Dopamine release from nerve endings induced by polysialogangliosides

Polysialogangliosides but not monosialoganlioside or a neutral glycosphingolipid induce release of [³H] -dopamine from synaptosomes in presence of Ca⁺⁺, presumably by exocytosis. This effect is discussed in relation to the ability of polysialogangliosides to induce membrane fusion in chicken erythrocytes and to their behaviour in lipid monolayers. It is suggested that characteristic interactions with phosphatidylcholine involving decreases of surface potential are participating in the polysialoganglioside-induced neurotransmitter release.     

Capacitance--voltage relationship in phospholipid bilayers containing gangliosides

Changes in the position of the minimum of the parabolic capacitance-voltage curve allow the measurement of the amount of ganglioside present in artificial bilayers made with phosphatidylcholine-ganglioside mixtures and asymmetrically shielded with Ca2+. The screening effect of the ionic solution must be considered. With ganglioside/phospholipid molar ratios of up to 15%, all glycolipids can be found at the membrane surfaces.     

Biophysics of sphingolipids II. Glycosphingolipids: An assortment of multiple structural information transducers at the membrane surface

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.     

Favorable and unfavorable lateral interactions of ceramide, neutral glycosphingolipids and gangliosides in mixed monolayers

Interactions among four natural neutral sphingolipids (ceramide, glucosyl-ceramide, lactosyl-ceramide and asialo-GM1) and six gangliosides (GM3, GM2, GM1, GD3, GD1a and GT1b) were studied in binary Langmuir monolayers at the air-buffer interface in terms of their molecular packing, compressibility, dipole potential and mixing behavior. The changes of surface organization can be grouped into three sets: (a) binary films of neutral GSLs, and of the latter with ceramide, exhibit thermodynamically unfavorable mixing with mean molecular area expansions and dipole moment hyperpolarization; (b) mixed monolayers of ceramide, or of GlcCer, and gangliosides occur with thermodynamically favorable interactions leading to mean molecular area condensation and depolarisation; (c) binary mixtures of LacCer or Gg4Cer with gangliosides, and all ganglioside species among them, revealed molecular immiscibility characterized by additive mean molecular area and dipole potential, with composition-independent constant collapse pressure. These results disclose basic tendencies of GSLs to molecularly mix or demix, leading to their surface segregation, which may underlay vectorial separation of their specific biosynthetic pathways.     

Calcium plays an important role in the regulation of hepatic branched-chain 2-oxo acid dehydrogenase activity

A fetal haemoglobin variant was noted in a healthy Jamaican infant of mixed African/Chinese extraction. A two-dimensional chromatogram of the soluble tryptic peptides (Tp) showed 2 ‘new’ ones/ One was composed of the last 4 residues of the usually insoluble Tpγ41–59. To permit a tryptic split this required a change of residue γ55 Met to Lys or Arg. The other new Tp contained arginine and was in the position expected for a Tpγ41–55 (55 Arg). As the material was limited it could not be analysed. When after more than 6 years no example of Hb F Kingston had become available it was decided to describe the variant on the basis of the present evidence.     

C-series polysialogangliosides are expressed on stellate neurons of adult human cerebellum

Until now c-series polysialogangliosides were known to exist in human brain only during development and in some pathological conditions like Alzheimers disease. Using thin-layer chromatography (TLC) and immunostaining with Q211 antibody (TLC-overlay technique) we have analysed c-series gangliosides in four human cerebella (age 20, 47, 52 and 54 years). Four distinct ganglioside bands, most probably corresponding to GT1c, GQ1c, GP1c and GH1c were found to exist in the analysed brains, which is convincing demonstration of the existence of c-series gangliosides in normal adult human brain. Immunohistochemical analysis was performed to locate polysialogangliosides in the analysed tissue. Q211 antibody was found to bind specifically to a single subpopulation of neurons in the molecular layer of adult cerebellum. According to their position and morphology these cells correspond to stellate neurons. © 1998 Rapid Science Ltd     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

Molecular Interactions of the Major Myelin Glycosphingolipids and Myelin Basic Protein in Model Membranes

The molecular organization, interactions, phase state and membrane-membrane interactions of model membranes containing cerebroside (GalCer), sulfatide (Sulf) and myelin basic protein (MBP) were investigated. Sulf shows a larger cross-sectional area than GalCer, in keeping with the lateral electrostatic repulsions in the negatively charged polar head group. The interactions of GalCer with different phospholipids are similar while those with Sulf depend on the phosphoryl choline moiety in the phospholipid. MBP induces a decrease of the phase transition temperature in both lipids but with Sulf this occurs at lower proportions of MBP. In mixtures of Sulf with phosphatidylcholine MBP induces phase separation among Sulf-rich and PC-rich domains. Extensive apposition of bilayers containing Sulf is induced by MBP while GalCer interferes with this process. Few membrane interactions proceed to bilayer merging or whole bilayer fusion and the glycosphingolipids help preserve the membrane integrity.     

Glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility

Glycosphingolipids (GSLs) GM3 (NeuAcα3Galβ4Glcβ1Cer) and GM2 (GalNAcβ4[NeuAcα3]Galβ4Glcβ1Cer) inhibit (i) cell growth through inhibition of tyrosine kinase associated with growth factor receptor (GFR), (ii) cell adhesion/motility through inhibition of integrin-dependent signaling via Src kinases, or (iii) both cell growth and motility by blocking “cross-talk” between integrins and GFRs. These inhibitory effects are enhanced when GM3 or GM2 are in complex with specific tetraspanins (TSPs) (CD9, CD81, CD82). Processes (i)-(iii) occur through specific organization of GSLs with key molecules (TSPs, caveolins, GFRs, integrins) in the glycosynaptic microdomain. Some of these processes are shared with epithelial-mesenchymal transition induced by TGFβ or under hypoxia, particularly that associated with cancer progression.     

