Fusion proteins containing the A2 domain of cholera toxin assemble with B polypeptides of cholera toxin to form immunoreactive and functional holotoxin-like chimeras.

Cholera enterotoxin (CT) is produced by Vibrio cholerae and excreted into the culture medium as an extracellular protein. CT consists of one A polypeptide and five B polypeptides associated by noncovalent bonds, and CT-B interacts with CT-A primarily via the A2 domain. Treatment of CT with trypsin cleaves CT-A into A1 and A2 fragments that are linked by a disulfide bond. CT-B binds to ganglioside GM1, which functions as the plasma membrane receptor for CT, and the enzymatic activity of A1 causes the toxic effects of CT on target cells. We constructed translational fusions that joined foreign proteins via their carboxyl termini to the A2 domain of CT-A, and we studied the interactions of the fusion proteins with CT-B. The A2 domain was necessary and sufficient to enable bacterial alkaline phosphatase (BAP), maltose-binding protein (MBP) or beta-lactamase (BLA) to associate with CT-B to form stable, immunoreactive, holotoxin-like chimeras. Each holotoxin-like chimera was able to bind to ganglioside GM1. Holotoxin-like chimeras containing the BAP-A2 and BLA-A2 fusion proteins had BAP activity and BLA activity, respectively. We constructed BAP-A2 mutants with altered carboxyl-terminal sequences and tested their ability to assemble into holotoxin-like chimeras. Although the carboxyl-terminal QDEL sequence of the BAP-A2 fusion protein was not required for interaction with CT-B, most BAP-A2 mutants with altered carboxyl termini did not form holotoxin-like chimeras. When holotoxin-like chimeras containing BAP-A2, MBP-A2, or BLA-A2 were synthesized in V. cholerae, they were found predominantly in the periplasm. The toxin secretory apparatus of V. cholerae was not able, therefore, to translocate these holotoxin-like chimeras across the outer membrane.