Biomedical subjects

I Braakman

Publications and source records attributed to I Braakman.

32 records · Page 2Linked to original sources

PubMed1994

Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.

Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.

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PubMed1993

Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells.

Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conversion to DTT resistance was therefore a pre-Golgi event. Reduction of folding intermediates was dependent on the intactness of the ER and on metabolic energy, suggesting cooperativity between DTT and ER folding factors. DTT did not inhibit most cellular functions, including ATP synthesis and protein transport within the secretory pathway. The results established DTT as an effective tool for analyzing the folding and compartmental distribution of proteins with disulfide bonds.

Adenosine Triphosphate↗

PubMed1993

Posttranslational folding of vesicular stomatitis virus G protein in the ER: involvement of noncovalent and covalent complexes.

In this study, we show that posttranslational folding of Vesicular Stomatitis virus G protein subunits can involve noncovalent, multimeric complexes as transient intermediates. The complexes are heterogeneous in size (4-21S20,W), contain several G glycopolypeptides, and are associated with BiP/GRP78. The newly synthesized, partially intrachain disulfide-bonded G proteins enter these complexes immediately after chain termination, and are released 1-4 min later as fully oxidized, trimerization-competent monomers. These monomers are properly folded, judging by their binding of conformation-specific mAbs. When the G protein is translated in the presence of DTT, it remains reduced, largely unfolded and aggregated in the ER, but it can fold successfully when the DTT is removed. In this case, contrary to normal folding, the aggregates become transiently disulfide cross-linked. We also demonstrated that the fidelity of the folding process is dependent on metabolic energy. Finally, we established that the G protein of the folding mutant of the Vesicular Stomatitis virus, ts045, is blocked at a relatively late step in the folding pathway and remains associated with oligomeric, BiP/GRP78-containing folding complexes.

Adenosine Triphosphate↗

PubMed1992

Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.

Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.

Adenosine Triphosphate↗

PubMed1992

Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum.

Addition of the reducing agent dithiothreitol (DTT) to the medium of living cells prevented disulfide bond formation in newly synthesized influenza hemagglutinin (HA0) and induced the reduction of already oxidized HA0 inside the ER. The reduced HA0 did not trimerize or leave the ER. When DTT was washed out, HA0 was rapidly oxidized, correctly folded, trimerized and transported to the Golgi complex. We concluded that protein folding and the redox conditions in the ER can be readily manipulated by addition of DTT without affecting most other cellular functions, that the reduced influenza HA0 remains largely unfolded, and that folding events that normally take place on the nascent HA0 chains can be delayed and induced post-translationally without loss in efficiency.

Adenosine Triphosphate↗

PubMed1992

The endoplasmic reticulum as a protein-folding compartment.

The lumen of the endoplasmic reticulum (ER) provides a dynamic and efficient environment for the folding of proteins destined for secretion and for a variety of cellular compartments and membranes. Usually, the folding process begins on the nascent chains and is completed minutes or hours later during assembly of oligomers. It is assisted by molecular chaperones and folding enzymes, some of which are unique to the ER. Quality control and selective degradation systems ensure only conformationally mature proteins are transported from the ER.

Journal Article↗

PubMed1991

Folding of influenza hemagglutinin in the endoplasmic reticulum.

The folding of influenza hemagglutinin (HA0) in the ER was analyzed in tissue culture cells by following the formation of intrachain disulfides after short (1 min) radioactive pulses. While some disulfide bonds were already formed on the nascent chains, the subunits acquired their final disulfide composition and antigenic epitopes posttranslationally. Two posttranslational folding intermediates were identified. In CHO cells constitutively expressing HA0, mature HA0 subunits were formed with a half time of 3 min and their folding reached completion at 22 min. The rate of folding was highly dependent on cell type and expression system, and thus regulated by factors other than the sequence of the protein alone. Exposure of cells to stress conditions increased the level of glucose regulated proteins, including BiP, and decreased the folding rate. The efficiency of folding and subsequent trimerization was not dependent on the rate of translation, nor on temperature between 37 and 15 degrees C; however, the rates of folding and trimerization decreased with decreasing temperature. Whereas the rate of folding was independent of expression level, trimerization was accelerated at higher levels of expression.

