Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites

PLoS One. 2014 Oct 23;9(10):e111170. doi: 10.1371/journal.pone.0111170. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD
  • Binding Sites
  • Botulinum Toxins / chemistry
  • Botulism / microbiology
  • Caco-2 Cells
  • Cadherins / chemistry*
  • Carbohydrates / chemistry*
  • Clostridium botulinum type B / chemistry*
  • Electric Impedance
  • Hemagglutinins / chemistry*
  • Humans
  • Intestinal Absorption
  • Intestines / microbiology
  • Mucins / chemistry
  • Neurotransmitter Agents / chemistry
  • Plasmids
  • Protein Binding

Substances

  • Antigens, CD
  • CDH1 protein, human
  • Cadherins
  • Carbohydrates
  • Hemagglutinins
  • Mucins
  • Neurotransmitter Agents
  • Botulinum Toxins

Grants and funding

Funding provided by the Funding Program for Next Generation World-Leading Researchers (NEXT Program) from the Japan Society for the Promotion of Science (JSPS) and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20790336, 21390128). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.