Effect of a pore-forming protein derived from Flammulina velutipes on the Caco-2 intestinal epithelial cell monolayer.
We have previously found a transepithelial electrical resistance (TEER)-decreasing protein derived from Flammulina velutipes, which was revealed to be identical to flammutoxin (FTX) that is known as a hemolytic pore-forming protein. This protein induced a rapid decrease in TEER and parallel increase in paracellular permeability in the intestinal epithelial Caco-2 cell monolayer without any cytotoxicity. An immunoblotting analysis revealed that the FTX-induced decrease in TEER was accompanied by the formation of a high-molecular-weight complex on the surface of Caco-2 cells. Intracellular Ca(2+) imaging showed that exposure to FTX caused a rapid Ca(2+) influx. It was observed by electron microscopy that FTX induced swelling of microvilli and expansion of the cellular surface. Staining with fluorescent phalloidin showed a marked change to filamentous actin in the FTX-treated cells. These results suggest that TEER reduction could sensitively detect small membrane pore formation by FTX in the intestinal epithelium which causes a morphological alteration and disruption of the paracellular barrier function.
Narai
Asako
A
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan. a-narai@nvau.ac.jp
Watanabe
Hirohito
H
Iwanaga
Toshihiko
T
Tomita
Toshio
T
Shimizu
Makoto
M
D000199:Actins / Q000378:metabolism; D001487:Basidiomycota / Q000737:chemistry*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004847:Epithelial Cells / Q000502:physiology; D005656:Fungal Proteins / Q000494:pharmacology*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000494:pharmacology*
D000199:Actins; D005656:Fungal Proteins; D009183:Mycotoxins; C011823:FTX protein, Flammulina velutipes; D002118:Calcium