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PubMed1991

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Acridine Orange↗

PubMed1989

Separation of hepatocytes of different acinar zones by flow cytometry.

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2-20 microM acridine orange (AO). After 10-60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or nonfluorescent (purity greater than 95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate-dehydrogenase, beta-glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.

Acridine Orange↗

PubMed1989

Zonal compartmentation of perfused rat liver: plasma reappearance of rhodamine B explained.

Rhodamine B (RB) fluorescence reappears in perfusion medium of a cyclically perfused rat liver after a rapid initial removal phase. At the same time the compound redistributes in the liver from acinar zone 1 toward zone 3. By analysis of the metabolic profile of RB, and by inhibition of glucuronidation (the main metabolic route) with salicylamide, we show in this paper that formation and secretion of RB-conjugates from liver into perfusate is not involved in the reappearance and redistribution phenomena. We therefore sought the explanation in a kinetic model, in which the acinar heterogeneity of the liver was simulated by several sequential liver compartments. Most kinetic parameters we used in the simulation were calculated from previous experiments with RB (Braakman et al., Hepatology 7: 849-855, 1987). This led to an accurate simulation of the measured RB curves in bile, medium and the acinar zones of the liver. In this study we show that a secondary rise in medium concentration of an injected compound is not necessarily caused by metabolism, but can be easily explained by considering the liver a sequence of compartments, instead of one well-stirred compartment. The conditions for the reappearance as well as for the intrahepatic redistribution are: a fast uptake into the liver, combined with a fast sinusoidal secretion and a slow biliary excretion of the injected substance.

Animals↗

PubMed1989

Zonal distribution of the cation lucigenin in rat liver: influence of taurocholate.

The yellow fluorescent cation lucigenin (LU) was used as a model compound to study acinar heterogeneity in transport of hydrophilic cations that enter the liver by adsorptive endocytosis. Hepatic uptake was fast and saturable. The extraction was about 50% in a cyclically perfused rat liver preparation in which endogenous bile salts were replaced by the infusion of taurocholate (TC). Fluorescence microscopy on 8-microns liver sections revealed a striking distribution pattern. LU appeared to be concentrated in micro- and macrovesicular structures in the cell. At the same time, LU skipped the first cells of the acinus, zone 1 in an antegrade and zone 3 in a retrograde perfusion. A downstream localization of the dye was the result. Single-pass perfusions with TC concentrations ranging from 0 to 180 microM showed that hepatic clearance of LU negatively correlated with the TC concentration (p less than 0.005). Clearance fell from 1.99 +/- 0.06 ml/min-g of liver(mean +/- SD) without TC to 1.61 +/- 0.21 with 45 microM TC and 1.65 +/- 0.12 with 180 microM TC. Moreover, in the absence of TC we observed a homogeneous distribution of LU. TC induced in the acinus a nonfluorescent upstream area that expanded with increasing TC concentration. We concluded that TC inhibited uptake of LU; a high medium concentration of TC in zone 1 (antegrade) was accompanied by a low uptake of LU in this zone, resulting in a downstream increasing acinar gradient. Hepatic uptake and acinar distribution of certain cationic drugs in vivo may, therefore, vary with the variable input of bile salts in the portal circulation and, hence, with nutritional status and the time of day.

Acridines↗

PubMed1989

Vesicular uptake system for the cation lucigenin in the rat hepatocyte.

