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2.
Biochimie ; 85(1-2): 65-73, 2003.
Article in English | MEDLINE | ID: mdl-12765776

ABSTRACT

The SPASIBA force field has been applied to the determination of the structure and dynamical properties of various disaccharides. It has been shown that the experimental properties (structure, dipole moment, conformational relative energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are confidently reproduced without specific terms for the alpha and beta anomers and the type of glycosidic linkages.


Subject(s)
Algorithms , Carbohydrate Conformation , Disaccharides/chemistry , Glycosides/chemistry , Software , Static Electricity , Stereoisomerism , Structure-Activity Relationship , Sulfhydryl Compounds/chemistry , Thermodynamics , Trehalose/chemistry
3.
Biochimie ; 85(1-2): 219-29, 2003.
Article in English | MEDLINE | ID: mdl-12765791

ABSTRACT

The potential role of glycoprotein N-glycans in the proliferation and adhesion of C6 glioblastoma cells was investigated using a set of N-glycosylation inhibitors (tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, swainsonine), and traffic (monensin). It was observed that both the proliferative and adhesive properties of C6 cells were dependent upon the expression at the cell surface of glycoproteins with oligomannosidic and hybrid type N-glycans, whereas the absence of N-glycans (tunicamycin) or the presence of glucosyl-oligomannosides (deoxynojirimycin and castanospermine) and the absence of glycoproteins at the cell surface (monensin) reduced both the proliferative and adhesive properties of C6 cells. Studies of the classical elements of signalling pathways indicated that the different inhibitors have a low impact on tyrosine phosphorylations and oncogene product expression (except the ras oncogene product), except on phosphorylations on other residues. An endogenous soluble lectin (CSL; J. Neurochem. 49 (1987) 1250), specific for oligomannosidic and hybrid type N-glycans, was present and externalised by the cells through a pinching-off of large intracellular vesicles, a mechanism that was not blocked by monensine; in contrast with the externalisation of its glycoprotein ligands. The inhibitory effect of anti-CSL Fab fragments on adhesion indicates that the polyvalent CSL acts as a bridging molecule for a family of surface glycoproteins expressed at the surface of C6 cells. The inhibitory effect of the same Fab fragments on the proliferation indicates that CSL is a mitogen for these cells, possibly involved in clustering its surface glycoprotein ligands. A mechanism for the loss of contact inhibition is discussed based on the over-expression of CSL ligands in C6 glioblastoma cells relative to normal cells.


Subject(s)
Oligosaccharides/metabolism , Polysaccharides/metabolism , Antibodies, Monoclonal/immunology , Cell Adhesion/drug effects , Cell Aggregation/drug effects , Cell Division/drug effects , Cell Line, Tumor/drug effects , Contact Inhibition , Glioblastoma , Glycoproteins/chemistry , Glycoproteins/metabolism , Glycosylation/drug effects , Humans , Immunoglobulin Fab Fragments/pharmacology , Indolizines/pharmacology , Lectins/metabolism , Ligands , Monensin/pharmacology , Oligosaccharides/chemistry , Oligosaccharides/pharmacology , Polysaccharides/chemistry , Tunicamycin/pharmacology
4.
Biochimie ; 85(3-4): 323-30, 2003.
Article in English | MEDLINE | ID: mdl-12770771

ABSTRACT

The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins.


Subject(s)
Glycoproteins/chemistry , Glycoproteins/metabolism , Animals , Biological Transport, Active , Cell Polarity , Epithelial Cells/metabolism , Glycosylation , Glycosyltransferases/metabolism , Humans , Mucins/chemistry , Mucins/metabolism , Signal Transduction , trans-Golgi Network/metabolism
5.
Biochem J ; 360(Pt 1): 179-88, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11696006

