Gangliosides GT1b Research Articles

Identification of a binding site for ganglioside on the receptor binding domain of tetanus toxin.

The carboxyl-terminal region of the tetanus toxin heavy chain (H(C) fragment) binds to di- and trisialylgangliosides on neuronal cell membranes. To determine which amino acids in tetanus toxin are involved in ganglioside binding, homology modeling was performed using recently resolved X-ray crystallographic structures of the tetanus toxin H(C) fragment. On the basis of these analyses, two regions in tetanus toxin that are structurally homologous with the binding domains of other sialic acid and galactose-binding proteins were targeted for mutagenesis. Specific amino acids within these regions were altered using site-directed mutagenesis. The amino acid residue tryptophan 1288 was found to be critical for binding of the H(C) fragment to ganglioside GT1b. Docking of GD1b within this region of the toxin suggested that histidine 1270 and aspartate 1221 were within hydrogen bonding distance of the ganglioside. These two residues were mutagenized and found also to be important for the binding of the tetanus toxin H(C) fragment to ganglioside GT1b. In addition, the H(C) fragments mutagenized at these residues have reduced levels of binding to neurites of differentiated PC-12 cells. These studies indicate that the amino acids tryptophan 1288, histidine 1270, and aspartate 1221 are components of the GT1b binding site on the tetanus toxin H(C) fragment.

Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H C) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H C, recombinant genes for the H C and two hybrid H C in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H C of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H C revealed that mutation of 23 residues in carboxy terminal half of H C (1029–1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.

Ganglioside Modulation Regulates Epithelial Cell Adhesion and Spreading via Ganglioside-specific Effects on Signaling

Gangliosides are implicated in regulating cell adhesion and migration on fibronectin by binding with the alpha(5) subunit of alpha(5)beta(1) integrin. However, the effects of gangliosides on cell spreading and related signaling pathways are unknown. Increases in gangliosides GT1b and GD3 inhibited spreading on fibronectin, concurrent with inhibition of Src and focal adhesion kinase. Although antibody blockade of GT1b or GD3 function and gene-modulated ganglioside depletion stimulated spreading and activated Src and focal adhesion kinase, the augmented spreading by disruption of GT1b function, but not by disruption of GD3 function, was inhibited by blockade of Src and focal adhesion kinase activation. In contrast, inhibitors of protein kinase C prevented the stimulation of spreading by GD3 functional inhibition, but not by GT1b functional blockade. Modulation of either GT1b or GD3 content affected phosphoinositol 3-kinase activation, and inhibition of this activation reversed the stimulation of cell spreading by anti-GD3 antibody, anti-GT1b antibody, and ganglioside depletion, suggesting that phosphoinositol 3-kinase is an intermediate in both the FAK/Src and protein kinase C pathways that lead to cell spreading. These studies demonstrate that epithelial cell ganglioside GT1b modulates cell spreading through alpha(5)beta(1)/FAK and phosphoinositol 3-kinase signaling, whereas GD3-modulated spreading appears to involve phosphoinositol 3-kinase-dependent protein kinase C signaling.

A colorimetric bead-binding assay for detection of intermolecular interactions.

We have developed a new technique that rapidly and reproducibly allows direct visualization of molecular interactions, including receptor-ligand binding. The technique can be easily applied to examine binding between proteins and glycoproteins, or proteins and glycolipids, including gangliosides. In this novel bead-binding assay, the suspected 'ligand' molecule is bound to 0.2- or 1.0- micro m colored FluoSphere beads. These coated beads are then mixed directly with 150- micro m Sepharose or 50- to 75- micro m agarose beads coated with the second 'target' molecule. Binding between molecules is easily detected by immunofluoresence microscopy as colored rosettes or aggregations formed by clustering of the smaller fluorescent beads around the larger non-fluorescent bead. The validity of this technique for glycolipid binding to protein was verified through demonstration of the known interaction between the beta subunit of cholera toxin with ganglioside GM1. The bead-binding technique facilitated the novel observations of interaction between ganglioside GT1b with the alpha5 subunit of alpha5beta1 integrin and the interaction of GM3 with the epidermal growth factor receptor. A modification of this technique, in which the coated beads are bound to protein fixed on plates, allows a quantifiable colorimetric assay of interaction. This versatile and rapid technique will have widespread applications for in vitro systems and may also be useful for in vivo analysis of binding to cell surface receptor molecules.

