A protein-based pentavalent inhibitor of the cholera toxin B-subunit

Abstract

Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pM for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies.

Keywords: carbohydrates; glycoproteins; multivalency; protein modifications; protein structures.

Figure 1

Strategy for re-engineering the CTB protein to prepare a pentavalent neoglycoprotein inhibitor for cholera toxin. The N termini of a nonbinding CTB mutant W88E are oxidized to give aldehydes that undergo oxime ligation with a carbohydrate ligand that bears an aminooxy function. The resulting neoglycoprotein has ligand groups arranged with optimal spacing to bind to the cholera toxin protein. In the 3D structure of CTB (PDB code: 3CHB),[24] the binding sites are marked in white, and N-terminal threonine residues are marked with black arrows.

Scheme 1

a) Synthesis of an oxime-modified GM1 oligosaccharide ligand 5; b) N-terminal oxidation of the CTB W88E protein and ligation with ligand 5.

Figure 2

Enzyme-linked lectin assay (ELLA) indicates the inhibitory potential of W88E(GM1) and analogous monovalent ligands 1 and 2. Error bars indicate the standard error of three measurements.

Figure 3

a) Dynamic light scattering (DLS) results showing the increase in the size of the particles when CTB is mixed at a ratio of 1:1 with W88E(4). b) Sedimentation coefficient distributions [c(s)] of CTB mixed with W88E(4) at different ratios and the appearance of a single species corresponding to a protein heterodimer.