ABSTRACT

Cytosolic and nuclear O-linked N-acetylglucosaminylation has been proposed to be involved in the nuclear transport of cytosolic proteins. We have isolated nuclear and cytosolic N-acetyl-d-glucosamine (GlcNAc)-specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc and identified these lectins, by a proteomic approach, as belonging to the heat-shock protein (HSP)-70 family (one of them being heat-shock cognate 70 stress protein). Two-dimensional electrophoresis indicated that the HSP-70 fraction contained three equally abundant proteins with molecular masses of 70, 65 and 55 kDa. The p70 and p65 proteins are phosphorylated and are themselves O-linked GlcNAc (O-GlcNAc)-modified. The HSP-70 associated into high molecular mass complexes that dissociated in the presence of reductive and chaotropic agents. The lectin(s) present in this complex was (were) able to recognize cytosolic and nuclear ligands, which have been isolated using wheat germ agglutinin affinity chromatography. These ligands are O-GlcNAc glycosylated as demonstrated by [(3)H]galactose incorporation and analysis of the products released by reductive beta-elimination. The isolated lectins specifically recognized ligands present in both the cytosol and the nucleus of human resting lymphocytes. These results demonstrated the existence of endogenous GlcNAc-specific lectins, identified as HSP-70 proteins, which could act as a shuttle for the nucleo-cytoplasmic transport of O-GlcNAc glycoproteins between the cytosol and the nucleus.


Subject(s)
Acetylglucosamine/metabolism , Cell Nucleus/metabolism , Cytosol/metabolism , Heat-Shock Proteins/metabolism , Lectins/chemistry , Liver/metabolism , Animals , Biotinylation , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Glycoproteins/metabolism , Isoelectric Focusing , Ligands , Lymphocytes/metabolism , Phosphorylation , Rats , Rats, Wistar , Sepharose/chemistry
6.
Front Biosci ; 6: D1235-44, 2001 Oct 01.
Article in English | MEDLINE | ID: mdl-11578961

ABSTRACT

To address the function of carbohydrates in mucins, GalNAcalpha-O-bn has been used in in vivo experiments on several human mucosal cultured cells as a potential competitor of the glycosylation of N-acetylgalactosamine residues. GalNAcalpha-O-bn is metabolized by glycosyltransferases expressed in the cell, and give rise to different internal derivatives starting in particular from the formation of the disaccharide Galalpha1-3GalNAcalpha-O-bn. In this line, GalNAcalpha-O-bn exposure inhibits peripheral glycosylation according a cell-type specific manner. The metabolic alterations are very important in HT-29 cell line, leading to a massive accumulation of GalNAcalpha-O-bn oligosaccharide derivatives and to a strong inhibition of the terminal elongation of O-glycans by alpha2,3 sialyltransferase ST3Gal I. GalNAcalpha-O-bn treatment also induced alterations at the cellular level, exhibiting a large scale in HT-29 cells, i.e. 1) an inhibition of mucin secretion, 2) a blockade in the targeting of some membrane glycoproteins (brush border glycoproteins such as dipeptidylpeptidase IV, carcinoembryonic antigen and the mucin-like glycoprotein MUC1, and the basolateral cell adhesion molecule CD44), 3) an inhibition in the processing of lysosomal enzymes. Morphological abnormalities have been evidenced in GalNAcalpha-O-bn treated cells, in particular the accumulation of numerous intracellular vesicles in HT-29 cells. Taken together, these data suggest that O-glycosylation might be involved in the regulation of the targeting of O-glycosylproteins through carrier vesicles.


Subject(s)
Acetylgalactosamine/analogs & derivatives , Acetylgalactosamine/pharmacology , Benzyl Compounds/pharmacology , Mucins/drug effects , Acetylgalactosamine/metabolism , Benzyl Compounds/metabolism , Biological Transport/drug effects , Caco-2 Cells , Glycosylation/drug effects , HT29 Cells , Humans , Lysosomes/drug effects , Lysosomes/enzymology , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Mucins/metabolism
7.
Glycobiology ; 11(8): 663-76, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11479277

ABSTRACT

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.


Subject(s)
Fluorocarbons/chemistry , Sialic Acids/chemistry , Acylation , Amines/chemistry , Animals , Gas Chromatography-Mass Spectrometry/methods , Lactones/chemistry , Methylation , N-Acetylneuraminic Acid/analogs & derivatives , N-Acetylneuraminic Acid/chemistry , Neuraminic Acids/chemistry , Propionates/metabolism , Sugar Acids/chemistry , Sulfates/chemistry
8.
J Cell Sci ; 114(Pt 8): 1455-71, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11282022

ABSTRACT

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcalpha-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G(-) and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G(-) cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcalpha-O-bn metabolites. We observed a strong inhibition of alpha2,3-sialylation of glycoproteins in HT-29 G(-) cells correlated to the high expression of alpha2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galbeta1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of alpha1,2-fucosylation was observed, in correlation with the high expression of alpha1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G(-) cells, GalNAcalpha-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcalpha-O-bn metabolites.