Botulinum Neurotoxin A Activity Is Dependent upon the Presence of Specific Gangliosides in Neuroblastoma Cells Expressing Synaptotagmin I

Botulinum neurotoxin A (BoNT/A) is the deadliest of all known biological substances. Although its toxicity makes BoNT/A a biological warfare threat, its biologic activity makes it an increasingly useful therapeutic agent for the treatment of muscular disorders. However, almost 200 years after its discovery, the neuronal cell components required for the activity of this deadly toxin have not been unequivocally identified. In this work, neuroblastoma cells expressing synaptotagmin I, a protein shown to be bound by BoNT/A, were used to determine whether specific gangliosides were necessary for BoNT/A activity as measured by synaptosomal-associated protein of 25 kDa (SNAP-25) cleavage. Ganglioside GT1b was found to support BoNT/A activity significantly more effectively than GD1a, which was far more effective than GM1 when added to ganglioside-deficient murine cholinergic Neuro 2a or to human adrenergic SK-N-SH neuroblastoma cells. Whereas both cell lines expressed synaptotagmin I, SNAP-25 cleavage was not observed in the absence of complex gangliosides. These results indicate that 1) gangliosides are required for BoNT/A activity, 2) synaptotagmin I in the absence of gangliosides does not support BoNT/A activity, and 3) Neuro 2a cells are an efficient model system for studying the biological activity of BoNT/A.

Ganglioside Loss Promotes Survival Primarily by Activating Integrin-Linked Kinase/Akt Without Phosphoinositide 3-OH Kinase Signaling

Keratinocyte gangliosides influence cellular functions, including proliferation, adhesion, migration, and differentiation. The effects of endogenous depletion of membrane gangliosides by gene transfection of a human ganglioside-specific sialidase on cell survival were investigated. Ganglioside depletion promotes survival of the human keratinocyte-derived SCC12 cell line through upregulated phosphorylation of beta1 integrin, and increased phosphorylation and activity of integrin-linked kinase, protein kinase B/Akt, and Bad, with resultant inhibition of caspase-9 activation. Ganglioside deficiency also increases expression of cyclins D1 and E, promoting cell cycle progression from G1 phase to S phase. Inhibition of either protein kinase B/Akt or integrin-linked kinase activity renders the ganglioside-deficient cells susceptible to triggers of apoptosis. Both serine-473 and threonine-308 sites of protein kinase B/Akt show increased phosphorylation in ganglioside-deficient cells, but the cell survival correlates with increased phosphorylation of the serine-473 site of Akt, not with increased phosphorylation of the threonine-308 site. Consistently, blockade of ganglioside GT1b function activates integrin-linked kinase and only the serine-473 site of protein kinase B/Akt. In contrast, antibody-induced blockade of GM3 function increases only threonine-308 phosphorylation of ganglioside-deficient cells. Whereas blockade of phosphoinositide 3-OH kinase function suppresses threonine-308 phosphorylation, it neither inhibits serine-473 phosphorylation nor triggers apoptosis. These data suggest that ganglioside depletion modulates cell survival primarily through protein kinase B/Akt stimulation by a pathway that does not require phosphoinositide 3-OH kinase and epidermal growth factor receptor signaling.

Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration.

Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.

The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho.

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. The receptor for MAG or signals that elicit morphological changes in neurons remained to be established. Here we show that the neurotrophin receptor p75 (p75NTR) is the signal transducing element for MAG. Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice carrying a mutation in the p75NTR gene are insensitive to MAG with regard to neurite outgrowth. MAG activates small GTPase RhoA, leading to retarded outgrowth when p75NTR is present. Colocalization of p75NTR and MAG binding is seen in neurons. Ganglioside GT1b, which is one of the binding partners of MAG, specifically associates with p75NTR. Thus, p75NTR and GT1b may form a receptor complex for MAG to transmit the inhibitory signals in neurons.

