Protein Sequence Analysis, Cloning, and Expression of Flammutoxin, a Pore-forming Cytolysin fromFlammulina velutipes
Journal of Biological Chemistry
Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal
... 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys 251 and Met 252 by trypsin. 1 The abbreviations used are: FTX, flammutoxin; rFTX, recombinant FTX; rFTX271, recombinant FTX consisting of 271 residues; rFTX251, recombinant FTX consisting of 251 residues; TPCK, N-tosyl-L-phenylalanyl-chloromethylketone; PE, S-pyridylethylated; HPLC, high performance liquid chromatography; RP-HPLC, reversed phase HPLC; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry.