title: Binding of Clostridium botulinum C3 exoenzyme to intact cells.
abstract: C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by mono-ADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentration-dependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.
authors.affiliation: Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany, rohrbeck.astrid@mh-hannover.de.
authors.forename: Astrid
authors.initials: A
authors.lastname: Rohrbeck
authors.forename: Leonie
authors.initials: L
authors.lastname: von Elsner
authors.forename: Sandra
authors.initials: S
authors.lastname: Hagemann
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D002417:Cattle; D003014:Clostridium botulinum / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding / Q000502:physiology
chemical_list: D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins