title: Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3.
abstract: Botulinum neurotoxin is conventionally divided into seven serotypes, designated A-G, and is produced as large protein complexes through associations with non-toxic components, such as hemagglutinin (HA) and non-toxic non-HA. These non-toxic 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. 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 pre-treated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to 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 expression of ST-I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. These clones became sensitive to HA/C activity when GM3 was expressed by transfection with the ST-I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing ganglioside synthesis genes whose products utilize GM3 as a substrate and consequently generate other a-series gangliosides, suggesting a GM3-specific mechanism. Our results demonstrate that HA/C affects cells in a GM3-dependent manner.
authors.affiliation: Laboratory of Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
authors.forename: Yo
authors.initials: Y
authors.lastname: Sugawara
authors.affiliation: Department of Biochemistry, Faculty of Science and Technology, Kinki University, Higashi-Osaka, Osaka, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Iwamori
authors.affiliation: Laboratory of Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Matsumura
authors.affiliation: Laboratory of Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yutani
authors.affiliation: Laboratory of Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Amatsu
authors.affiliation: Laboratory of Infection Cell Biology, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Fujinaga
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003014:Clostridium botulinum / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005679:G(M3) Ganglioside / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000172:deficiency / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D015820:Cadherins; D005679:G(M3) Ganglioside; D006388:Hemagglutinins; D011994:Recombinant Proteins; D012794:Sialic Acids; D012799:Sialyltransferases
keywords: GM3; botulinum toxin; ganglioside; hemagglutinin; sialic acid