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 pemphigoid 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 exceeded that of the free synthetic epitopes and was further increased with the number of epitopic blocks in the multimer fusion products. This has been explained 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 also contribute to the above phenomenon. Circular dichroism (CD) and Fourier transform infrared (FTIR) absorption spectroscopic studies revealed the importance of glutathione-S-transferase to induce and stabilize ordered secondary structures of the epitopic peptides. The free monomer and multimer peptides in aqueous buffer were present as a mixture of unordered and beta-sheet conformation, while binding them to the fusion partner the proportion of ordered secondary structures increased in parallel with the number of antigenic epitopes. The most prominent changes in the conformational state of the monomers in the fusion form were the increase of alpha-helical and beta-sheet and the decrease of unordered conformation, while in the case of oligomeric peptides the adoption of a helical conformation was accompanied by the decrease of beta-sheet structure. An outstanding alpha-helix content (46%) was detected in the case of the trimeric BP1 in its recombinant fusion form.
Laczkó
I
I
Institute of Biophysics, Biological Research Center, HAS, Szeged, Hungary. laczko@nucleus.szbk.u-szeged.hu
Vass
E
E
Tóth
G K
GK
Marczinovits
I
I
Kiss
M
M
Husz
S
S
Molnár
J
J
D001324:Autoantigens / Q000737:chemistry* / Q000276:immunology; D002352:Carrier Proteins; D003094:Collagen / Q000737:chemistry* / Q000276:immunology; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000175:diagnosis* / Q000276:immunology; D011487:Protein Conformation; D000097805:Collagen Type XVII
D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D005982:Glutathione Transferase; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII; C486268:DST protein, human