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R Cacan

Publications and source records attributed to R Cacan.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of an oligosaccharide-pyrophosphodolichol pyrophosphatase activity in yeast.

Use of Triton X-114 allowed us to develop a new method to separate hydrophilic oligosaccharidic material from hydrophobic oligosaccharide pyrophosphodolichols (oligosaccharide-PP-Dol). Taking advantage of this procedure we characterize, in yeast microsomal membranes, an enzymic activity that hydrolyses oligosaccharide-PP-Dol into oligosaccharidic material. H.p.l.c. analysis together with alkaline-phosphatase- and endo-N-acetyl-beta-D-glucosaminidase-susceptibility demonstrate that the oligosaccharidic released material is mainly composed of oligomannosides containing a chitobiose phosphate at the reducing end. The enzymic activity requires bivalent cations and is inhibited by pyrophosphate, NAD+ and bacitracin. As other, commercially available, pyrophosphatases have no action on lipid intermediates, the described pyrophosphatase activity appears to be the specific enzyme for oligosaccharide-PP-Dol. This enzymic splitting of the pyrophosphate bond might be the primary event in the catabolism of lipid intermediates.

Bacitracin↗
PubMed1987

Catabolic pathway of oligosaccharide-diphospho-dolichol. Study of the fate of the oligosaccharidic moiety in mouse splenocytes.

Metabolic labelling of mouse splenocytes with radioactive mannose indicates that the glycosylation process is accompanied by the release of soluble oligomannoside material. Chase experiments with an excess of unlabelled mannose indicate that the radioactivity is mainly chased from oligosaccharide-PP-Dol (PP-Dol = diphosphodolichol): 10% is recovered as (Man)9(GlcNAc)2-P, (Man)9(GlcNAc)2, (Man)9GlcNAc and (Man)5 GlcNAc, and 90% is rapidly degraded further. Tunicamycin inhibits both oligosaccharide-PP-Dol synthesis and the formation of the oligosaccharide material to the same extent. The results thus indicate that these soluble oligomannoside structures represent the main steps of the oligosaccharide-PP-Dol catabolic pathway, starting with the cleavage of the diphosphate bond. However, it cannot be excluded that part of this material is released from newly formed glycoproteins. The soluble oligomannoside material does not contain glucose residues despite the fact that part of the oligosaccharide-PP-Dol is glucosylated and it was shown, by the use of glucosidase I inhibitors (castanospermine, deoxynojirimycin) that, after cleavage, the glycan moiety of glucosylated oligosaccharide-PP-Dol is first rapidly deglucosylated. These experiments provide a physiological basis to our previous results obtained in vitro and allow the definition of further steps in the catabolic pathway of oligosaccharide-PP-Dol.

1-Deoxynojirimycin↗
PubMed1987

Membrane transport of sugar donors to the glycosylation sites.

The assembly of N-linked glycoproteins in eukaryotic cells begins with the segregation of these molecules within the lumen of intracellular vesicles. Since the sugar nucleotides are cytoplasmic molecules, translocation of the sugar moiety across the membrane appears as a crucial event in the glycoprotein synthesis. This N-glycosylation process occurs in two different cytological sites: in the rough endoplasmic reticulum, the stepwise synthesis of a large lipid-linked oligosaccharide takes place, as well as its transfer to protein; then after trimming the immature glycoprotein is further elongated in the Golgi apparatus. In this paper, a brief review will be given of the present knowledge on the sugar donor transport across the membrane barrier to the glycosylation site. Based upon the transmembrane orientation of oligosaccharide lipid intermediates and on the localization of the glycosyltransferase active sites, the different processes required to translocate the sugar moieties during the preassembly of the dolichyl-pyrophosphate-oligosaccharides will be examined. Combining the different results, obtained in several laboratories, it is suggested that the Man5-GlcNAc2-lipid is synthesized on the cytoplasmic side directly from the sugar-nucleotides and then translocated to the lumenal face where the Glc3-Man9-GlcNAc2-lipid is completed using Man-P-Dol and Glc-P-Dol as transmembrane carriers of these sugars. Concerning the elongation process leading to assembly of the antennae of N-acetyllactosamine type oligosaccharides, specific carriers for sugar nucleotides have been described as Golgi markers. Several authors have characterized such carriers for UDP-Gal, GDP-Fuc, CMP-NeuAc, UDP-GlcNAc and UDP-Glc using microsomal vesicles and similar results have been obtained in our laboratory using plasma membrane permeabilized cells. This carrier-mediated process leads to the formation of an intralumenal pool whose biological significance will be discussed. The translocation process of sugar donors occurring in the rough endoplasmic reticulum via lipid intermediates as well as in the Golgi apparatus via specific carriers would represent a regulation step based on the availability of the substrates for the glycosylation.

