glycoprotein occurs at the ribosomal level rather shortly after the nascent peptide chain has reached the cisternal lumen of the endoplasmic reticulum.

Acetylgalactosamine↗
PubMed1979

Effect of fasting and feeding on synthesis and intracellular transport of proteins in the frog exocrine pancreas.

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.

Animals↗
PubMed1978

Site of synthesis, intracellular transport and secretion of glycoprotein in exocrine cells.

The site of attachment of the first sugar, N-acetylgalactosamine, to the seryl and threonyl residues of the protein chain is unknown in exocrine cells. The subsequent sugars of the carbohydrate side chains, galactose and N-acetylglucosamine alternately, and the end-group sugars, galactose, N-acetylgalactosamine and fucose, are attached in the Golgi complex. Sulphate too is attached in that structure. In the stomach, sulphate is probably transferred in the most mature cisterna of the Golgi stacks, galactose and fucose in other cisternae, suggesting a gradient in transferase activities along the stack. The possibilities of regulating the amount and relative sugar composition of the glycoproteins are discussed. The secretory product is stored in granules. Their polygonal, large and swollen appearance and complex formation by loss of bordering membranes, as observed in many kinds of glycoprotein-secreting cells ('mucous cells') might be caused by ineffective fixation techniques. Direct vascular perfusion results in a picture no different from what is found in non-mucous cells. Whether secretion is merely exocytotic, as in non-mucous cells, or whether it is accompanied by a loss of membrane and even cytoplasm needs thorough investigation, with the effects of various fixation techniques being compared.

Acetylgalactosamine↗
PubMed1976

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.

Animals↗