The hepatic transport mechanism for the fluorescent bivalent hydrophilic organic cation lucigenin (LU) was characterized employing kinetic and morphological methods. The extraction of LU by the perfused rat liver was 50% and uptake was saturable. LU did not inhibit the carrier-mediated hepatic uptake of the model organic cationic compounds tributylmethyl ammonium (type 1) and vecuronium (type 2) in isolated hepatocytes, whereas the uptake of LU in the perfused liver was not affected by either type of cation or by the cardiac glycoside cymarin, a potent type 2 inhibitor. The cytoskeleton-disrupting agents cytochalasin B and nocodazole, however, significantly lowered hepatic uptake of LU. In the intact liver, LU did not stimulate fluid phase endocytosis, as indicated by a lack of effect on the internalization of horseradish peroxidase. These kinetic data point to adsorptive endocytosis as the most probable uptake mechanism. This was confirmed by the inhibitory effect of neomycin and the polycation poly(L-lysine) on LU uptake. Fluorescence microscopy revealed that LU accumulated in the hepatocytes in discrete vesicular structures. Partial co-localization of rhodamine-dextran and acid phosphatase with LU indicated that part of the LU fluorescence was present in lysosomes, although not all lysosomes contained LU. Taken together, we conclude that we identified a novel vesicular pathway for uptake of organic cations by hepatocytes.

Acridines↗

PubMedDate not supplied

Acinar redistribution and heterogeneity in transport of the organic cation rhodamine B in rat liver.

We studied a possible acinar heterogeneity in the transport of organic cations, using rhodamine B as model compound. Employing perfusions of isolated rat livers in the ante- and retrograde mode and quantitative fluorescence microscopy, Zones 1 and 3 were shown to be equally efficient in taking up rhodamine B. Ten minutes after injection in an antegrade perfusion, 95% of the dose was localized in the portal half of the acinus. Fifty minutes later, however, the amount of rhodamine B in Zone 1 had been reduced to 23%; 30 and 31% were in Zones 2 and 3, respectively, and the medium concentration was doubled. Thus, unchanged rhodamine B appeared to be transported downstream within the liver, either via the medium or directly from cell to cell, finally resulting in a relatively higher rhodamine B concentration in Zone 3. To obtain additional data, we designed a perfusion setup in which the zones could be studied separately. In both zones, the amount excreted into the medium was about 30 times the amount excreted into bile. Intracellular sequestration of rhodamine B and the rate constant for sinusoidal secretion were higher in Zone 3, while the sinusoidal uptake rates were equal; biliary excretion was higher in Zone 1. Acinar distribution changed with time because rhodamine B, primarily accumulated in Zone 1, was secreted into the sinusoids and taken up again by downstream cells. The finally higher rhodamine B concentration in Zone 3 was caused by a zonal heterogeneity in intracellular sequestration and sinusoidal secretion of rhodamine B.

Animals↗

PubMedDate not supplied

Heterogeneous acinar localization of the asialoglycoprotein internalization system in rat hepatocytes.

Desialylated glycoprotein is rapidly cleared from plasma by a receptor-mediated endocytic mechanism located on hepatocytes. We studied the hepatic acinar distribution of this asialoglycoprotein transport system with the ligand 125I-asialoorosomucoid using rat liver perfused in either antegrade or retrograde direction in combination with quantitative light microscopic autoradiography. Grain distribution along the acinus appeared dependent on the perfusion direction. A rather shallow zone 1 to zone 3 gradient was observed if livers were perfused in the normal direction. However, a statistically significantly steeper zone 3 to zone 1 gradient was detected in retrograde perfusions. Kinetic analysis of perfusate clearance profiles yielded a hepatic clearance of 21.6 +/- 1.3 ml per min in antegradely perfused liver. Hepatic extraction was calculated to be 60.1 +/- 7.4%. Biliary secretion of radioactivity amounted to 1.89 +/- 0.18% of the dose within 1 hr after injection and consisted of intact material (1.39 +/- 0.25%) and radioactive low-molecular-weight degradation products (0.52 +/- 0.08%), of which more than 90% could be accounted for by 125I-. Apart from a minor difference regarding biliary secretion of an unidentified glycopeptide (less than 0.1% of the injected dose), transport data for the retrogradely perfused livers were identical to those obtained with livers perfused in antegrade direction, emphasizing the functional equivalence of both groups of livers. The autoradiographic data indicate that zone 3 hepatocytes take up 125I-asialoorosomucoid more avidly than zone 1 cells. The kinetic and biochemical data indicate that further processing in the hepatocytes is virtually similar in the two zones.(ABSTRACT TRUNCATED AT 250 WORDS)

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