Animals↗
PubMed1986

Mechanism of UDP-sugar transport into intracellular vesicles. Occurrence of UDP-GlcNAc/UDP and UDP-Gal/UDP antiports.

The mechanism of translocation of UDP-GlcNAc, UDP-Gal and UDP-Glc into intracellular vesicles has been studied using thymocytes whose plasma membranes have been permeabilized with isotonic ammonium chloride. It has been previously shown that the intracellular vesicles have specific carriers for UDP-GlcNAc and UDP-Gal. We now report that the translocation of these two sugar nucleotides occurs via UDP-GlcNAc/UDP and UDP-Gal/UDP antiports. The entry of UDP-GlcNAc or UDP-Gal into vesicles was specifically dependent on the exit of UDP from these vesicles. In contrast, no antiport mechanism has been recovered with UDP-Glc for which no transport and accumulation into intracellular vesicles were observed.

Animals↗
PubMed1986

Dilatation of Golgi vesicles by monensin leads to enhanced accumulation of sugar nucleotides.

Incubation of mouse thymocytes with 10 microM monensin for 1 hour induces morphological alterations characterized by the extensive dilatation and vacuolization of the Golgi complex. This effect is used to study the transport and utilization of labelled sugar nucleotides into intracellular vesicles by using thymocytes whose plasma membrane has been permeabilized by ammonium chloride treatment. It is demonstrated that monensin stimulates the incorporation of labelled sialyl, fucosyl, galactosyl, and N-acetylglucosaminyl residues. This enhanced incorporation is not due to a direct effect of monensin on glycosyltransferase activities themselves but is a consequence of a higher entry and accumulation of labelled sugar nucleotides in the dilated vesicles.

Ammonium Chloride↗
PubMed1985

Accumulation of UDP-GlcNAc into intracellular vesicles and occurrence of a carrier-mediated transport. Study with plasma-membrane-permeabilized mouse thymocytes.

Metabolic labelling of mouse thymocytes with radioactive mannose or glucosamine leads to the formation of labelled GDP-Man and UDP-GlcNAc. Using isotonic ammonium chloride treatment which renders the plasma membrane of thymocytes permeable to sugar nucleotides, we demonstrate that, in contrast to GDP-Man, a pool of UDP-GlcNAc remains associated with the cells after plasma membrane permeabilization. These observations are confirmed in experiments in which permeabilized thymocytes are incubated with exogenous labelled GDP-Man and UDP-GlcNAc, and we show that only UDP-GlcNAc is accumulated into sealed intracellular vesicles. This accumulation is a saturable process which can be inhibited by UDP, demonstrating the occurrence of a specific carrier. This transport mechanism can be blocked by covalent attachment of a non-permeant inhibitor UDP-dialdehyde without affecting the N-acetylglucosaminyltransferase itself. The fact that this carrier-mediated transport is not inhibited by tunicamycin indicates that this translocation process of UDP-GlcNAc does not involve lipid intermediates.

Animals↗
PubMed1984

Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes.

Treatment with NH4Cl of mouse thymocytes renders their plasma membrane permeable to sugar nucleotides both inwards and outwards. Using this model, we studied the entry and utilization of CMP-NeuAc, GDP-Fuc and UDP-Gal into intracellular vesicles in situ. It is shown that CMP-NeuAc and GDP-Fuc enter the vesicles in a manner indicating a carrier-mediated transport (substrate saturation curve, inhibition by substrate analogues, temperature dependence) and are entrapped in their uncleaved form. This leads to the formation of an intralumenal pool of these precursors which can be further utilized by the sialyltransferases and fucosyltransferases. The occurrence of an endogenous pool of CMP-NeuAc and GDP-Fuc is demonstrated by the fact that, when the vesicles are disrupted by detergent, the release of the endogenous sugar nucleotides causes an isotopic dilution of the labelled precursors added to measure the glycosyltransferase activities. In contrast, no accumulation of UDP-Gal has been detected, suggesting that transport and transfer reaction are simultaneous events. However, experiments with UDP 2',3'-dialdehyde indicate that UDP-Gal is not transported through the membrane by galactosyltransferase action but by a distinct carrier molecule.

Ammonium Chloride↗
PubMed1984

Viral neuraminidase and cellular ectosialyltransferase in human lymphoblastoid cells infected with influenza virus.

