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Amyloid formation in rat transthyretin: effect of oxidative stress
Takahiro Tajiri1, Yukio Ando, Kanako Hata
1Department of Pharmacology and Therapeutics, Graduate School of Clinical Pharmacy, Kumamoto University, 5-1 Oe-honmachi, 860-0973, Kumamoto, Japan.
Background:
Transgenic mice carrying a human mutant transthyretin (TTR) gene are too small for in vivo experiments. It is necessary to have rat TTR protein and its antibody to overcome this problem.
Methods:
Posttranslational modification of purified TTR was analyzed by means of matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). Production of amyloid fibrils in vitro was confirmed by thioflavin T test and electron microscopy. Amyloidogenicity of rat TTR from rats with or without challenging paraquat was compared in vitro by thioflavin T test.
Results:
MALDI/TOF-MS for rat TTR revealed three major modified forms-sulfate-conjugated, Cys-conjugated and glutathione-conjugated-in addition to the unconjugated (free) form of TTR. Although rat TTR in buffer of pH 7.0 could not make amyloid fibrils, rat TTR at pH 2.0-3.5 significantly formed amyloid fibrils, as confirmed by the thioflavin T test and electron microscopy. TTR purified from rats administered 4 mg/kg of paraquat formed much more amyloid fibrils than that from normal rats at pH 2.0-3.5 and significant amyloid fibrils were confirmed even at pH 7.0.
Conclusions:
Rat TTR may be a valuable experimental tool for examination of the amyloidogenicity of senile systemic amyloidosis (SSA) as well as familial amyloidotic polyneuropathy (FAP) both in vitro and in vivo.