Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.
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The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly. (+info)
Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.
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The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals. (+info)
Modulation of phospholipase A2 by electrostatic fields and dipole potential of glycosphingolipids in monolayers.
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Phospholipase A2 activity against mixed monolayers of dilauroylphosphatidic acid or dilauroylphosphatidylcholine with glycosphingolipids can be reversibly modulated by external constant electrostatic fields. The changes of enzymatic activity are correlated to the depolarization or hyperpolarization of the film caused by specific dipolar properties of glycosphingolipids. Hyperpolarizing fields enhance the enzymatic activity against pure dilauroylphosphatidic acid while depolarizing fields induce a decrease of activity. Compared to the pure substrate, the interface of mixed films containing neutral glycosphingolipids or gangliosides is already partially depolarized and the magnitude of activation induced by an external hyperpolarizing field is decreased; conversely, depolarizing fields cause an increased inhibition of activity. Differing from gangliosides, sulfatides bring about a hyperpolarization of the mixed lipid monolayer and external hyperpolarizing or depolarizing fields cause enhanced activation and reduced inhibition, respectively. The effects of glycosphingolipids depend on their relative proportion in the monolayer. Results were similar with dilauroylphosphosphatidylcholine but the field effects were less than half of those found with dilauroylphosphatidic acid. Our work shows that the activity of phospholipase A2 in addition to responding reversibly to external electrostatic fields, is directly modulated by the polarity and magnitude of the lipid polar head group dipole moments. (+info)
Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.
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Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant. (+info)
Modulation of nanotube formation by structural modifications of sphingolipids.
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Galactosylceramides (GalCers) containing nervonoyl (24:1(Delta15(cis))) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (. Biophys. J. 69:1976-1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1((X-9)). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1(Delta15(cis)) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25-30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1(Delta15)GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1(Delta15)Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1(Delta15)GalCer. 22:1(Delta13)GalCer dispersions consisted of long, smooth tubules (35-40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1(Delta11)GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30-35 nm, with helical pitches of 80-90 nm. 18:1(Delta9)GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation. (+info)
Ca2+-induced fusion of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles.
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The fusogenic properties of sulfatide-containing 1,2-dioleoyl-3-sn -phosphatidylethanolamine (DOPE) small unilamellar vesicles (SUVs) in the presence of CaCl2 were studied by mixing membrane lipids based on an assay of fluorescence resonance energy transfer (FRET). Fusion of the vesicles was also confirmed by mixing aqueous contents with the Tb/dipicolinate (DPA) assay. The half-times of lipid mixing revealed that the fusion rate decreased with increasing molar concentration of sulfatide. This inhibitory effect was more obvious at sulfatide concentrations higher than 30 mol%, where hydration at the membrane surface reached its maximum and the fusion was no longer pH-sensitive in the range of pH 6.0 - 9.0. Similar inhibitory effect was also observed in Ca2+-induced fusion of DOPE/ganglioside GM1 vesicles but at a lower concentration of the glycosphingolipid (20 mol%). In contrast, increasing the concentration of phosphatidylserine (PS) in DOPE/PS SUVs resulted in an increase in the rate of Ca2+-induced lipid mixing and the pH sensitivity of this system was not affected. These results are consistent with an increasing steric hindrance to membrane fusion at higher molar concentration and larger headgroup size of the glycosphingolipids. Interestingly, the pH sensitivity of the sulfatide-containing liposomes was retained when they were allowed to fuse with synaptosomes in the absence of Ca2+ by a mechanism involving protein mediation. (+info)
In vitro production of cytokines is influenced by sulfatide and its precursor galactosylceramide.
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Effects of sulfatide and its precursor galactosylceramide (gal-cer) on the kinetics of production of cytokines were studied. In human mononuclear leucocytes, gal-cer but not sulfatide induced significantly increased amounts of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF) mRNA. In phytohemagglutinin-stimulated cultures, gal-cer increased the levels of IL-1beta and IL-6 mRNA and secreted IL-1beta and IL-6, while sulfatide decreased the amounts of IL-6 mRNA and secreted IL-6. Gal-cer also increased TNF secretion. In lipopolysaccharide-stimulated cells, sulfatide but not gal-cer decreased the secretion of IL-1beta and IL-10, a potent suppressor of production of many cytokines. Thus, sulfatide and gal-cer affect cytokine production differently, most likely at the level of gene expression. This may have implications in diseases where inflammatory cytokines play a pathogenic role. (+info)
Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.
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An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms. (+info)