A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Detecção de cinco novas mutações em pacientes brasileiros com gangliosidose GM1

A Gangliosidose GM1 é um Erro Inato do Metabolismo (EIM) causado pela deficiência da enzima B-galactosidase ácida. Essa doença é caracterizada pelo acúmulo de metabólitos não degradados, principalmente gangliosídeo GM1, nos lisossomos de vários tipos celulares. Baseado na idade de início e na atividade residual da enzima, a Gangliosidose GM1 é classificada em três diferentes tipos: infantil, juvenil e adulto. O gene da B-galactosidase ácida (GLB1, GeneBank M27507) está situado no cromossomo 3 e possui mais de 60 kb, contendo 16 exons. Cerca de 50 mutações associadas à doença estão descritas na literatura. No sul do Brasil, há uma alta freqüência dessa doença (1:17.000 nascidos vivos). Neste trabalho, vinte pacientes diagnosticados no Hospital de Clínicas de Porto Alegre (Brasil) tiveram o gene GLB1 investigado por SSCP (Single Strand Conformational Polymorphism) usando DNA extraído de sangue periférico. Através desta triagem foram encontradas 52 alterações de mobilidade do DNA, indicando a presença de mutações. As amostras relativas aos exons 2 e 15 foram submetidas a sequenciamento direto com seqüenciador ABI31O(Applied Biosystens) utilizado kit BigDye 3.1. Cinco novas mutações no gene GLB1 (F63Y, R38G, Y36S, Y64F e R59C) e duas mutações já descritas (R59H e 1622-1627insG) foram encontradas. Este trabalho possibilitou a genotipagem completa de 6 pacientes e parcial de 5, e direcionou a investigação de mutações, contribuindo diretamente no diagnóstico da enfermidade e permitindo a realização de estudos de correlação genótipo/fenótipo destes pacientes.

Augmentation de l’absorption intestinale à l’aide de promédicaments se liant aux gangliosides GM1

Le but de ce projet est de développer une approche biomimétique pour augmenter l'absorption de molécules actives peu perméables. Cette approche réplique le mécanisme d'internalisation de la toxine du choléra par sa liaison au récepteur GM1 à la surface des cellules intestinales. La technologie proposée est de synthétiser des promédicaments composés d'une molécule active, d'un espaceur et d'un peptide ayant de l'affinité pour le GM1. Les hypothèses de ce projet sont que le peptide faisant partie du promédicament augmentera l'absorption intestinale de médicaments peu perméables et que le complexe sera métabolisé rapidement après son absorption. Des prototypes de promédicaments ont été synthétisés et des essais de stabilité, d'affinité et de perméabilité sur les peptides et les promédicaments ont été développés et réalisés. Les résultats de cette étude ont démontré la possibilité de synthétiser des promédicaments à partir d'un peptide liant le GM1 et d'une molécule thérapeutique ayant une faible biodisponibilité. Les essais de stabilité, d'affinité et de perméabilité ont été optimisés et implémentés avec succès. Les études in vitro initiales ont rapporté des résultats prometteurs concernant les propriétés de liaison du peptide utilisé et d'un des promédicaments préparés. Par contre, les résultats des essais de stabilité ont montré un métabolisme partiel des promédicament dans le plasma ainsi qu'une instabilité des solutions mères. De plus, les essais de perméabilité se sont avérés non concluants. Les prochaines études porteront sur la préparation de nouveaux promédicaments par une approche similaire ainsi que sur l'investigation du mécanisme d'internalisation du peptide.

Augmentation de l’absorption intestinale à l’aide de promédicaments se liant aux gangliosides GM1