Ganglioside reactive antibodies in the neuropathy associated with celiac disease

We tested patients with celiac disease (CD) for the presence of serum anti-ganglioside antibodies. Six of twenty-seven patient sera were reactive against brain gangliosides by an agglutination immunoassay. Neurological examination in all six revealed the presence of distal sensory loss, consistent with the diagnosis of peripheral neuropathy. When tested by ELISA for antibodies to isolated GM1, GM2, GD1a, GD1b, GT1b, and GQ1b gangliosides, all six were positive for IgG antibodies to at least one. The neuropathy of celiac disease may be autoimmune and associated with anti-ganglioside antibodies. The presence of IgG reactivity furthermore implicates a T cell-mediated response to ganglioside antigens.

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B.

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.

Detection of anti-ganglioside antibodies in Guillain-Barré syndrome and its variants by the agglutination assay

Sera from 40 patients with Guillain-Barré syndrome (GBS), including the subtypes acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), and Miller Fisher syndrome (MFS) were examined for the presence of anti-ganglioside antibodies using the ganglioside agglutination assay, and the enzyme-linked immunosorbent assay (ELISA). In the ELISA system, sera were tested for IgM and IgG antibodies to GM1, GM2, GD1a, GD1b, GT1b, and GQ1b gangliosides. Antibodies to gangliosides were detected in 21 (53%) of the GBS patients by agglutination assay and in 17 (43%) of the patients by ELISA. Some of the sera reacted with more than one ganglioside. Antibodies were not found in the control sera that were studied. The agglutination assay may be useful for rapid screening of GBS sera for antibodies to multiple gangliosides.

Different metabolic effects of ganglioside GM1 in brain synaptosomes and phagocytic cells.

The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe2+-H2O2 system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of 45Ca2+ influx and oxidative inactivation of Na+,K+-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10(-11)-10(-8) M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10(-11)-10(-10) M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection.

Characterization of receptor-mediated signal transduction by Escherichia coli type IIa heat-labile enterotoxin in the polarized human intestinal cell line T84.

Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl(-) secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl(-) secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl(-) secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl(-) secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E. coli.

Inhibition of Integrin-linked Kinase/Protein Kinase B/Akt Signaling: MECHANISM FOR GANGLIOSIDE-INDUCED APOPTOSIS

Ganglioside GT1b inhibits keratinocyte attachment to and migration on a fibronectin matrix by binding to alpha(5)beta(1) and preventing alpha(5)beta(1) interaction with fibronectin. The role of gangliosides in triggering keratinocyte apoptosis, however, is unknown. Addition of GT1b to keratinocyte-derived SCC12 cells, grown in serum-free medium but exposed to fibronectin, suppressed Bad phosphorylation, activated caspase-9, and inhibited cyclin D and E expression, resulting in cell cycle arrest at G(1) phase and initiation of apoptosis. The mechanism of GT1b activation of caspase-9 involved inhibition of beta(1) integrin serine/threonine phosphorylation and decreased phosphorylation of both integrin-linked kinase and protein kinase B/Akt at its Ser-473 site, leading to cytochrome c release from mitochondria. Consistently, blockade of GT1b function with anti-GT1b antibody specifically activated the Ser-473 site of Akt, markedly suppressing apoptosis. The ganglioside-induced inhibition of Akt phosphorylation was GT1b-specific and was not observed when cells were treated with other keratinocyte gangliosides, including GD3. These studies suggest that the modulation of keratinocyte cell cycle and survival by GT1b is mediated by its direct interaction with alpha(5)beta(1) and resultant inhibition of the integrin/integrin-linked kinase/protein kinase B/Akt signaling pathway.

Intravenous immunoglobulin for cranial polyneuropathy associated with Campylobacter jejuni infection

This study reports the efficacy of IV immunoglobulin in a patient with cranial polyneuropathy resulting from Campylobacter jejuni infection who had high titers of serum IgG antibodies against gangliosides GD1a and GT1b in the acute phase. Treatment with IV immunoglobulin (400 mg/kg/day × 5 days) led to rapid partial resolution of his neurologic manifestations, but complete recovery was not obtained until 6 months later. The present case suggests that IV immunoglobulin therapy prevents further progression of the disease but that it may not shorten the clinical course of cranial polyneuropathy in some cases associated with Campylobacter jejuni infection.

