Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration.

Gangliosides, sialylated glycosphingolipids which are the predominant glycans on vertebrate nerve cell surfaces, are emerging as components of membrane rafts, where they can mediate important physiological functions. Myelin associated glycoprotein (MAG), a minor constituent of myelin, is a sialic acid binding lectin with two established physiological functions: it is involved in myelin-axon stability and cytoarchitecture, and controls nerve regeneration. MAG is found selectively on the myelin membranes directly apposed to the axon surface, where it has been proposed to mediate myelin-axon interactions. Although the nerve cell surface ligands for MAG remain to be established, evidence supports a functional role for sialylated glycoconjugates. Here we review recent studies that reflect on the role of gangliosides, sialylated glycosphingolipids, as functional MAG ligands. MAG binds to gangliosides with the terminal sequence 'NeuAc alpha 3Gal beta 3GalNAc' which is found on the major nerve gangliosides GD1a and GT1b. Gangliosides lacking that terminus (e.g., GM1 or GD1b), or having any biochemical modification of the terminal NeuAc residue fail to support MAG binding. Genetically engineered mice lacking the GalNAc transferase required for biosynthesis of the 'NeuAc alpha 3Gal beta 3GalNAc' terminus have grossly impaired myelination and progressive neurodegeneration. Notably the MAG level in these animals is dysregulated. Furthermore, removal of NeuAc residues from nerve cells reverses MAG-mediated inhibition of neuritogenesis, and neurons from mice lacking the 'NeuAc alpha 3 Gal beta 3GalNAc' terminus have an attenuated response to MAG. Cross-linking nerve cell surface gangliosides can mimic MAG-mediated inhibition of nerve regeneration. Taken together these observations implicate gangliosides as functional MAG ligands.

Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers.

Lateral assemblies of sphingolipids, glycosphingolipids and cholesterol, termed rafts, are postulated to be present in biological membranes and to function in important cellular phenomena. We probed whether rafts are heterogeneous by determining the relative distribution of two gangliosides, GM1 and GD3, in artificial supported monolayers, in intact rat primary cerebellar granule neurones, and in membrane rafts isolated from rat cerebellum. Fluorescence resonance energy transfer (FRET) using fluorophore-labelled cholera toxin B subunit (which binds GM1) and mAb R24 (which binds GD3) revealed that GM1 spontaneously self-associates but does not co-cluster with GD3 in supported monolayers and on intact neurones. Cholera toxin and immunocytochemical labelling of isolated membrane rafts from rat cerebellum further demonstrated that GM1 does not co-localise with GD3. Furthermore, whereas the membrane raft resident proteins Lyn and caveolin both co-localise with GD3 in isolated membrane rafts, GM1 appears in separate and distinct aggregates. These data support prior reports that membrane rafts are heterogeneous, although the mechanisms for establishing and maintaining such heterogeneity remain to be determined.

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.

Glycosaminoglycans bind to homologous cardiotoxins with different specificity.

We have reported that some cardiotoxins (CTXs), homologous basic polypeptides of cobra venom, bind strongly to heparin. Herein we show that CTXs from spitting cobra venom bind avidly to chondroitin-6-sulfate (CS6) and dermatan sulfate (DS), the glycosaminoglycans (GAGs) abounding in the cornea and elsewhere. We compared the binding strength of Tgamma, a major component of spitting cobra, Naja nigricollis, venom with that of CTX A3, a major component of Naja atra venom to various GAGs including CS6, chondroitin-4-sulfate (CS4), DS, keratan sulfate (KS), hyaluronan (HYA), and heparin. The binding strength of Tgamma followed the order CS6 > KS > HYA > DS > CS4 > heparin, whereas that of CTX A3 was heparin > KS > CS4 > DS > CS6 > HYA. The binding specificity displayed by different CTXs toward GAGs is impressive, given the high homology among CTXs and among GAGs. Strong binding of Tgamma to CS6, rather than to the highly anionic and versatile cousin, heparin, implies specific interaction with CS6. Heparin, at high concentration, displaced CS6 from CS6-Tgamma and CS6-A3 complexes. We also show that corneal CS/DS likely allow Tgamma to bind to corneal epithelium. CTXs of spitting cobra venom are known to cause corneal opacity and/or blindness. Taken together with these observations, our results suggest that corneal CS/DS play a role in the action of CTX in the eye. Most importantly, the present results establish CTXs as cationic, readily available, avidly binding ligands of CS/DS.

Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif.

