A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

Jobling, Michael G.; Yang, ZhiJie; Kam, Wendy R.; Lencer, Wayne I.; Holmes, Randall K.

1B) . The heterogenous mixture of holotoxins was further separated into its components by anion-exchange chromatography. The GSH6 tag not only changed the size of the B monomer but also changed its predicted pI from 8.24 for native CTB to 6.21 for CTB-GSH6. The respective predicted pIs for the CTB-G33D variant changed from 7.31 for the native length to 5.91 for the GSH6-tagged variant. All holotoxins bound to the anion exchange matrix, and at least five peaks could be discerned following elution with a salt gradient (Fig. 1C) . Samples from individual fractions were analyzed by SDS-PAGE either without (Fig. 1D , upper) or after (Fig. 1D , lower) boiling in sample loading buffer. The unboiled samples in lanes 4 to 13 showed a single band corresponding to assembled holotoxin (AB 5 ), with a small amount of contaminating protein of slower mobility in lanes 2 and 3. The major fractions from each peak are homogenous and are consistent with each peak containing a single species CTB-G33D-Linker-His 6 , pAra BAD , Cm r , Ori p15a This study of variant holotoxin, The assembled holotoxins in the unboiled samples bind less SDS than if they were fully denatured and therefore do not migrate in direct proportion to their molecular weights. The relative molecular weight (M r ) for each holotoxin variant is increased by the presence of one or more tagged CTB subunits. Each of these holotoxins completely disassembles into its component polypeptides after boiling (Fig. 1D , lower panel; CTA, 29 kDa; CTB-GSH6-tagged monomer, 14.4 kDa; and native CTB-G33D monomer, 11.5 kDa). The holotoxin in peak 0 ( Fig. 1C ) has homopentameric CTB-G33D subunits and no functional GM 1 BS, while holotoxins in peaks 1 and 2 have a mixture of wt CTB-GSH6 and CTB-G33D pentamers with 1 and 2 GM 1 BS, respectively. The fractions loaded in lanes 2 to 5 contain CTA plus 5 CTB-G33D (peak 0), and those in lanes 6 to 9 contain CTA plus 1 wt CTB-GSH6 and 4 CTB-G33D (peak 1), and those in lanes 10 to 13 contain CTA plus 2 wt CTB-GSH6 and 3 CTB-G33D (peak 2). Only these first three peaks were well enough separated to be purified efficiently from the extract of production strain AMBT. The fractions corresponding to each peak were pooled, dialyzed, and rerun over the same anion-exchange column to achieve a very high degree of purity. To make holotoxins with 3 and 4 native GM 1 BS, an expression and purification run was done using strain ABMT, which produced three peaks similar to those shown in Fig. 1C , containing holotoxins with zero, one, or two GSH6tagged CTB-G33D subunits and five, four, or three native GM 1 BS, respectively. Finally, as noted above, strain ABBT was used for expression and purification of wt control holotoxins, all with 5 GM 1 BS and zero, one, or two GSH6-tagged CTB subunits. Analysis of the eight final highly purified holotoxin preparations by SDS-PAGE and Coomassie blue staining is shown in Fig. 2 . The upper panel shows a gel run with unboiled samples, and the lower panel shows a gel run with samples boiled in sample buffer to dissociate the components. Without boiling, holotoxins with untagged or tagged wt CTB pentamers (Fig. 2 , upper panel, lanes 2 to 4) dissociated into free CTA and stable pentameric B subunits that ran near the 45-kDa marker (lane 1). Interestingly, the presence of one or more CTB-G33D polypeptides increased stability of the heterohexameric AB 5 holotoxins. Consequently, unboiled holotoxins with three or more CTB-G33D polypeptides (Fig. 2