TY - JOUR T1 - Structural Features of the Botulinum Neurotoxin Molecule That Govern Binding and Transcytosis across Polarized Human Intestinal Epithelial Cells JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 633 LP - 641 DO - 10.1124/jpet.104.066845 VL - 310 IS - 2 AU - Andrew B. Maksymowych AU - Lance L. Simpson Y1 - 2004/08/01 UR - http://jpet.aspetjournals.org/content/310/2/633.abstract N2 - Experiments were done to help localize the minimum essential domain within the botulinum toxin molecule that is necessary for binding and transport across human gut epithelial cells. The data demonstrated that the neurotoxin alone, in the absence of auxiliary proteins, undergoes transcytosis. The neurotoxin by itself was examined in the single chain (unnicked serotype B) and dichain (nicked serotype B, nicked serotype A) forms, and all displayed the ability to bind and penetrate epithelial barriers. In addition, the single chain and dichain molecules were examined in the oxidized and reduced states, and again all forms were transported. To further define the minimum essential domain, experiments were done with two toxin fragments: 1) the heavy chain, which was derived from native toxin, and 2) the carboxy-terminal portion of the heavy chain, which was generated by recombinant techniques. Interestingly, both fragments were fully competent in crossing epithelial barriers. These data suggest that a polypeptide derived from the toxin could be used as a carrier domain to transport other molecules across epithelial cells. In related experiments, physiological (i.e., potassium depletion) and pharmacological (i.e., chlorpromazine) manipulations were used to implicate clathrin-coated pits/vesicles as the structures responsible for endocytosis of toxin. The American Society for Pharmacology and Experimental Therapeutics ER -