Subject(s)
Fucosyltransferases/genetics , Galactose/analogs & derivatives , Galactose/administration & dosage , Galactose/metabolism , Glycosylation/drug effects , Protein Transport/physiology , Sialyltransferases/genetics , Caco-2 Cells/metabolism , Cell Differentiation , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/drug effects , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Enzyme Activation/physiology , Epitopes/immunology , Epitopes/metabolism , Fucosyltransferases/metabolism , Gene Expression/genetics , HT29 Cells/metabolism , Humans , Hyaluronan Receptors/drug effects , Hyaluronan Receptors/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Monosaccharides/chemistry , Monosaccharides/metabolism , Polysaccharides/immunology , Polysaccharides/metabolism , Protein Transport/drug effects , Sialyltransferases/metabolism , Tumor Cells, Cultured
9.
FEBS Lett ; 488(3): 160-4, 2001 Jan 19.
Article in English | MEDLINE | ID: mdl-11163764

ABSTRACT

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three-fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long-chain bases found in free ceramides were d18:1 sphingosine, d18:0 3-ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3-ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long-chain base of glucosylceramide of outer membranes.


Subject(s)
Antigens, CD , Ceramides/analysis , Ceramides/chemistry , Mitochondria, Liver/chemistry , Neutral Glycosphingolipids/analysis , Neutral Glycosphingolipids/chemistry , Sphingosine/analogs & derivatives , Animals , Fatty Acids/analysis , Gas Chromatography-Mass Spectrometry , Glucosylceramides/analysis , Glucosylceramides/chemistry , Intracellular Membranes/chemistry , Lactosylceramides/analysis , Lactosylceramides/chemistry , Rats , Sphingosine/analysis
10.
J Biol Chem ; 276(8): 5685-91, 2001 Feb 23.
Article in English | MEDLINE | ID: mdl-11050099

ABSTRACT

A method was developed for the determination of putative lectin activities of cytokines. It involved the immunoblotting measurement of the quantity of these cytokines unbound to a series of different immobilized glycoconjugates and displacement of the bound cytokines with oligosaccharides of known structures. This method allows demonstrating that the following interleukins specifically recognize different oligosaccharide structures in a calcium-independent mechanism: interleukin-1alpha binds to the biantennary disialylated N-glycan completed with two Neu5Acalpha2-3 residues; interleukin-1beta to a GM4 sialylated glycolipid Neu5Acalpha2-3Galbeta1-Cer having very long and unusual long-chain bases; interleukin-4 to the 1,7 intramolecular lactone of N-acetyl-neuraminic acid; interleukin-6 to compounds having N-linked and O-linked HNK-1-like epitopes; and interleukin-7 to the sialyl-Tn antigen. Because the glycan ligands are rare structures in human circulating cells, it is suggested that such activities could be essential for providing specific signaling systems to cells having both the receptors and the oligosaccharide ligands of the interleukin at their cell surface.


Subject(s)
Carbohydrate Metabolism , Interleukin-1/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Interleukin-7/metabolism , Animals , Binding Sites , Bufo bufo , CD57 Antigens/metabolism , Carbohydrate Sequence , Gangliosides/metabolism , Glucuronates/metabolism , Glycoconjugates/metabolism , Humans , Lectins/metabolism , Molecular Sequence Data , Mucins/metabolism , Oligosaccharides/metabolism , Protein Binding , Recombinant Proteins/metabolism
11.
Biochem J ; 352 Pt 1: 37-48, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11062056

ABSTRACT

On the basis of the detection of expressed sequence tag ('EST') similar to the rat N-acetylgalactosamine alpha2,6-sialyltransferase (ST6GalNAc) III cDNA, we have identified a novel member of the human ST6GalNAc family. We have isolated a cDNA clone containing an open reading frame that codes for a type II membrane protein of 302 amino acids with a seven-amino-acid cytoplasmic domain, an 18-amino-acid transmembrane domain and the smallest described catalytic domain of 277 amino acids. This predicted sialyltransferase sequence is similar to the rat ST6GalNAc III (46.6%), but was found to be even more similar to the recently reported mouse ST6GalNAc IV (88.1%) on the basis of amino acid sequence identity. Northern-blot analysis showed that the newly identified gene is expressed constitutively in various adult human tissues as a 2.2kb transcript, but was also found to be expressed at lower levels in brain, heart and skeletal muscle as a 2.5kb transcript. Expression of the hST6GalNAc IV gene was investigated by reverse transcription PCR in various human cancer cells, and was found to be present in the majority of cell types with the exception of the carcinoma cell line T47D and pro-monocyte THP cells. The transient expression in COS-7 cells of the full-length cDNA led to the production of an active enzyme sharing the acceptor specificity of the ST6GalNAc family towards Neu5Ac alpha 2-3Gal beta 1-3GalNAc alpha-O-R (where 'R' denotes H, benzyl, or a peptidic chain). Detailed analysis in vitro of substrate specificity revealed that the enzyme required the trisaccharide Neu5Ac alpha 2-3Gal beta1-3GalNAc found on O-glycans and arylglycosides. In addition, we have clarified the genomic organization of ST6GalNAc IV gene.