Heparin and heparan sulfate have recently been shown to bind to snake cardiotoxin (CTX) and to potentiate its penetration into phospholipid monolayer under physiological ionic conditions. Herein we analyze the heparin-binding domain of CTX using 10 CTXs from Taiwan and African cobra venom. We also performed computer modeling to obtain more information of the binding at molecular level. The results provide a molecular model for interaction of CTX-heparin complex where the cationic belt of the conserved residues on the concave surface of three finger beta-sheet polypeptides initiates ionic interaction with heparin-like molecules followed by specific binding of Lys residues near the tip of loop 2 of CTX. The dissociation constants of CTXs differ by as much as 4 orders of magnitude, ranging from approximately 140 microM for toxin gamma to approximately 20 nM for CTX M3, depending on the presence of Lys residues near the tip of loop 2. High affinity heparin binding becomes possible due to the presence of Arg-28, Lys-33, or the so-called consensus heparin binding sequence of XKKXXXKRX near the tip of the loop. The well defined three-finger loop structure of CTX provides an interesting template for the design of high affinity heparin-binding polypeptides with beta-sheet structure. The finding that several cobra CTXs and phospholipase A2 bind to heparin with different affinity may provide information on the synergistic action of the two venom proteins.

Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target for cardiotoxin action.

Cardiotoxins (CTXs) from cobra venom show cytotoxicity toward several cell types. They cause systolic heart arrest and severe tissue necrosis. Their interaction with phospholipids is established but by itself fails to explain the specificity of these toxins; other component(s) of membrane must, therefore, intervene to direct them toward their target. We herein show, for the first time, that sulfated glycosaminoglycans, heparin, heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), interact with CTX A3, a major component of Taiwan cobra venom, by use of affinity chromatography, circular dichroism, absorbance, and fluorescence intensity and anisotropy measurements. The relative strength of binding, determined by the NaCl concentration required to dissociate the CTX-glycosaminoglycan complex, varied as follows: heparin > DS > CS > HS. In physiological buffer (8 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, 138 mM NaCl, pH 7.4), however, only heparin and HS bound to CTX, with respective dissociation constants of 1.4 and 16 microM, while CS and DS failed to exhibit well defined binding behavior, as indicated by fluorescence measurements. We estimate that CTX makes 3-4 ionic contacts with heparin based on a salt-dependent binding study and that approximately 40% of binding free energy is derived from purely electrostatic interactions under physiological conditions. Sulfated pentasaccharide may be sufficient to bind to CTX. We also found that heparin accentuates the penetration of CTX into phospholipid membranes as analyzed by Langmuir monolayer measurement. In view of these results we propose that heparin-like moieties of the cell surface may modulate the action of CTX.

Expression of glutathione S-transferase-cardiotoxin fusion protein in Escherichia coli.

We report here the construction of cardiotoxin V gene, from cobra snake venom (Naja naja atra), by chemically synthesized oligonucleotides and its expression as a glutathione S-transferase-cardiotoxin fusion protein in the inclusion bodies of Escherichia coli. The expression of cardiotoxin fusion protein in protein with a yield of about 35 mg/liter culture was confirmed by highly specific anti-peptide antibodies generated against the unique amino acid residues located at the tip of loop II of cardiotoxin V. Since the fusion protein can be easily treated by CNBr to free the toxin moiety, as revealed by immunoblotting of the cleaved protein, the results provide an avenue for future structural and functional studies of cardiotoxin molecules.

Two distinct types of cardiotoxin as revealed by the structure and activity relationship of their interaction with zwitterionic phospholipid dispersions.

Cardiotoxins (CTXs) are a group of homologous proteins found in cobra snake venom and consist of 60-62 amino acid residues. Although CTXs are known to consist of three extended beta-sheet loops similar to neurotoxins, the target and interaction of CTXs with membranes unlike those of neurotoxins are not well understood. Herein, we report comparative studies of 10 CTXs purified from Taiwan cobra (Naja naja atra) and Mozambique spitting cobra (Naja mossambica mossambica) snake venoms with respect to their interactions with zwitterionic phospholipids. Based on the CTX-induced mixing of sphingomyelin vesicles and the binding of CTX to lysophosphatidylcholine micelles, two distinct types of CTX, i.e. P- and S-type CTX, are identified. P-type CTXs are characterized by the presence of Pro-31 within a putative phospholipid binding site near the tip of loop 2; whereas S-type CTXs are characterized by the presence of Ser-29 within the same but more hydrophilic region. Although binding of all CTXs to phospholipid membranes involves a phospholipid binding site at loop 1, P-type CTXs exhibit higher fusion and binding activity than S-type CTXs, presumably due to the additional phospholipid binding site at loop 2. The binding modes of P- and S-type CTX are thus different. Analysis of the primary structures of 46 CTXs from the genus Naja indicates that these two types of CTXs exist in all species examined. Reasonable structure/activity correlation can be detected for the effects of CTXs on muscle and red blood cells, although notable exceptions are also found. S-type CTXs are generally found to exhibit higher muscle cell depolarization activity, whereas P-type CTXs are found to possess a higher hemolytic activity. Thus the mechanism of action of CTXs seems to involve CTX-membrane interactions and depends on the type of the cell membrane and CTX molecules under study. The two lipid binding sites in P-type CTXs and one lipid binding site in S-type CTXs show large variation in their amino acid residues, but they do display some common distribution of residue type. Analogous to the signal sequences for protein import, these regions are characterized by the coexistence of an exposed hydrophobic surface flanked on either side by a cationic residue. A hypothesis is proposed to explain the general cytotoxic and specific cardiotoxic effect of CTXs based on the two CTX subtypes in snake venom.