Purification and cDNA cloning of a protein derived from Flammulina velutipes that increases the permeability of the intestinal Caco-2 cell monolayer.
A new protein that decreases transepithelial electrical resistance (TEER) in the human intestinal Caco-2 cell monolayer was found in a water-soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER-decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water-soluble marker substances such as Lucifer Yellow CH (Mr 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F. velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the apparent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N-terminal amino-acid sequence of purified TDP is identical to the recently reported N-terminal sequence of flammutoxin, a membrane-perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J. 333, 24794-24799]. The cDNA coding for TDP was cloned by 5' and 3' rapid amplification of cDNA ends. The ORF encodes a protein with 272 amino-acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure-function relationships of membrane pore-forming toxins.
Watanabe
H
H
Department of Applied Biological Chemistry, The University of Tokyo, Japan.
Narai
A
A
Shimizu
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000737:chemistry* / Q000235:genetics; D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000302:isolation & purification; D017097:Electric Impedance; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009183:Mycotoxins
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D009183:Mycotoxins; C011823:FTX protein, Flammulina velutipes