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G J Strous

Publications and source records attributed to G J Strous.

At least 73 records · Page 4Linked to original sources
PubMed1989

Sulphation causes heterogeneity of gastric mucins.

The synthesis of mucus glycoprotein in rat stomach was studied in stomach segments, which were pulse-labelled with both [3H]galactose and [35S]sulphate and chased for various times. The radioactive glycoproteins were analyzed by CsCl centrifugation and by agarose gel electrophoresis. After a pulse-labelling for 15 min with [3H]galactose, a possible intermediate with an Mr of 200,000 and a buoyant density of 1.60 g/ml could be demonstrated. Following chase periods of 1 and 4 h, [3H]galactose and [35S]sulphate were present in glycoproteins with a mean buoyant density of 1.50 g/ml. This is clearly different from the main density of glycoproteins isolated from mucosal scrapings (1.46 g/ml). Another difference is the high electrophoretic mobility on gel electrophoretic analysis of newly synthesized glycoproteins compared to that of the major portion of the glycoproteins from mucosal scrapings. When sulphation of glycoproteins was inhibited by sodium chlorate, electrophoretic mobility and buoyant density both decreased. Sodium chlorate had no effect on glycoprotein synthesis nor on glycoprotein secretion. We conclude from our data that the heterogeneity in electrophoretic mobility and buoyant density can be attributed to a different degree of sulphation of the same glycoprotein.

Animals↗
PubMed1989

Release of soluble resident as well as secretory proteins from HepG2 cells by partial permeabilization of rough-endoplasmic-reticulum membranes.

Secretory proteins migrate from the rough endoplasmic reticulum (ER) to the Golgi complex at different rates. Selective retention of specific proteins to rough-ER membrane constituents could explain this phenomenon. We have permeabilized HepG2 cells with low concentrations of saponin. Release of newly synthesized proteins was studied after brief labelling in the presence of [35S]methionine. The efflux of several secretory proteins was studied at various saponin concentrations; a 2-fold higher saponin concentration was required to release transferrin compared with that required to release albumin and orosomucoid. Glucosidase II, a soluble resident protein of the ER, is released at the same saponin concentration as albumin. Saponin did not destroy the membrane skeleton structure; at the concentrations used, the integral membrane protein G of vesicular-stomatitis virus remained fully associated with the cells.

Albumins↗
PubMed1989

Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells.

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.

Asialoglycoprotein Receptor↗
PubMed1988

Metalloendoprotease inhibitors block protein synthesis, intracellular transport, and endocytosis in hepatoma cells.

Fusion of membrane vesicles has been implicated in many intracellular processes including the transport of proteins destined for secretion or storage. Vesicular transport coupled with membrane fusion has been demonstrated for rough endoplasmic reticulum to Golgi and Golgi to plasma membrane transport as well as receptor mediated endocytosis and receptor recycling. Recent studies with inhibitors suggest that metalloendoproteases may mediate a wide variety of intracellular fusion events. Thus, in order to examine the potential role of metalloendoproteases in both transport/secretion and endocytosis/recycling we have used selected dipeptide substrates to probe these processes in human HepG2 cells. Using pulse-chase labeling, immunoprecipitation, and polyacrylamide gel electrophoresis we show that transport and secretion of newly synthesized proteins along the exocytotic route were completely inhibited by substrate dipeptides (e.g. Cbz-Gly-Phe-amide, where Cbz is benzyloxycarbonyl) but not by irrelevant dipeptides (e.g. Cbz-Gly-Gly-amide). The effect was rapid, reversible, and specific. The secretory pathway was blocked between the rough endoplasmic reticulum and Golgi as well as Golgi and plasma membrane as judged by the status of N-glycosylation intermediates. In addition, these inhibitors specifically inhibited protein synthesis without alterations in cellular ATP concentrations. However, cell-free amino acid incorporation was not inhibited. Receptor-mediated uptake of asialoglycoproteins was specifically and reversibly inhibited by dipeptide substrates. This effect appears to be secondary to inhibition of recycling as neither ligand binding nor internalization were affected. Thus the present observations suggest that metalloendoprotease activity may be involved in the regulation of multiple intracellular pathways perhaps at the level of vesicular fusion events.

Biological Transport↗
PubMed1988

In vitro biosynthesis of two human galactosyltransferase polypeptides.

