High-avidity binding drives nucleation of amyloidogenic transthyretin monomer
Li Gao, Xinfang Xie, Pan Liu, Jing Jin
Li Gao, Xinfang Xie, Pan Liu, Jing Jin
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Research Article Aging Cardiology

High-avidity binding drives nucleation of amyloidogenic transthyretin monomer

Abstract

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursors. Transthyretin (TTR) naturally folds into a stable tetramer, whereas conditions and mutations that foster aberrant monomer formations facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the early assembly of oligomers by WT TTR compared with its V30M and V122I variants. We monitored time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20–40 TTR subunits via engineered biotin-streptavidin (SA) interactions. As expected, these multimer seeds rapidly nucleated TTR monomers into larger complexes, while having less effect on dimers and tetramers. In vivo, SA-induced multimers formed TTR-like deposits in the heart and the kidney following i.v. injection in mice. While all 3 variants prominently deposited glomerulus in the kidney, only V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized in the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR engaging in high-avidity interactions with tissue amyloids. Our multimeric induction approach provides a model for studying the initiation of TTR deposition in the heart.

Authors

Li Gao, Xinfang Xie, Pan Liu, Jing Jin

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Figure 1

Biotin-TTR fusion and SA-induction model.

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Biotin-TTR fusion and SA-induction model. (A) Crystal structure of tetra...
(A) Crystal structure of tetrameric TTR (PDB 5H0V) (58). The 2 sets of antiparallel β-strands that form the β-sheets of DAGH and CBEF were marked in 1 of the subunits (upper-right). Biotin moieties (represented by spheres) were added to the N-termini of all TTR subunits. (B) Schematics of site-specific biotinylation by BirA and subsequent multimeric induction by SA. Full-length TTR variants (asterisks) of WT, V30M, and V122I were each fused with an N-terminus AviTag, which was biotinylated. In the presence of SA (lower left: follow arrow), 4 biotin-TTR monomers formed SA-TTR tetramers. In the absence of SA (right panel: follow arrow), soluble TTR formed a mixture of monomer (M), dimer (D), and tetramer (T), in which the broken circle represents the natural tetrameric fold of TTR. Following SA induction of these mixed TTR forms (Bottom right: follow arrow), larger TTR complexes were formed, jointed by monomer, dimer, and tetrameric TTRs (in box). (C) WT, V122I, and V30M TTRs in the presence or absence of SA were resolved by reducing SDS PAGE, and subsequently stained with Coomassie blue. Without induction by SA, all variants existed predominantly as SDS-resistant dimers with the presence of less abundant monomers. Following SA induction, TTR formed larger complexes of greater than 600 kDa. (D) Biotinylated TTRWT in 1M urea pH 7.6 solution formed 3 distinct complexes of M, D, and T as revealed by SEC (left panel). Following induction by SA (right panel), TTR in multimeric complexes with SA gained molecular size (the red line for after induction compared with the solid black line for before induction; SA alone: dotted line). The results of V30M and V122I TTRs with SA induction are in Supplemental Figure 1.