Regeneration of active receptor recognition domains on the B subunit of cholera toxin by formation of hybrids from chemically inactivated derivatives

In order to test the hypothesis that binding sites of cholera toxin for its receptor, the monosialoganglioside GM1, are shared between adjacent beta-polypeptide chains, two inactive chemical derivatives of the B subunit of cholera toxin (CTB) were prepared and were subsequently used for the construction of hybrid CTB pentamers. One inactive derivative consisted of CTB specifically modified in the single essential Trp-88 residue of each beta-chain. This residue was modified by formylation, a treatment preserving the structural integrity of CTB. The other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site. Using [1,4-14C]succinic anhydride for the site-specific succinylation and analysis of radiolabeled tryptic fragments of S-carboxymethylated [14C]sssCTB revealed that the amino groups specifically modified were the alpha-amino group of Thr-1 and the epsilon-amino groups of respectively Lys-34 and Lys-91. Upon submitting equal amounts of formylated CTB and site-specific succinylated CTB to a denaturation-renaturation cycle, hybrid pentamers were formed which in contrast to the parental compounds were able to bind GM1. The affinity of hybrid CTB for GM1, as estimated by a competitive solid-phase radiobinding assay was unexpectedly high and only 2.5-fold lower than that of its native counterpart. The number of active binding sites on hybrid CTB was determined from: (i) titration with the oligosaccharide moiety of GM1 (oligo-GM1) and monitoring the reversal of the Trp fluorescence quenching by iodide ions and (ii) rapid gel filtration over a superdex HR column of a mixture of hybrid CTB and an excess of 3H-labeled oligo-GM1. The data are in agreement with the formation of one active binding per four reconstituted binding sites in hybrid CTB, which is consistent with a random association of CTB monomers during the denaturation-renaturation cycle.