Subject(s)
Sialyltransferases/biosynthesis , Sialyltransferases/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Western , COS Cells , Catalytic Domain , Chromosomes, Human, Pair 9 , Cloning, Molecular , Cytoplasm/metabolism , DNA, Complementary/metabolism , Expressed Sequence Tags , Fluorescent Antibody Technique , Humans , Kinetics , Mice , Microscopy, Fluorescence , Models, Genetic , Molecular Sequence Data , Open Reading Frames , Protein Structure, Tertiary , RNA/metabolism , Rats , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sialyltransferases/metabolism , Substrate Specificity , Tissue Distribution , Transfection , Tumor Cells, Cultured
12.
Anal Biochem ; 284(2): 201-16, 2000 Sep 10.
Article in English | MEDLINE | ID: mdl-10964402

ABSTRACT

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.


Subject(s)
Fluorocarbons/chemistry , Gas Chromatography-Mass Spectrometry/methods , Glycolipids/analysis , Animals , Bacteria/chemistry , Esters/analysis , Fatty Acids/chemistry , Glycolipids/chemistry , Hydroxylation , Rats , Yeasts/chemistry
13.
Glycobiology ; 10(6): 565-75, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10814698

ABSTRACT

Permanent exposure of differentiated HT-29 cells to the sugar analogue, 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha- O -bn) leads to marked effects upon the phenotypic properties of mucin-secreting or enterocyte-like HT-29 cells: an inhibition in the secretion of mucins, a blockade in the apical targeting of membrane brush border glycoproteins and a swelling of cells with intracellular accumulation of numerous vesicles. Folch extraction and partition of treated enterocyte-like HT-29 cells revealed a very important accumulation of orcinol and/or resorcinol reactive material in the upper phase (usually containing gangliosides), as compared with untreated HT-29 cells and with treated and untreated Caco-2 cells. Structural analysis indicated the accumulation of a series of GalNAcalpha- O -bn derived oligosaccharides, most of them with the common core Galbeta1-3GalNAcalpha- O -bn. These oligosaccharides contained residues of GlcNAc, Gal, Neu5Ac, or Fuc. In particular, the tagged sialyl-Lewis(x)was identified, as well as more complex sialylated derivatives, including the sialyl-Lewis(x)substituted by an additional Neu5Ac residue. The benzylated oligosaccharides were not significantly detected in the culture medium except for Galbeta1-3GalNAcalpha- O -bn. Upon reversion of the treatment, these derivatives dis-appeared from the cells within few days, however were not recovered as such in the culture medium. Intracellular degradation occurred with desialylation and defucosylation as the first steps. The spectacular accumulation of benzylated oligosaccharides in HT-29 cell, permanently exposed to GalNAcalpha- O -bn very likely plays an important role in the alterations of cellular processes previously described in this cell line. The HT-29 cell culture system also appears to be an efficient source of several tagged oligosaccharides.


Subject(s)
Enterocytes/drug effects , Enterocytes/metabolism , Galactose/analogs & derivatives , Mucins/metabolism , Oligosaccharides/biosynthesis , Cell Differentiation , Chromatography, High Pressure Liquid , Culture Media, Conditioned/chemistry , Enterocytes/ultrastructure , Galactose/pharmacology , Gas Chromatography-Mass Spectrometry , HT29 Cells , Humans , Magnetic Resonance Spectroscopy , Membrane Glycoproteins/metabolism , Methylation , Microvilli/drug effects , Microvilli/metabolism , N-Acetylneuraminic Acid/metabolism , Oligosaccharides/analysis , Oligosaccharides/metabolism , Sialyl Lewis X Antigen
14.
Biochim Biophys Acta ; 1472(1-2): 71-81, 1999 Oct 18.
Article in English | MEDLINE | ID: mdl-10572927