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells

Neuroblastoma and glioma cells were grown in the presence of [³H]galactose, and the incorporation of ³H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of ³H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance, but not low temperature.     

Blood group H active glycolipid from rat ascites hepatoma AH 7974F.

A new type of fucose-containing glycolipid exhibiting blood group H activity was isolated from rat ascites hepatoma cell AH 7974F. As a result of studying its structure by partial acid hydrolysis, enzymatic degradation and immuno-precipitation reaction, the structure was tentatively proposed as Fuc(1 → 2)Gal(1 → 3)GalNAc(1 → 4)Gal(1 → 4)Glc(1 → 1)Cer.     

Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV.

Extracts of cultured skin fibroblasts derived from patients with mucolipidosis IV showed a marked increase and altered distribution of GM₃ and GD₃ gangliosides. GD₃ is elevated 1.5–2 times that of normal whereas GM₃ is elevated to a lesser extent. No abnormalities were found in the neutral glycolipids. These two gangliosides apparently comprise most of the accumulated lipid-like material observed on ultrastructural analysis in this disease.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

Endo-beta-galactosidase of Escherichia freundii. Hydrolysis of pig colonic mucin and milk oligosaccharides by endoglycosidic action.

The purified keratansulfate degrading enzyme from $\text{Eschericia freundii}$ could hydrolyze desialyzed pig colonic mucin and milk oligosaccharides. Desialyzed pig colonic mucin was digested to produce GlcNAcβ(1→3)Gal, GlcNAc-6Sβ(1→3)Gal and resistant polymer. Lacto-N-tetraose and lacto-N-tetraitol were hydrolyzed endoglycosidically to release glucose and sorbitol, respectively. Therefore, this enzyme was found to be an endo-β-galactosidase of rather wide specificity.     

Biosynthesis of yeast mannan : Diversity of mannosyltransferases in the mannan-synthesizing enzyme system from yeast

1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed.2. Of the compounds tested besides mannose, only those containing an α-linked mannosyl unit at the nonreducing position of their moleculae were effective as receptors. Monodeoxyanalogues of mannose as well as α-mannose phosphates did not serve as receptors in the above reaction.3. The structure of the product formed with mannose as receptor was determined to be O-α-D-mannosyl-(1→2)-mannose; with αMan(1→Man(1→6)mannose as the acceptor, the product was αMan(1→6)αMan(1→6)mannose and with αMan-(1→2)mannose the product was tentatively characterized as a mixture of αMan-(1→3)αMan(1→2)mannose and αMan(1→2)αMan(1→2)mannose.4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation.5. Radioactive disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysacchride to a minor extent under the same conditions.     

Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles,monomolecular films and UV-absorbing surface-coated substrate

Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10?ng of enzymes, against 100?ng to 10?μg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.     

Molecular Cloning and Crystal Structural Analysis of a Novel β-N-Acetylhexosaminidase from Paenibacillus sp. TS12 Capable of Degrading Glycosphingolipids

We report the molecular cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 Å; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 Å; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 Å. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.     

Interaction of 1-anilinonaphthalene 8-sulfonic acid with interfaces containing cerebrosides, sulfatides and gangliosides

The fluorescence lifetime, quantum yield and emission spectra of 1-anilinonaphthalene 8-sulfonic acid (ANS) associated with interfaces of pure dipalmitoylphosphatidylcholine or its mixtures with phosphatidylserine, galactosylceramide, sulfatide or gangliosides GM1 and GD1a were studied at low and high ionic strength. Modification of the molecular organization of the lipid interfaces in the presence of the probe was also studied with mixed lipid monolayers. ANS has little affect on the intermolecular packing of the lipids but influences their surface potential, consistent with a location of ANS in the polar head group region of the interface. ANS senses a more polar microenvironment when associated with interfaces containing anionic glycosphingolipids at low ionic strength but, except for interfaces containing phosphatidylserine, it detects approximately the same polarity for neutral or anionic interfaces in 0.25 M NaCl.     

Enzymatic fragmentation of carbohydrate moieties of radish arabinogalactan-protein and elucidation of the structures

We investigated the structures of L-arabino-galactooligosaccharides released from the sugar moieties of a radish arabinogalactan-protein (AGP) by the action of exo-β-(1→3)-galactanase. We detected a series of neutral β-(1→6)-linked galactooligosaccharides forming branches of one to up to at least 19 consecutive Gal groups, together with corresponding acidic derivatives terminating in 4-O-methyl-glucuronic acid (4-Me-GlcA) at the non-reducing end. Some oligosaccharide chains of degree of polymerization (dp) higher than 3 for neutral, and 4 for acidic oligomers were modified with L-Araf residues. The acidic tetrasaccharide 4-Me-β-GlcA-(1→6)[α-L-Araf-(1→3)]-β-Gal-(1→6)-Gal was detected as an abundant L-Araf-containing oligosaccharide among these neutral and acidic oligomers. A pentasaccharide containing an additional L-Araf group attached to the L-Ara in the tetrasaccharide through an α-(1→5)-linkage was also found. We observed L-arabino-galactooligosaccharides substituted with single or disaccharide L-Araf units at different Gal residues along these neutral and acidic β-(1→6)-galactooligosaccharide chains, indicating that these side chains are highly variable in length and substituted variously with L-Araf residues.