In human lymphoblastoid cells, infected with an influenza virus, Fowl Plague Virus (FPV), glycoproteins (such as secreted IgM) are hyposialylated, through the action of viral neuraminidase. In this study, the modulation of the cellular ectosialyltransferase activity during viral infection was investigated. This activity was detectable in FPV-infected cells, was shown to be 2.5-fold higher than that of uninfected cells, and to be able to restore, at least partially, the level of sialylation of the cell surface acceptors.

Cell Line↗
PubMed1983

Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes.

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.

Ammonium Chloride↗
PubMed1983

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration.

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.

Animals↗
PubMed1982

Effect of bis-(p-nitrophenyl) phosphate on the biosynthesis and the utilization of lipid-intermediates.

Incubations of rat spleen lymphocytes with the required labelled nucleotide sugars lead to the formation of the various lipid-intermediates involved in the N-glycosylation of proteins. The effect of bis-(p-nitrophenyl) phosphate on the different reactions involved in the dolichol pathway has been studied. Although dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl diphosphate N-acetylglucosamine synthesis is not affected at all by bis-(p-nitrophenyl) phosphate (20 mM), this product inhibits completely the addition of the second N-acetylglucosamine residue on the dolichyl diphosphate N-acetylglucosamine acceptor. The addition of the five innermost mannose residues from GDP-mannose as donor is also strongly abolished. However, the addition of the more distal sugars, i.e. the four mannose residues using dolichyl phosphate mannose as donors and the additional glucose residues are only slightly affected. The reactions involved in the utilization of dolichyl diphosphate oligosaccharide, i.e. transfer to the proteins or degradation into soluble phospho-oligosaccharides, are also strongly inhibited. Thus bis-(p-nitrophenyl) phosphate appears to affect only the reactions involving the presence of dolichyl diphosphate sugar as substrate.

Animals↗
PubMed1982

Discrimination between activity of (alpha 2-3)-sialyltransferase and (alpha 2-6)-sialyltransferase in human platelets using p-nitrophenyl-beta-D-galactoside as acceptor.

Exogenous asialo-glycoproteins and endogenous acceptors are both sialylated by incubating cytidine 5'-monophosphate N-[14C]acetylneuraminic acid (CMP [14C]NeuAc) with a lysate of human platelets but their respective incorporation levels vary with the divalent cation concentration. P-Nitrophenyl-beta-D-galactoside has also been demonstrated to be an acceptor of sialyl residues, and two different sialyl derivatives are synthesized according to the concentration of divalent cations. P-Nitrophenyl-beta-D-[6-3H]galactoside has been prepared by reduction with tritiated borohydride of the compound previously oxidized by galactose oxidase. Using this labelled p-nitrophenyl-beta-D-galactoside as acceptor and unlabelled CMP-NeuAc as donor, the two sialyl derivatives have been identified by methylation analysis as alpha-sialosyl-(2-3)-p-nitrophenyl-beta-D-galactoside and alpha-sialosyl-(2-6)-p-nitrophenyl-beta-D-galactoside. In addition to their different responses to divalent cation requirements, the sialyltransferase activities responsible for the synthesis of the two sialylgalactoside isomers have been clearly distinguished by their temperature and pH optimal values. They also exhibit different susceptibilities to dithioerythritol and different stabilities. These results demonstrate the presence in human platelets of two sialyltransferases: a CMP-NeuAc: galactoside (alpha 2-3)-sialyltransferase and a CMP-NeuAc: galactoside (alpha 2-6)-sialyltransferase.

Blood Platelets↗
PubMed1982

Dolichol-dependent synthesis of chitobiosyl proteins and their further mannosylation. A second route for glycosylation of proteins in rat-spleen lymphocytes?

Incubation of whole lymphocytes with UDP-N-acetyl [3H]glucosamine used as the only precursor leads to the formation of dolichyl diphosphate [3H]chitobiose, DolPP-(GlcNAc)2, and dolichyl diphosphate N-acetyl-[3H]glucosamine, DolPP-GlcNAc. Although very few dolichyl diphosphate oligosaccharides are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [3H]chitobiosyl and N-acetyl[3H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DolPP-(GlcNAc)1-2 and the incorporation of the N-acetyl[3H] glucosaminyl residues onto proteins, indicating that these carbohydrate units are transferred onto proteins acceptors from their dolichol derivatives. Chase experiments have indicated that, in fact, the DolPP-(GlcNAc)1-2 were utilized in two ways: either their transfer onto proteins or their degradation into water-soluble saccharidic material. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DolPP-(GlcNAc)1-2 is not recovered on proteins. This fact allows to show that part of the [3H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP-mannose. This direct mannosylation of chitobiosyl-proteins may represent a second route for the N-glycosylation of proteins.

Animals↗
PubMed1981

Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides.

