Conformational consequences of coupling bullous pemphigoid antigenic peptides to glutathione-S-transferase and their diagnostic significance

Recombinant epitopic peptides BP1 and BP2 representing the Bullous pemphigo id autoantigens of BP230 and BP180 bound to the fusion partner glutathione- S-transferase (pGEX-4T-2, Pharmacia) have been previously shown to increase the efficacy of diagnosis of the disease. Using glutathione-S-transferase- bound monomer peptides, the sensitivity of the immunological reaction excee ded that of the free synthetic epitopes and was further increased with the number of epitopic blocks in the multimer fusion products. This has been ex plained by the avidity effect of the fusion partner dimer formation and the high ligand affinity due to the tandem repetitions of epitopic sequences. However, a beneficial conformation of the bound epitopic peptides might als o contribute to the above phenomenon. Circular dichroism (CD) and Fourier t ransform infrared (FTIR) absorption spectroscopic studies revealed the impo rtance of glutathione-S-transferase to induce and stabilize ordered seconda ry structures of the epitopic peptides. The free monomer and multimer pepti des in aqueous buffer were present as a mixture of unordered and beta-sheet conformation, while binding them to the fusion partner the proportion of o rdered secondary structures increased in parallel with the number of antige nic epitopes. The most prominent changes in the conformational state of the monomers in the fusion form were the increase of alpha-helical and beta-sh eet. and the decrease of unordered conformation, while in the case of oligo meric peptides the adoption of a helical conformation was accompanied by th e decrease of beta-sheet structure. An outstanding alpha-helix content (46% ) was detected in the case of the trimeric BP1 in its recombinant fusion fo rm. Copyright (C) 2000 European peptide Society and John Wiley & Sons, Ltd.