Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus

Nobuki Sakurai, Jun Kaneko, Yoshiyuki Kamio, Toshio Tomita

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)

Abstract

Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Here, we cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. The genomic regions encoding pleurotolysin A and B contained two and eight introns, respectively, and putative promoter sequences. The complementary DNA (cDNA) for pleurotolysin A encoded 138 amino acid residues, and the predicted product was identical with natural pleurotolysin A, except for the presence of the first methionine. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively, which were unfolded with 8 M urea and refolded by 100-fold dilution with 10 mM Tris-HCl buffer, pH 8.5. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell as efficiently as the natural proteins at nanomolar concentrations. In contrast, the precursor of pleurotolysin B was much less hemolytically active than mature pleurotolysin B in the presence of pleurotolysin A.

Original languageEnglish
Pages (from-to)65-73
Number of pages9
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1679
Issue number1
DOIs
Publication statusPublished - 2004 Jul 13

Keywords

  • Basidiomycete
  • Complementary DNA
  • Genomic DNA
  • PCR
  • Ply
  • Pore-forming protein
  • SDS-PAGE
  • SDS-polyacrylamide gel electrophoresis
  • Sphingomyelin
  • Two-component hemolysin
  • base pair
  • bp
  • cDNA
  • complementary DNA
  • pleurotolysin
  • polymerase chain reaction

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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