Le but de ce projet est de développer une approche biomimétique pour augmenter l'absorption de molécules actives peu perméables. Cette approche réplique le mécanisme d'internalisation de la toxine du choléra par sa liaison au récepteur GM1 à la surface des cellules intestinales. La technologie proposée est de synthétiser des promédicaments composés d'une molécule active, d'un espaceur et d'un peptide ayant de l'affinité pour le GM1. Les hypothèses de ce projet sont que le peptide faisant partie du promédicament augmentera l'absorption intestinale de médicaments peu perméables et que le complexe sera métabolisé rapidement après son absorption. Des prototypes de promédicaments ont été synthétisés et des essais de stabilité, d'affinité et de perméabilité sur les peptides et les promédicaments ont été développés et réalisés. Les résultats de cette étude ont démontré la possibilité de synthétiser des promédicaments à partir d'un peptide liant le GM1 et d'une molécule thérapeutique ayant une faible biodisponibilité. Les essais de stabilité, d'affinité et de perméabilité ont été optimisés et implémentés avec succès. Les études in vitro initiales ont rapporté des résultats prometteurs concernant les propriétés de liaison du peptide utilisé et d'un des promédicaments préparés. Par contre, les résultats des essais de stabilité ont montré un métabolisme partiel des promédicament dans le plasma ainsi qu'une instabilité des solutions mères. De plus, les essais de perméabilité se sont avérés non concluants. Les prochaines études porteront sur la préparation de nouveaux promédicaments par une approche similaire ainsi que sur l'investigation du mécanisme d'internalisation du peptide.

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

Uptake of oxLDL and IL-10 production by macrophages requires PAFR and CD36 recruitment into the same lipid rafts

Macrophage interaction with oxidized low-density lipoprotein (oxLDL) leads to its differentiation into foam cells and cytokine production, contributing to atherosclerosis development. In a previous study, we showed that CD36 and the receptor for platelet-activating factor (PAFR) are required for oxLDL to activate gene transcription for cytokines and CD36. Here, we investigated the localization and physical interaction of CD36 and PAFR in macrophages stimulated with oxLDL. We found that blocking CD36 or PAFR decreases oxLDL uptake and IL-10 production. OxLDL induces IL-10 mRNA expression only in HEK293T expressing both receptors (PAFR and CD36). OxLDL does not induce IL-12 production. The lipid rafts disruption by treatment with βCD reduces the oxLDL uptake and IL-10 production. OxLDL induces co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein flotillin-1, and colocalization with the lipid raft-marker GM1-ganglioside. Finally, we found colocalization of PAFR and CD36 in macrophages from human atherosclerotic plaques. Our results show that oxLDL induces the recruitment of PAFR and CD36 into the same lipid rafts, which is important for oxLDL uptake and IL-10 production. This study provided new insights into how oxLDL interact with macrophages and contributing to atherosclerosis development.

Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund’s adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon γ levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

Ganglioside GM1 binds to the Trk protein and regulates receptor function.

Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a > 3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk.

Different IVIG glycoforms affect in vitro inhibition of anti-ganglioside antibody-mediated complement deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.

Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis.

BACKGROUND: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. RESULTS: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. CONCLUSIONS: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.

Heat-labile enterotoxin: beyond G(m1) binding.

Enterotoxigenic Escherichia coli (ETEC) is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside G(M1), the toxin's host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor

It is recognised that cholera toxin (Ctx) is a significant cause of gastrointestinal disease globally, particularly in developing countries where access to uncontaminated drinking water is at a premium. Ctx vaccines are prohibitively expensive and only give short-term protection. Consequently, there is scope for the development of alternative control strategies or prophylactics. This may include the use of oligosaccharides as functional mimics for the cell-surface toxin receptor (GM I). Furthermore, the sialic acid component of epithelial receptors has already been shown to contribute significantly to the adhesion and pathogenesis of Ctx. Here, we demonstrate the total inhibition of Ctx using GM1-competitive ELISA with 25 mg mL(-1) of a commercial preparation of sialyloligosaccharides (SOS). The IC50 value was calculated as 5.21 mg mL(-1). One-hundred percent inhibition was also observed at all concentrations of Ctx-HRP tested with 500 ng mL(-1) GM1-OS. Whilst SOS has much lower affinity for Ctx than GM1-OS, the commercial preparation is impure containing only 33.6% carbohydrate; however, the biantennary nature of SOS appears to give a significant increase in potency over constituent monosaccahride residues. It is proposed that SOS could be used as a conventional food additive, such as in emulsifiers, stabilisers or sweeteners, and are classified as nondigestible oligosaccharides that pass into the small intestine, which is the site of Ctx pathogenesis. (C) 2008 Elsevier Ltd. All rights reserved.

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved