Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance

Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosph atidylinositol (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 cont actin with similar rate constants and affinities. Enzymatic removal of N-li nked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin, Ae rolysin 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 smal l lobe bound the GPI-anchored protein at least 1000-fold more weakly than t he intact toxin, Mutation analyses provided further evidence that both lobe s were involved in GPI anchor binding, with certain single amino acid subst itutions in either domain leading to reductions in affinity of as much as 1 00-fold. A variant with single amino acid substitutions in both lobes of th e protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weak ly to immobilized proaerolysin, suggesting that interactions with cell-surf ace carbohydrate structures other than GPI anchors may partially mediate to xin binding to host cells.