title: Secretion and properties of the large and small lobes of the channel-forming toxin aerolysin.
abstract: Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25 degreesC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, Box 3055, Victoria, BC, Canada, V8W 3P6.
authors.forename: D B
authors.initials: DB
authors.lastname: Diep
authors.forename: T S
authors.initials: TS
authors.lastname: Lawrence
authors.forename: J
authors.initials: J
authors.lastname: Ausio
authors.forename: S P
authors.initials: SP
authors.lastname: Howard
authors.forename: J T
authors.initials: JT
authors.lastname: Buckley
mesh_terms: D000333:Aeromonas / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D052899:Pore Forming Cytotoxic Proteins; D051381:Rats; D013053:Spectrophotometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017261:Glycosylphosphatidylinositols; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; C107096:proaerolysin; C032676:aerolysin