Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors.

The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography-mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.

Reduced diversity of the human erythrocyte membrane sialic acids in polycythemia vera. Absence of N-glycolylneuraminic acid and characterisation of N-acetylneuraminic acid 1,7 lactone.

Sialic acids from the erythrocyte (RBC) membrane of a patient suffering from polycythemia vera, a malignant orphan disorder of hematopoietic cells, was studied using GC/MS. We found that the sialic acid diversity of these membranes was drastically reduced since only four entities were identified: Neu5Ac (91.5%) and its 1,7 lactone Neu5Ac1,7L (7.5%) which is absent in normal RBC, Neu4,5Ac(2) (0.50%) and Neu4,5Ac(2) 9Lt (0.50%); in normal RBC, Neu5,7Ac(2), Neu5,9Ac(2), Neu5Ac9Lt, Neu5Ac8S and Neu, as well as traces of Kdn, were also present. Neu5Gc and its O-alkylated or O-acetylated derivatives, which are considered by various authors as cancer markers, were not detected.

Localisation and distribution of O-acetylated N-acetylneuraminic acids, the endogenous substrates of the hemagglutinin-esterases of murine coronaviruses, in mouse tissue.

Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac(2)) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac(2)). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac(2) was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac(2)9Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac(2), relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac(2) was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.

Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells.

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAc alpha-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4-depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins.

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper, it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C((2))-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.

Induction of a storage phenotype and abnormal intracellular localization of apical glycoproteins are two independent responses to GalNAcalpha-O-bn.

Our previous studies on an inhibitor of O-glycosylation of glycoproteins, GalNAcalpha-O-bn, in the model of enterocytic HT-29 cells, have shown at the cellular level an alteration of the normal localization of apical glycoproteins, and at the biochemical level an in situ synthesis and storage of sialylated GalNAcalpha-O-bn oligosaccharides. The purpose of this study was to examine if a relation existed between these two events, using different cell lines. Intracellular storage of GalNAcalpha-O-bn metabolites occurred in HT-29 and CAPAN-1 cells but not in Caco-2 cells. On the other hand, an accumulation of endosomal/lysosomal compartments was observed in HT-29 and CAPAN-1 cells but not in Caco-2 cells. These data focused on a GalNAcalpha-O-bn-derived storage phenotype in HT-29 and CAPAN-1 cells. The apical membrane glycoproteins MUC1 and CEA showed an abnormal localization inside intracytoplasmic vesicles in HT-29 cells, whereas they kept their normal localization in Caco-2 and CAPAN-1 cells. Studies on the glycosylation of these apical glycoproteins showed that GalNAcalpha-O-bn inhibited the glycosylation in a cell-specific manner. The alteration in the apical targeting of glycoproteins, and the appearance of a GalNAcalpha-O-bn-derived storage phenotype are two independent and cell type-specific events. The former depends on the inhibition pattern of the glycosylation of endogenous glycoproteins, whereas the latter is connected to the intracellular accumulation of GalNAcalpha-O-bn metabolites.

Lectin activities of cytokines: functions and putative carbohydrate-recognition domains.

The discovery that some cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain (CRD), which is localized at the opposite of the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity, which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase/phosphatase involved in receptor phosphorylation/dephosphorylation and in specific signal transduction. As a correlate, a cytokine can act only on cells possessing both the receptor and the ligand. Two cytokines using the same receptor can have different target cells and functions because of their different lectin activities. Based on a few examples, the CRD can be predicted based on the 3-D structures of the molecules.

Gas-chromatography/mass-spectrometry analysis of human skin constituents as heptafluorobutyrate derivatives with special reference to long-chain bases.

The composition of the constituents (monosaccharides, long-chain bases, and fatty acids) found in an ethanol extract of the human skin could be determined, without time-consuming steps of purification, after acid-catalyzed anhydrous methanolysis, followed by the formation of volatile derivatives with heptafluorobutyric anhydride and gas-chromatography/mass-spectrometry analysis. Despite the extreme heterogeneity of such extracts, the electron impact analysis of the constituents allowed qualitative and quantitative determinations of monosaccharides, long-chain bases, fatty acids, and alkyl-glycerols. Throughout the different long-chain bases, sphingenines (Sphes), sphinganines, phytosphingosines, and 6-hydroxy-Sphes (6oh-Sphes) can be identified and quantified. Long-chain bases with a chain-length up to 28 carbon atoms can be identified through specific fragmentation patterns in the electron impact mode. Particular attention was drawn to the behavior of compounds of the family of 6oh-Sphes upon acid-catalyzed methanolysis.

Function and molecular modeling of the interaction between human interleukin 6 and its HNK-1 oligosaccharide ligands.

Interleukin 6 (IL-6) is endowed with a lectin activity for oligosaccharide ligands possessing the HNK-1 epitope (3-sulfated glucuronic acid) found on some mammalian glycoprotein N-glycans (Cebo, C., Dambrouck, T., Maes, E., Laden, C., Strecker, G., Michalski, J. C., and Zanetta, J. P. (2001) J. Biol. Chem. 276, 5685-5691). Using high affinity oligosaccharide ligands, it is demonstrated that this lectin activity is responsible for the early dephosphorylation of tyrosine residues found on specific proteins induced by interleukin 6 in human resting lymphocytes. The gp130 glycoprotein, the signal-transducing molecule of the IL-6 pathway, is itself a molecule possessing the HNK-1 epitope. This indicates that IL-6 is a bi-functional molecule able to extracellularly associate its alpha-receptor with the gp130 surface complex. Computational modeling indicates that the lower energy conformers of the high affinity ligands of IL-6 have a common structure. Docking experiments of these conformers suggest that the carbohydrate recognition domain of IL-6 is localized in the domain previously identified as site 3 of IL-6 (Somers, W., Stahl, M., and Seehra, J. S. (1997) EMBO J. 16, 989-997), already known to be involved in interactions with gp130.