Detailed structural analysis of amyloidogenic wild-type transthyretin using a novel purification strategy and mass...

Wild-type transthyretin (TTR), normally a soluble plasma-circulating protein, can be amyloidogenic, i.e., form tissue-deposited fibrillar material in the extracellular matrix of various organs throughout the body. Senile systemic amyloidosis (SSA) is one such pathology and features TTR-containing amyloid deposits that are found primarily in the heart. The cause for this transition from soluble to insoluble protein in SSA is yet to be determined as specific structural features that might favor TTR fibrillogenesis have not yet been identified. The precise characterization of ex vivo fibril deposits might provide insight, but structural analyses of TTR from amyloid deposits have been hindered thus far by the lack of purification strategies that overcome the insolubility of the tissue-derived protein without degrading it. Consequently, the true biochemical nature of deposited TTR remains in question. In this study, we provide detailed analyses of both the soluble (serum) and deposited (tissue) forms of TTR from cases of SSA. In the serum, a distribution of mixed disulfides, specifically S-sulfonated and S-cysteinylated forms of TTR, as well as the unmodified protein were identified. The relative levels of the three TTR species in the SSA group were comparable to amounts present in sera from age-matched control groups. For characterization of the amyloid deposited TTR, we investigated cardiac tissue samples obtained from three separate cases of SSA. We report a novel chromatographic purification strategy performed under nonreducing conditions (to maintain cysteine disulfide status) and the use of this procedure in conjunction with detailed mass spectrometric analysis of TTR from the amyloid deposits. A series of C-terminal TTR fragments with N-termini ranging from amino acids 46 to 55 were identified. We also determined that the deposits in all samples contained Cys10 disulfide-linkedhomodimers composed of full-length TTR monomers. This last finding suggests an important role for Cys10 conjugation in the transition from soluble TTR to the pathological amyloid fibril.