ABSTRACT

O-Linked N-Acetylglucosamine (O-GlcNAc) is a major form of post-translational modification found in nuclear and cytoplasmic proteins. Several authors have advanced the hypothesis according to which phosphorylation and O-GlcNAc glycosylation are reciprocally related to one another [1,2]. In order to test this hypothesis we have investigated the effect of a broad spectrum phosphatase inhibitor, okadaic acid (OA), generally used to induce protein hyperphosphorylation, on the GlcNAc content of cellular glycoproteins. We demonstrate that in neuronal cells lines OA decreases the level of O-GlcNAc in both nuclear and cytoplasmic proteins with a greater effect in the nuclear fraction. This phenomenon was demonstrated by the use of three different procedures for the detection of O-GlcNAc in conjunction with a systematic treatment with PNGase F. O-Linked GlcNAc was characterized using respectively lectin staining with WGA, galactosyltransferase labeling and metabolic labeling of cultured cells with [3H]glucosamine. Although the effects on individual proteins varied, a less pronounced effect was observed on HeLa or COS cell total homogenates. When Kelly cells were treated with OA, the major observation was a decrease in O-GlcNAc content of nuclear proteins. The measurement of the UDP-GlcNAc level clearly demonstrates that the decrease on the O-GlcNAc level in the neuroblastoma cell line after treatment with okadaic acid is not a consequence of the modification of the UDP-GlcNAc pool.


Subject(s)
Acetylglucosamine/metabolism , Neuroblastoma/metabolism , Okadaic Acid/pharmacology , Animals , COS Cells , Galactose/metabolism , HeLa Cells , Humans , Neuroblastoma/pathology , Subcellular Fractions/metabolism , Tritium , Tumor Cells, Cultured
15.
Anal Biochem ; 267(2): 300-8, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-10036134

ABSTRACT

The most common method used for the liberation of monosaccharides from glycoprotein N-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides as O-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively the N-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave the N-glycosidic bond (96%), liberating glucosamine (and not its O-methylglycosides) and other minor reaction products which were identified. Because other N-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as the O-methylglycosides of glucosamine, the GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.


Subject(s)
Glycoproteins/analysis , Monosaccharides/analysis , Chromatography, Gas , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Polysaccharides/analysis
16.
Glycobiology ; 9(3): 255-66, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10024663

ABSTRACT

We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved.


Subject(s)
Chromatography, Gas/methods , Fluorocarbons/chemistry , Glycolipids/chemistry , Glycoproteins/chemistry , Methylglycosides/chemistry , Monosaccharides/isolation & purification , Acylation , Carbohydrate Sequence , Gas Chromatography-Mass Spectrometry/methods , Molecular Sequence Data
17.
Glycoconj J ; 16(10): 617-27, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10972140

ABSTRACT

A major impediment in the analysis of glycosaminoglycans is the difficulty to cleave quantitatively the glycosidic bonds because of the stabilisation of glycosidic bonds and of the relative instability of the liberated constituents. This manuscript describes a modified procedure of methanolysis in the presence of barium acetate, reducing the destruction of uronic acids and increasing the cleavage yield. The reaction products could be identified and analysed quantitatively by GC and GC/MS of the heptafluorobutyrate derivatives of O-methyl glycosides of monosaccharides (for keratan sulphate and chondroitin sulphate B), or as a mixture of O-methyl glycosides of monosaccharides and of disaccharides (for the other sulphated glycosaminoglycans). Quantitative molar ratio between the different monosaccharide constituents (including the linkage region constituents) could be obtained, even when proteoglycans also contain classical N-glycans or O-glycans.


Subject(s)
Fluorocarbons/metabolism , Glycosaminoglycans/chemistry , Glycosaminoglycans/metabolism , Methanol/metabolism , Proteoglycans/chemistry , Proteoglycans/metabolism , Acylation , Artifacts , Barium/metabolism , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/metabolism , Chromatography, Thin Layer , Disaccharides/analysis , Gas Chromatography-Mass Spectrometry/methods , Heparin/chemistry , Heparin/metabolism , Keratan Sulfate/chemistry , Keratan Sulfate/metabolism , Methylglycosides/chemistry , Methylglycosides/metabolism , Monosaccharides/analysis , Polysaccharides/analysis , Polysaccharides/chemistry , Uronic Acids/metabolism
18.
Acta Anat (Basel) ; 161(1-4): 180-95, 1998.
Article in English | MEDLINE | ID: mdl-9780358

