Yo Sugawara

Osaka University, Suika, Ōsaka, Japan

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Publications (11)47.98 Total impact

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    ABSTRACT: Botulinum neurotoxin is conventionally divided into seven serotypes designated A-G, and is produced as large protein complexes through associations with nontoxic components, such as hemagglutinin (HA) and non-toxic non-HA. These nontoxic proteins dramatically enhance the oral toxicity of the toxin complex. HA is considered to have a role in toxin transport through the intestinal epithelium by carbohydrate binding and epithelial barrier-disrupting activity. Type A and B HAs disrupt E-cadherin-mediated cell adhesion and, in turn, the intercellular epithelial barrier. Meanwhile, type C HA (HA/C) disrupts the barrier function by affecting cell morphology and viability, the mechanism of which remains unknown. In this study, we identified GM3 as the target molecule of HA/C. We found that sialic acid binding of HA is essential for the activity. It was abolished when cells were pretreated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to GM3 and a-series gangliosides in a glycan array. In parallel, we isolated clones resistant to HA/C activity from a susceptible mouse fibroblast strain. These cells lacked the expression of ST-I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. In addition, these clones became sensitive to HA/C activity when GM3 was expressed by transfecting the ST-I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing the ganglioside synthesis genes that utilize GM3 as a substrate and consequently generated a-series gangliosides, suggesting a GM3-specific mechanism. Our results demonstrate that HA/C affects cells in a GM3-dependent manner. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 06/2015; DOI:10.1111/febs.13346
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    ABSTRACT: To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.
    Nature Communications 02/2015; 6:6255. DOI:10.1038/ncomms7255
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    ABSTRACT: Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.
    PLoS ONE 10/2014; 9(10):e111170. DOI:10.1371/journal.pone.0111170
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    ABSTRACT: Clostridium botulinum hemagglutinin (HA) is a component of the large botulinum neurotoxin (BoNT) complex, and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and which are mainly made up of β-strands. Here, we demonstrate structural and functional reconstitution of the whole HA, and present the complete structure of HA of serotype B, determined by X-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.
    Journal of Biological Chemistry 10/2013; 288(49). DOI:10.1074/jbc.M113.521179
  • Y. Sugawara, T. Matsumura, Y. Fujinaga
    Toxicon 06/2013; 68:98. DOI:10.1016/j.toxicon.2012.07.109
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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. DOI:10.1007/978-3-642-33570-9_3
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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. DOI:10.1107/S1744309111027412
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    Yo Sugawara, Yukako Fujinaga
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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. DOI:10.4161/cam.5.1.13574
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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. DOI:10.1083/jcb.200910119
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    ABSTRACT: Food-borne botulinum neurotoxin (BoNT) in the gastrointestinal lumen must cross an epithelial barrier to reach peripheral nerves to mediate its toxicity. The detailed mechanism by which BoNT traverses this barrier remains unclear.We found that hemagglutinin (HA) proteins of type B BoNT complex play an important role in the intestinal absorption of BoNT, disrupting the paracellular barrier of intestinal epithelium, which facilitates transepithelial delivery of BoNT both in vitro and in vivo (Matsumura, T., et al., 2008. Cell. Microbiol. 10, 355–364). We also found that type A HA proteins have a similar disrupting activity with a greater potency than type B HA proteins in the human intestinal epithelial cell lines Caco-2 and T84. 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. These results may indicate that types A and B HA contribute to develop the food-borne human botulism by facilitating the intestinal transepithelial delivery of BoNTs.
    Toxicon 10/2009; DOI:10.1016/j.toxicon.2008.11.014
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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. DOI:10.1099/mic.0.021246-0