Binding and transcytosis of botulinum neurotoxin by polarized human colon carcinoma cells.
T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxic in vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8-10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 microM brefeldin A, decreased 42% when microtubules were disrupted by 10 microM nocodazole, and decreased 74% at 18 degreesC. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not.
Maksymowych
A B
AB
Departments of Medicine and of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Simpson
L L
LL
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D001905:Botulinum Toxins / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D020126:Brefeldin A; D018938:Caco-2 Cells; D016764:Cell Polarity; D003110:Colonic Neoplasms / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D004064:Digestive System / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D018942:Macrolides; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D015739:Nocodazole / Q000494:pharmacology; D011485:Protein Binding; D013696:Temperature; D013744:Tetanus Toxin / Q000378:metabolism
D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D018942:Macrolides; D008744:Methylamines; D013744:Tetanus Toxin; C096323:rimabotulinumtoxinB; D020126:Brefeldin A; C040929:bafilomycin A1; C027451:methylamine; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096324:botulinum toxin type C; D015739:Nocodazole