Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.
Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.
MacKenzie
C R
CR
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
Hirama
T
T
Buckley
J T
JT
D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D058966:Contactins; D004252:DNA Mutational Analysis; D006021:Glycophorins / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006461:Hemolysis; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D018800:Thy-1 Antigens / Q000378:metabolism
D001427:Bacterial Toxins; D015816:Cell Adhesion Molecules, Neuronal; D058966:Contactins; D006021:Glycophorins; D017261:Glycosylphosphatidylinositols; D007473:Ion Channels; D052899:Pore Forming Cytotoxic Proteins; D011956:Receptors, Cell Surface; D018800:Thy-1 Antigens; C107096:proaerolysin; C032676:aerolysin