title: Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.
abstract: BACKGROUND
  How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear.
  
  
  RESULTS
  BoNT/C utilizes dual gangliosides as host cell receptors.
  
  
  CONCLUSIONS
  BoNT/C accesses gangliosides on the plasma membrane.
  
  
  CONCLUSIONS
  Plasma membrane accessibility of the dual ganglioside receptors suggests synaptic vesicle exocytosis may not be necessary to expose BoNT/C receptors. Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A-G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Andrew P-A
authors.initials: AP
authors.lastname: Karalewitz
authors.forename: Zhuji
authors.initials: Z
authors.lastname: Fu
authors.forename: Michael R
authors.initials: MR
authors.lastname: Baldwin
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Barbieri
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005732:Gangliosides; D011956:Receptors, Cell Surface; D001905:Botulinum Toxins; C096324:botulinum toxin type C