HeLa cell galactosyltransferase is synthesized as two precursor polypeptides of Mr = 45,000 and Mr = 47,000. The enzyme is present in the Golgi complex as a (mature) Mr = 54,000 glycoprotein. If cells are treated with tunicamycin, two precursor polypeptides are synthesized without N-linked oligosaccharides with molecular weights of 42,000 and 44,000, respectively. To investigate whether the two precursor polypeptides are synthesized on different mRNAs total RNA from HeLa cells was translated in a wheat germ cell-free system. Galactosyltransferase polypeptides were isolated by immunoprecipitation and compared to the polypeptides synthesized in vivo in the presence of tunicamycin. The two in vitro translated polypeptides co-migrate exactly with the polypeptides made in the cells in the presence of tunicamycin, indicating two different mRNAs for galactosyltransferase. The results also indicate that translocation of galactosyltransferase through the membrane of the rough endoplasmic reticulum is not followed by signal peptide cleavage.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Recycling of 5'-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.

5'-Nucleotidase↗
PubMed1988

The influence of pre-incubation on the rate of iron uptake and of transferrin synthesis by intact isolated rat seminiferous tubules.

It has been reported recently that major kinetic differences exist in the rate of iron uptake and of transferrin synthesis between intact seminiferous tubules and cultured Sertoli cells. To investigate possible causes of these differences, intact isolated rat seminiferous tubules were isolated and pre-incubated overnight. Then the rates of iron uptake and of transferrin synthesis were compared with those of freshly isolated tubules. We found that overnight pre-incubation increased the rate of both processes. Iron depletion was not the cause of these changes, since pre-incubation under iron excess gave comparable results. Possible explanations of these phenomena are discussed.

Animals↗
PubMed1988

The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum.

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome-associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1-anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.

Biological Transport↗
PubMed1988

Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles.

The intracellular distributions of the cation-independent mannose 6-phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgp120) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgp120 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes. lgp120, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgp120 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR+/lgp120-, MPR+/lgp120+, MPR-/lgp120+, and vacuoles nonimmunolabeled for MPR and lgp120. Tracer first reached MPR+/lgp120-, then MPR+/lgp120+, and finally MPR-/lgp120+ vacuoles, which are assumed to represent lysosomes. To study the kinetics of appearance of endocytic tracers in MPR-and/or lgp120-containing pools in greater detail, cells were allowed to endocytose horse-radish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgp120 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgp120. The combined data suggest that MPR+/lgp120+ vacuoles are endocytic vacuoles, intermediate between MPR+/lgp120-endosomes and MPR-/lgp120+ lysosomes, and represent the site where MPR is sorted from lgp120 destined for lysosomes. We propose that MPR is sorted from lgp120 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgp120 in the vacuoles and the net transport of lgp120 to lysosomes.

Animals↗
PubMed1988

Kinetics of transferrin endocytosis and iron uptake by intact isolated rat seminiferous tubules and Sertoli cells in culture.

The receptor-mediated endocytotic cycle of rat and human transferrin has been studied in intact, isolated rat seminiferous tubules and Sertoli cells in culture. Double-labeled [( 59Fe125I]) transferrin has been used to study the fate of transferrin and iron. Diferric transferrin binds to the tubules and the cultured Sertoli cells and is internalized. The iron remains inside, while the transferrin recycles and is released into the medium. Although, as reported before (Wauben-Penris et al., 1986), "extra" binding sites for human transferrin exist as compared to rat transferrin, this does not result in extra uptake of transferrin or iron. Both rat and human transferrin transport iron into the cells and recycle back to the surface, and do so with identical kinetics. A striking difference has been found between the mean efficient recycling times of the transferrin receptors in intact tubules (90 min) and in Sertoli cells in culture (21 min). Possible explanations of this difference are discussed. Light-microscopic autoradiography of [125 I]-labeled transferrin has revealed that the transferrin protein is excluded from the adluminal compartment, even after 21 h of incubation. This indicates that externally added transferrin itself does not deliver iron to the postmeiotic germ cells in intact, isolated rat seminiferous tubules.

Animals↗
PubMed1987

Golgi galactosyltransferase contains serine-linked phosphate.

In HeLa and HepG2 cells the Golgi complex enzyme galactosyltransferase became phosphorylated following incubation with 32Pi-Analysis on sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed incorporation of 32P into the mature 54-kDa form. This phosphorylation was independent of protein synthesis. Serine was identified as the sole phosphorylated amino acid; no radioactive phosphate was detected on N-linked oligosaccharide. The phosphate-labelled galactosyltransferase has the same turnover as [35S]methionine-labelled polypeptides (t1/2 = 20 h). Soluble enzyme, released by the cells, contained very little phosphate relative to that which remained cell-associated. Charge heterogeneity arising from phosphorylation contributes in part to the heterodispersed appearance of the enzyme on two-dimensional gels, as the degree of radioactive phosphate differs among the different iso-enzymes.

