Search for intermediate structures in transthyretin fibrillogenesis: soluble tetrameric Tyr78Phe TTR expresses a specific epitope present only in amyloid fibrils.
Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of TTR into an insoluble beta-sheet. The TTR tetramer is thought to dissociate into monomeric intermediates and subsequently polymerise into the pathogenic amyloid form. The biochemical mechanism behind this transformation is unknown. We characterised intermediate TTR structures in the in vitro amyloidogenesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the TTR tetrameric fold, based on the known crystallographic structure of a Leu55Pro transthyretin variant. We generated a soluble tetrameric form of TTR that is recognised by a monoclonal antibody, previously reported to react only with highly amyloidogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M(-1). We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway.
Redondo
C
C
Amyloid Unit, Institute for Molecular and Cell Biology, Portugal.
Damas
A M
AM
Olofsson
A
A
Lundgren
E
E
Saraiva
M J
MJ
D019943:Amino Acid Substitution / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006860:Hydrogen Bonding; D007106:Immune Sera / Q000276:immunology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D058225:Plaque, Amyloid / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012995:Solubility; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007106:Immune Sera; D011228:Prealbumin