Incubation of rat-spleen lymphocytes with UDP-glucose together with GDP-mannose and UDP-N-acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligosaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non-glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p-nitrophenyl-alpha-D-glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.

Animals↗
PubMed1980

Fate of oligosaccharide-lipid intermediates synthesized by resting rat-spleen lymphocytes.

Using conditions to avoid the utilization of labelled precursors by intracellular glycosyltransferases, experiments are described demonstrating that intact rat-spleen lymphocytes are capable of utilizing exogenous GDP-mannose and UDP-N-acetylglucosamine to synthesize dolichyl monophosphate mannose and dolichyl diphosphate oligosaccharides. Kinetic and chase experiments show that dolichyl diphosphate oligosaccharides are either utilized for the transfer of their carbohydrate moieties to protein acceptors or further degraded. Since glycosylation of proteins is limited in resting lymphocytes, the degradation pathway appears as a major event in the fate of the dolichyl diphosphate oligosaccharides synthesized in vitro. These dolichyl diphosphate oligosaccharides are degraded into phospho-oligosaccharides and oligosaccharides which are released in the medium. This enzymatic cleavage of the phosphodiester bond is inhibited by bacitracin. The phospho-oligosaccharides are susceptible to alkaline phosphatase giving neutral oligosaccharides and they are cleaved by endo-N-acetyl-beta-D-glucosaminidase H leaving N-acetylglucosamine 1-phosphate and neutral oligosaccharides. These data suggest that splitting of the phosphodiester bond of colichyl diphosphate oligosaccharides, dephosphorylation and/or endo-N-acetyl-beta-D-glucosaminidase hydrolysis of the phosphorylated oligosaccharides could represent the beginning of the catabolic pathway of dolichyl diphosphate oligosaccharides.

Acetylglucosaminidase↗
PubMed1980

Metabolism of lipid-linked oligosaccharide intermediates in rat spleen lymphocytes. Evidence for ectoglycosyltransferase activities.

Double-labelling experiments show that intact lymphocytes as well as lymphocyte homogenates can utilize GDP-[14C]mannose and UDP-N-[3H]acetylglucosamine to synthesize lipid-linked oligosaccharide intermediates. However, the intermediates formed are quantitatively and qualitatively different in the two systems. The amount of dolichyl diphosphate oligosaccharides synthesized in both cases was calculated by using external labelling by sodium boro[3H]hydride reduction of the glycan moiety obtained after mild acid treatment of [14C]mannose-labelled dolichyl diphosphate oligosaccharides. This showed that, due to the liberation of intracellular enzymes, a larger amount of dolichyl diphosphate oligosaccharides was synthesized by homogenate. However, this higher glycosyltransferase activity was not detected by the direct measurement of incorporation of labelled GDP-[14C]mannose and UDP-N-[3H]acetylglucosamine, due to isotopic dilution caused by both endogenous soluble UDP-N-acetylglucosamine and membrane-bound dolichyl phosphate mannose accumulated during the homogenization process. In addition, endogenous UDP-glucose allowed the formation, by homogenate, of glucosylated dolichyl diphosphate oligosaccharides which were not observed with intact cells unless exogenous UDP-glucose was added. These striking differences between the lipid intermediates synthesized by homogenate or by intact cells exclude the possibility that intracellular glycosyltransferases could account for the glycosyltransferase activities observed with whole lymphocyte suspensions. This allows us to conclude that ectoglycosyltransferases involved in the dolichol cycle are present at the outer surface of lymphocytes.

Animals↗
PubMed1979

Detection of ectosiallyltransferase activity using whole cells. Correction of misleading results due to the release of intracellular CMP-N-acetylneuraminic acid.

An inhibitory effect due to broken cells is observed when sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) is measured with mixture of intact and homogenized lymphocytes. This intracellular inhibitory factor ib purified and characterized as CMP-N-acetylneuraminic acid (CMP-NeuNAc) by its behavior in various chromatographic and electrophoretic systems and by its susceptibility to CMP-NeuNAc hydrolase. This endogenous CMP-NeuNAc leads to an isotopic dilution of the exogenous labelled CMP-NeuNAc explaining the apparently lower activity of homogenate when compared to whole cells. Consequently, the radioactivity bound to acceptors may not be related to a known number of sialyl residues transferred, calling into question the validity of comparing the incorporation of [14C]NeuNAc by homogenate and whole cells in order to assign sialyltransferase activity to ectoenzyme. A new approach is developed to detect ectoglycosyltransferases with whole cells, taking into account that both intracellular enzymes and endogenous precursor may be introduced by the small percentage of broken cells.

Animals↗