Amino Acids↗
PubMed1987

Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron.

Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed.

Cell Line↗
PubMed1987

Glucosidase II, a protein of the endoplasmic reticulum with high mannose oligosaccharide chains and a rapid turnover.

Glucosidase II is regarded as a resident protein of the endoplasmatic reticulum. The enzyme removes alpha-1-3-linked glucose from high mannose oligosaccharides N-linked to asparagine residues of glycoproteins. Monospecific antibodies raised against the pig kidney enzyme are used to study the metabolism of the enzyme in a rat hepatoma cell line. These antiglucosidase II antibodies specifically immune precipitate glucosidase II as a 100,000-Da species from [35S]methionine-labeled cells. In addition, protein blotting and immune staining of cell extracts from both rat liver and human and rat hepatoma cell lines show identity in apparent Mr (100,000). Glucosidase II synthesized in the presence of tunicamycin is approximately 94,000 Da, indicating the presence of one or more N-linked oligosaccharide chains. Cell-free protein synthesis of rat hepatoma total RNA demonstrates that glucosidase II is synthesized as a slightly higher molecular weight species as compared to the polypeptide synthesized in whole cells in the presence of tunicamycin, indicating that the enzyme has a cleavable signal sequence. Using a pulse-chase protocol, the apparent molecular weight does not change upon longer chase periods. In addition, the 100,000-Da protein remains sensitive to endo-beta-N-acetylglucosaminidase H regardless of prolonged chase periods. The cells incorporate [3H]mannose into the enzyme; after release with endo-beta-N-acetylglucosaminidase H, most of the radioactivity comigrates with Glc1-Man9-GlcNAc on a gel filtration column. Phase separation in Triton X-114 shows a partition between the aqueous and the Triton phase, the major portion being separated in the aqueous phase. In rat hepatoma cells glucosidase II has a half-life of 50 min. This value is not altered if the cells are grown in the presence of monensin nor of methyl-deoxynoijirimycin. However, tunicamycin and low concentrations or primaquine (raising the pH of acidic compartments) causes a 100% increase in half-life of glucosidase II. We conclude that glucosidase II is a hydrophilic, probably not a transmembrane membrane, protein with a short half-life. It is the first example of an oligosaccharide-processing enzyme not being an integral membrane protein.

Animals↗
PubMed1987

Effect of 16,16-dimethyl prostaglandin E2 on mucus glycoprotein biosynthesis in rat stomach and duodenal glands.

The present study describes the effect of 16,16-dimethyl prostaglandin E2 (16,16-dmPGE2) on mucus glycoprotein biosynthesis in rat stomach and duodenal glands. After in vivo treatment with 16,16-dmPGE2 (10 micrograms/kg subcutaneously) for 1 h, the incorporation rate of [3H]galactose, [3H]glucosamine, and [3H]serine in the ex vivo vascularly perfused stomach was determined by light microscopic autoradiography. As was previously found by us for the surface mucous cells in the fundus of 16,16-dmPGE2-treated rats, the incorporation of [3H]galactose and [3H]glucosamine (indicative of mucus glycoprotein synthesis) in the isthmus was increased two- to fourfold. Small if any increases were detected in the mucous cells near the base of the glands of the fundus (neck cells), the mucous cells in the antrum and the mucous cells in the duodenal glands. Total protein synthesis as measured by [3H]serine incorporation was not increased in any of these cells. We conclude that 16,16-dmPGE2 has different effects on mucus glycoprotein biosynthesis in various regions of the rat stomach. Increased biosynthesis in the fundus points to a role for mucus in the prostaglandin-induced protection of the gastric mucosa against injury.

16,16-Dimethylprostaglandin E2↗
PubMed1987

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells.

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'-diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake.

Asialoglycoprotein Receptor↗
PubMed1987

Ligand- and weak base-induced redistribution of asialoglycoprotein receptors in hepatoma cells.

The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34% of the total cellular receptors on the plasma membrane, 37% in compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell surface receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 microM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42%. ASGPR labeling of CURL increased only to 47%. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed.

Asialoglycoprotein Receptor↗