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ABSTRACT: Foodborne and intestinal botulism are the most common forms of human botulism; both result from the absorption of botulinum neurotoxin (BoNT) from the digestive tract into the circulation. BoNT is a large protein toxin (approximately 150 kDa), but it is able to pass through the epithelial barrier in the digestive tract. Recent cellular and molecular biology studies have begun to unravel the mechanisms by which this large protein toxin crosses the intestinal epithelial barrier. This review provides an overview of current knowledge relating to the absorption of botulinum toxins (BoNT and BoNT complex) from the gastrointestinal tract, with particular emphasis on the interaction of these toxins with the intestinal epithelial barrier.
Current topics in microbiology and immunology 01/2013; 364:45-59. · 4.93 Impact Factor
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ABSTRACT: The haemagglutinin subcomponent HA3 of the type B botulinum neurotoxin complex, which is important in toxin absorption from the gastrointestinal tract, has been expressed, purified and subsequently crystallized in two crystal forms at different pH values. Form I belonged to space group R32, with unit-cell parameters a = b = 357.4, c = 249.5 Å, α = β = 90, γ = 120°. Form II belonged to space group I4(1)32, with unit-cell parameters a = b = c = 259.0 Å, α = β = γ = 90°. Diffraction data were collected from these crystals to a resolution of 3.0 Å for both form I and form II.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2011; 67(Pt 10):1244-6. · 0.51 Impact Factor
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ABSTRACT: Botulinum neurotoxin (BoNT) causes the disease botulism, which is characterized by flaccid paralysis, in humans and animals. The metalloprotease activity of BoNT inhibits neurotransmitter release at neuro-muscular junctions. In most cases, poisoning occurs when BoNT is ingested. Therefore, BoNT must pass through the epithelial barrier of the gastrointestinal tract to enter the systemic circulation and reach the target site. BoNT forms large protein complexes by associating with non-toxic components referred to as non-toxic non-hemagglutinin (NTNH) and hemagglutinin (HA). These proteins protect BoNT from the low pH and proteases in the digestive tract. We recently determined that HA has an unexpected function of disrupting the intercellular epithelial barrier by directly binding to E-cadherin. HA binds to E-cadherin and disrupts its function in a species-specific manner, and this interaction is essential to disrupt tight junctions. This activity is thought to facilitate the absorption of BoNT through the paracellular route of the intestinal epithelium in susceptible species.
Cell adhesion & migration 01/2011; 5(1):34-6. · 1.82 Impact Factor
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ABSTRACT: Botulinum neurotoxin is produced by Clostridium botulinum and forms large protein complexes through associations with nontoxic components. We recently found that hemagglutinin (HA), one of the nontoxic components, disrupts the intercellular epithelial barrier; however, the mechanism underlying this phenomenon is not known. In this study, we identified epithelial cadherin (E-cadherin) as a target molecule for HA. HA directly binds E-cadherin and disrupts E-cadherin-mediated cell to cell adhesion. Although HA binds human, bovine, and mouse E-cadherin, it does not bind rat or chicken E-cadherin homologues. HA does not interact with other members of the classical cadherin family such as neural and vascular endothelial cadherin. Expression of rat E-cadherin but not mouse rescues Madin-Darby canine kidney cells from HA-induced tight junction (TJ) disruptions. These data demonstrate that botulinum HA directly binds E-cadherin and disrupts E-cadherin-mediated cell to cell adhesion in a species-specific manner and that the HA-E-cadherin interaction is essential for the disruption of TJ function.
The Journal of Cell Biology 05/2010; 189(4):691-700. · 10.26 Impact Factor
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ABSTRACT: Orally ingested botulinum neurotoxin (BoNT) causes food-borne botulism, but BoNT must pass through the gut lining and enter the bloodstream. We have previously found that type B haemagglutinin (HA) proteins in the toxin complex play an important role in the intestinal absorption of BoNT by disrupting the paracellular barrier of the intestinal epithelium, and therefore facilitating the transepithelial delivery of BoNT. Here, we show that type A HA proteins in the toxin complex have a similar disruptive activity and a greater potency than type B HA proteins in the human intestinal epithelial cell lines Caco-2 and T84 and in the canine kidney epithelial cell line MDCK I. In contrast, type C HA proteins in the toxin complex (up to 300 nM) have no detectable effect on the paracellular barrier in these human cell lines, but do show a barrier-disrupting activity and potent cytotoxicity in MDCK I. These findings may indicate that type A and B HA proteins contribute to the development of food-borne botulism, at least in humans, by facilitating the intestinal transepithelial delivery of BoNTs, and that the relative inability of type C HA proteins to disrupt the paracellular barrier of the human intestinal epithelium is one of the reasons for the relative absence of food-borne human botulism caused by type C BoNT.