Gangliosides GD1b, GT1b, and GQ1b Suppress the Growth of Human Melanoma by Inhibiting Interleukin-8 Production: the Inhibition of Adenylate Cyclase

We studied the effects of various gangliosides on in vitro growth of human metastatic melanoma WM266-4. GD1b, GT1b, and GQ1b inhibited 3H-thymidine uptake and growth rate of WM266-4 whereas the other gangliosides were ineffective. The growth inhibition by GD1b, GT1b, and GQ1b was counteracted by interleukin-8 but not by the other growth factors. The growth inhibition by gangliosides was not detected in the presence of anti-interleukin-8 antibody. GD1b, GT1b, and GQ1b reduced the constitutive interleukin-8 secretion and mRNA levels in WM266-4. Transient transfection showed that GD1b, GT1b, and GQ1b inhibited the constitutive chloramphenicol acetyltransferase expression driven by interleukin-8 promoter in WM266-4. Transfection with a series of 5'-deleted mutants demonstrated that the sequences between -98 and -62 bp on interleukin-8 promoter may be involved in the transcriptional repression by these gangliosides. Cyclic AMP analog dibutyryl cAMP counteracted GD1b, GT1b, and GQ1b-induced inhibition of interleukin-8 production at the levels of protein secretion, mRNA expression, and promoter activity. GD1b, GT1b, and GQ1b reduced cAMP level and protein kinase A activity in WM266-4. These gangliosides suppressed adenylate cyclase activity without altering that of cyclic nucleotide phosphodiesterase in WM266-4. The data indicate that GD1b, GT1b, and GQ1b may suppress the growth of melanoma by inhibiting interleukin-8 production via the inhibition of adenylate cyclase.

Sensorimotor demyelinating neuropathy with IgM antibody against gangliosides GD1a, GT1b and GM3

We report a patient with sensorimotor demyelinating neuropathy with high-titer IgM antibody against gangliosides GD1a, GT1b and GM3. The patient was a 65-year-old male who was hospitalized with chief complaints of muscular weakness of all limbs and numbness of the hands and feet. Nerve-conduction studies revealed reduced conduction velocities of the motor nerves with increased temporal dispersion and loss of sensory nerve action potentials. Treatment with steroids was ineffective. IgM antibody against GD1a, GT1b and GM3, which are known to be the ligands for myelin-associated glycoprotein (MAG), might have played a role in the demyelination in this patient by inhibiting adhesion between myelin and axonal membrane.

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 ‘NeuAcα3Gal β3GalNAc’ 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 ‘NeuAcα3Gal β3GalNAc’ 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 ‘NeuAcα3Gal β3GalNAc’ 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.

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin. Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1). The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells. The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1). The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.

Fine specificity of anti-GQ1b IgG and clinical features

Anti-GQ1b IgG frequently is present in sera of patients with Miller Fisher syndrome (MFS), Guillain-Barré syndrome (GBS) with ophthalmoplegia, Bickerstaff's brainstem encephalitis (BBE), and acute ophthalmoparesis (AO) in the acute phase. Why various clinical signs develop under these conditions, however, has yet to be clarified. We investigated the fine specificity of anti-GQ1b IgG and its clinical correlation in sera from 82 patients: 56 with MFS, 11 with GBS, 13 with BBE, and 2 with AO. Anti-GQ1b IgG antibodies were absorbed by GT1a in 80 (98%) of the 82 sera, by GD1b in 11 (13%), and by the other b-series gangliosides GD3, GD2, or GT1b in 24 (29%). The most frequent pattern of fine specificity was the cross-reaction with GT1a alone, seen in 56 (68%) samples. Of the 11 patients with anti-GQ1b IgG, cross-reacting with GD1b, 6 (55%) had impaired deep sense, and the association was significant ( p=0.02). This is the first study to show that the fine specificity of anti-GQ1b IgG is heterogeneous and that the difference is correlated with the presence of a particular clinical sign.