title: Clostridium botulinum C2 toxin: binding studies with fluorescence-activated cytometry.
abstract: Clostridium botulinum C2 enterotoxin consists of two unlinked proteins designated as C2II, which recognizes a cell-surface glycoprotein and translocates an ADP-ribosyltransferase, C2I, into the cytosol of a targeted cell. Fluorescence-activated cytometry was used to study the cellular interactions of Alexa488-labeled C2I (C2I-A488) and proteolytically activated C2II (C2IIa-A488). The binding of C2IIa-A488 (4 degrees C/10 min) to Chinese hamster ovary (CHO) and African green monkey kidney (Vero) cells yielded a signal/noise ratio of 7:1 and 4:1, respectively. C2I-A488 binding required C2IIa and resulted in a 4:1 (CHO) and 10:1 (Vero) signal/noise ratio that was readily competed by unlabeled C2I. Neither C2I nor C2IIa bound to a CHO line (RK14), lacking the receptor for C2IIa. C2I-A488 did not dock with the heterologous cell-binding component (iota b) of Clostridium perfringens iota toxin, a binary toxin closely related to C2. Pretreatment of wild-type CHO or Vero cells with pronase or papain (37 degrees C/30 min) prevented a cell-associated C2IIa-specific signal. However, CHO and Vero cells pretreated with papain at 25 degrees C had a 1.5- to 2.3-fold increase in C2IIa-specific fluorescence versus untreated cells incubated with C2IIa-A488. Overall, these studies further demonstrated the utility of fluorescence-activated cytometry for studying the binding characteristics of bacterial binary toxins like C2.
authors.affiliation: Department of Immunology and Molecular Biology, US Army Medical Research Institute of Infectious Diseases, Frederick MD 21702-5011, USA. bradley.stiles@amedd.army.mil
authors.forename: Bradley G
authors.initials: BG
authors.lastname: Stiles
authors.forename: Dagmar
authors.initials: D
authors.lastname: Blöcker
authors.forename: Martha L
authors.initials: ML
authors.lastname: Hale
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Guetthoff
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D016466:CHO Cells; D002522:Chlorocebus aethiops; D003016:Clostridium perfringens / Q000378:metabolism; D006224:Cricetinae; D005434:Flow Cytometry; D009154:Mutation; D010206:Papain / Q000494:pharmacology; D011402:Pronase / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D014709:Vero Cells
chemical_list: D011994:Recombinant Proteins; D010206:Papain; D011402:Pronase; D001905:Botulinum Toxins; C096324:botulinum toxin type C