Microbiology 02/2009; 155(Pt 1):35-45. · 3.06 Impact Factor
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ABSTRACT: The heterotrimeric G protein alpha q subunit (Galphaq) mediates a variety of cell functions by activating the effector molecule phospholipase Cbeta. Galphaq activity is regulated by G protein betagamma subunits, G protein-coupled receptors, RGS proteins, and Ric-8. In this study, we identified the lipid raft resident proteins, flotillin-1/reggie-2 and flotillin-2/reggie-1, as Galphaq-binding proteins. The interactions of Galphaq and flotillins were independent of the nucleotide-binding state of Galphaq, and the N-terminal portion of flotillins was critical for the interaction. A short interfering RNA-mediated knockdown of flotillins, particularly flotillin-2, attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK) but not that of ERK1/2. The activation of p38 MAPK was inhibited by the Src family tyrosine kinase inhibitor PP2 and the cholesterol-depleting agent methyl-beta-cyclodextrin, which is generally used for the disruption of lipid rafts. In contrast, the activation of ERK1/2 was not inhibited by these compounds. These lines of evidence suggested that a Gq-coupled receptor activates specifically p38 MAPK through lipid rafts and Src kinase activation, in which flotillins positively modulate the Gq signaling.
Cellular Signalling 07/2007; 19(6):1301-8. · 4.06 Impact Factor
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ABSTRACT: RIC-8 was originally found by genetic studies on C. elegans mutants that were resistant to inhibitors of acetylcholinesterase and reported to act in vitro as a guanine nucleotide exchange factor for G protein alpha subunits. However, the physiological role of a mammalian homolog Ric-8A on G protein-coupled receptor signaling in intact cells is largely unknown. We isolated Ric-8A using a yeast two-hybrid system with Galphaq and examined the role of Ric-8A on Gq-mediated signaling. The small interfering RNA of Ric-8A diminished the Gq-coupled receptor-mediated ERK activation and intracellular calcium mobilization in 293T cells. Ric-8A was translocated to the cell membrane in response to the Gq-coupled receptor stimulation. The expression of the myristoylation sequence-conjugated Ric-8A mutant was located in the membranes and shown to enhance the Gq-coupled receptor-mediated ERK activation. Moreover, this enhancement on ERK activation and the guanine nucleotide exchange activity of Ric-8A for Galphaq were inhibited by Gq selective inhibitor YM-254890. These results suggested that Ric-8A potentiates Gq-mediated signal transduction by acting as a novel-type regulator in intact cells.
Genes to Cells 06/2006; 11(5):487-98. · 2.68 Impact Factor
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ABSTRACT: Cell migration is a complex phenomenon that is stimulated by chemoattractive factors such as chemokines, a family of ligands for G protein-coupled receptors (GPCRs). In contrast, factors that suppress cell migration, and the mechanism of their action, remain largely unknown. In this study, we show that endothelin, a GPCR ligand, inhibits cell motility in a manner dependent upon signaling through the c-Jun N-terminal kinase (JNK) pathway. We further demonstrate that this effect is dependent upon Src kinase and small GTPases Rac1 and Cdc42. These findings provide new insight into GPCR-mediated regulation of cell migration.
FEBS Letters 10/2002; 527(1-3):284-8. · 3.54 Impact Factor
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ABSTRACT: We previously reported that the alpha1B-adrenergic receptor leads to activation of Rho family small GTPases, and in turn, c-Jun N-terminal kinase (JNK), which results in the inhibition of cell proliferation. Here, we show the involvement of the Rho family guanine nucleotide exchange factor (GEF) Dbl's Big Sister (Dbs) in the signaling pathway. Transfection of a Dbl-homology (DH) and pleckstrin-homology (PH) domain-deficient form of Dbs into cells blocked the alpha1B-adrenergic receptor-induced activation of JNK. Conversely, transfection of an isolated DH domain of Dbs induced JNK activation. Stimulation of the alpha1B-adrenergic receptor enhanced an intrinsic Cdc42-GEF activity of Dbs in a manner dependent on Src family tyrosine kinases. Additionally, DH and PH domain deficient Dbs blocked the receptor-induced inhibition of cell proliferation, while DH domain of Dbs inhibited cell proliferation via the JNK-dependent pathway. Taken together, Dbs may play an important role in the anti-mitogenic JNK pathway downstream of the alpha1B-adrenergic receptor.
Biochemical and Biophysical Research Communications 09/2002; 296(1):85-92. · 2.48 Impact Factor
