title: Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.
abstract: Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom. m.bokori-brown@exeter.ac.uk
authors.forename: Monika
authors.initials: M
authors.lastname: Bokori-Brown
authors.forename: Maria C
authors.initials: MC
authors.lastname: Kokkinidou
authors.forename: Christos G
authors.initials: CG
authors.lastname: Savva
authors.forename: Sérgio
authors.initials: S
authors.lastname: Fernandes da Costa
authors.forename: Claire E
authors.initials: CE
authors.lastname: Naylor
authors.forename: Ambrose R
authors.initials: AR
authors.lastname: Cole
authors.forename: David S
authors.initials: DS
authors.lastname: Moss
authors.forename: Ajit K
authors.initials: AK
authors.lastname: Basak
authors.forename: Richard W
authors.initials: RW
authors.lastname: Titball
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002470:Cell Survival; D003015:Clostridium Infections / Q000382:microbiology*; D003016:Clostridium perfringens / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004285:Dogs; D054884:Host-Pathogen Interactions; D061985:Madin Darby Canine Kidney Cells; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D011956:Receptors, Cell Surface