ABSTRACT

There is increasing evidence that lectins are widely distributed in mammalian tissues, including the nervous tissue. Based on histochemical techniques using neoglycoproteins, different lectin activities specific for different monosaccharides or glycans have been identified (fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid and heparin). Most of them showed a cellular specificity and developmental regulation in the central nervous system. Several lectins isolated from the nervous tissue seem to play an essential role during ontogenetic processes, especially as far as cell adhesion and cell recognition mechanisms are concerned (axonal growth and fasciculation, neuron migration, synaptogenesis, myelination). But some of them seem to be involved in signaling events both intracellularly (nuclear lectins) or at the cell surface by autocrine and paracrine mechanisms. This review discusses the structure and the identified functions of these important constituents of the nervous tissue.


Subject(s)
Central Nervous System/chemistry , Lectins/chemistry , Lectins/physiology , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/physiology , Peripheral Nervous System/chemistry , Animals , Axons , Cell Adhesion/physiology , Cell Differentiation/physiology , Cell Movement , Cerebellum/chemistry , Cytokines/metabolism , Galectins , Hemagglutinins/analysis , Hemagglutinins/chemistry , Hemagglutinins/physiology , Heparin/metabolism , Immunohistochemistry , Lectins/analysis , Mannose/metabolism , Nerve Tissue Proteins/analysis , Synapses
19.
Glycobiology ; 8(3): 221-5, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9451031

ABSTRACT

The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor ss, IL-2 associates the latter which the CD3/TCR complex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al. , 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R beta by ++p56(lck) , the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2-dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous "amplifier of activation signals" (Zanetta et al. , 1995, Biochem. J., 311, 629-636).


Subject(s)
Candida albicans/metabolism , Glioblastoma/metabolism , Interleukin-2/metabolism , Lectins/metabolism , Biotinylation , CD3 Complex/metabolism , Candida albicans/pathogenicity , Cell Wall/chemistry , HIV Envelope Protein gp160/metabolism , Humans , Kluyveromyces/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mannans/metabolism , Membrane Glycoproteins/metabolism , Phosphorylation , Receptors, Antigen, T-Cell/metabolism , Receptors, Interleukin-2/metabolism , Recombinant Proteins/metabolism , Saccharomyces/metabolism , Schizosaccharomyces/metabolism , Tumor Cells, Cultured
20.
J Biol Chem ; 272(45): 28622-9, 1997 Nov 07.
Article in English | MEDLINE | ID: mdl-9353328

ABSTRACT

We have cloned a novel protein kinase from human cerebellum and named it LZK (leucine zipper-bearing kinase). The LZK cDNA encoded a 966-amino acid polypeptide that contains a kinase catalytic domain and double leucine/isoleucine zippers separated by a short spacer region. The amino acid sequence of the kinase catalytic domain was a hybrid between those in serine/threonine and tyrosine protein kinases, indicating that LZK belongs to the subfamily of the mixed lineage kinase (MLK) family. The kinase catalytic domain of LZK was most similar to DLK (Holtzman, L. B., Merritt, S.E., and Fan, G. (1994) J. Biol. Chem. 269, 30808-30817), MUK (Hirai, S., Izawa, M., Osada, S., Spyrou, G., and Ohno, S. (1996) Oncogene 12, 641-650), and ZPK (Reddy, U. R., and Presure, D. (1994) Biochem. Biophys. Res. Commun. 202, 613-620), which belong to the same subfamily of the MLK family. However, besides the kinase catalytic domain and double leucine/isoleucine zippers, there was no significant homology with known proteins. The recombinant LZK autophosphorylated in the presence of ATP and divalent cations, and exhibited serine/threonine kinase catalytic activity. Northern blot analysis revealed that LZK is expressed most strongly in the pancreas, with a pattern that differs from other MLKs. Expression of LZK in COS7 cells induced phosphorylation of c-Jun and activation of JNK-1, indicating the association of LZK in the c-Jun amino-terminal kinase/stress-activated protein kinase pathway. The expressed LZK was detected primarily in the membrane fraction, suggesting that LZK interacts with other cellular components in vivo.


Subject(s)
Brain/enzymology , Leucine Zippers , MAP Kinase Kinase Kinases , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Catalysis , Cloning, Molecular , DNA, Complementary/chemistry , Humans , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/biosynthesis , Protein Structure, Secondary , Sequence Alignment
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