Fig 1: Correlation of solvent excluded surface area of tetrameric and monomeric TTR with peptide inhibitor segments and the misTTR epitope.The solvent excluded surface area (SES) of monomeric and the tetramer subunit form of TTR was calculated using Chimera molecular visualization program47 using the coordinate file 3KGU. The peptide inhibitors are those developed by Saelices et al.43. Note the correlation of the locations of the misTTR epitope and inhibitor peptides with changes in SES associated with monomerization of TTR.
Fig 2: (A) Proposed model for the inhibition of TTR fibrillogenesis by the misTTR antibody. During the fibril formation process, native tetrameric TTR first dissociates into an altered monomeric intermediate. This altered monomeric intermediate self assembles into stable nuclei, which allows for fibril formation to proceed. Substoichiometric amounts of the misTTR antibody may suppress TTR fibril formation by binding misfolded conformations of the TTR that comprise the critical nuclei, which are likely present at very low concentrations. The misTTR antibody may also act to inhibit TTR fibrillogenesis by binding to the unfolded monomers and/or to the extremities of fibrils, essentially capping the ends of preformed fibrils to prevent fibril growth. (B) Surface representation of native tetrameric TTR (green) with buried misTTR epitope in red. (C) Surface representation of monomeric TTR (green) with exposed epitope in red. Structures were generated using PDB ID 1DVQ in Deep View (Swiss-PBD Viewer 3.7).
Fig 3: Misfolded TTR antibody labels TTR amyloids in vivo.Formalin fixed paraffin embedded (FFPE) cardiac tissue sections from a patient with I84S TTR mutation were labeled using misTTR (dark brown, A), a non-specific isotype control antibody (dark brown, B), or the amyloid specific dyes Thioflavin T or Congo Red (green C, red D, green E).
Fig 4: MisTTR antibody selectively binds monomeric, misfolded conformations of TTR.(A) Indirect ELISA using monomeric TTR (0.01 mg/mL TTR incubated at 25 °C , pH 3, 44 h), guanidine unfolded TTR (TTR in 6 M guanidine , incubated overnight at 25 °C), folded native TTR and BSA. (B) Competition ELISA using guanidine unfolded TTR as bound antigen, with monomeric and tetrameric TTR as competitors, and misTTR as the antibody. IC50 = 63 ± 20 nM for monomeric TTR’s binding affinity for misTTR. (C) Native and SDS-PAGE analysis was performed on wild-type TTR purified from human plasma (purchased from Sigma-Aldrich). For native PAGE analysis, three pairs of sample lanes were run. The first lane consisted of molecular weight markers while the second lane consisted of 2 mg of wild-type TTR. The three pairs of lanes were excised from the gel. The first excised lane pair was stained with Coomassie Blue dye. The second pair was transferred to PDVF membrane and immunoblotted with misTTR antibody. The third pair was similarly transferred to PDVF membrane but immunoblotted with commercial anti-TTR antibody. The same procedure was repeated for an SDS-PAGE counterpart. In the native gels, misTTR did not recognize any species, while anti-TTR recognized putative dimers and higher molecular weight oligomers. In SDS gels misTTR only recognized monomers, while anti-TTR recognized monomers and putative dimers.
Fig 5: Recognition of TTR amyloid fibrils by misTTR antibody.(A) Representative TEM image of amyloid fibrils produced from 1 mg/mL TTR. (B) Competition ELISA with misTTR antibody showing selective binding of TTR amyloid fibrils and antigenic peptide.
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