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 6

SA-TTR multimer formed renal deposits in mice.

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SA-TTR multimer formed renal deposits in mice. (A) A total of 25 mice (n...
(A) A total of 25 mice (n = 5/group) received 7 i.v. doses of buffer, uninduced TTRWT, or SA-induced TTRWT, TTRV30M, or TTRV122I at 15 mg/kg BW per dose for 7 consecutive days (arrows). The mice were harvested (arrowhead) 3 hours after the last injection on day 7. Kidney, liver, and heart specimens were stained with anti-TTR antibody by IF. (B) IF images of the kidney sections showed no TTR signal from the buffer and only a trace amount of uninduced TTR in some, but not all, glomeruli (top panels). In contrast, all SA-induced TTR variants in their multimeric forms showed prominent TTR signals as puncta of varying sizes exclusively in the glomerulus (arrowheads and circled areas). All glomeruli were stained positive, whereas renal tubules only showed background-level signals with no TTR puncta (bottom panels). (C) Inset of SA-induced V30M image from bottom center panel in the boxed area of A. (Images from induced TTRWT and TTRV122I injections are in Supplemental Figure 5). CD31 marked glomerular capillaries. Cross sections of the capillary loops are pointed by arrowheads (left panel of CD31 staining). Strong SA-TTR deposition was observed with aggregates varying in size and shape (middle panel). Composite image of the glomerulus showed TTR deposits were not in the capillary lumen (arrowheads). Instead, the deposits were located to the glomerulus mesangial areas. (D) The liver of SA-V30M–injected mice showed scattered TTR puncta that also appeared to be outside of vascular lumina (marked with CD31). Scale bar: 30 μm.