Amyloid formation in rat transthyretin: effect of oxidative stress.
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.
Department of Pharmacology and Therapeutics, Graduate School of Clinical Pharmacy, Kumamoto University, 5-1 Oe-honmachi, 860-0973, Kumamoto, Japan.
Takahiro
T
Tajiri
Yukio
Y
Ando
Kanako
K
Hata
Kaeko
K
Kamide
Motonori
M
Hashimoto
Masaaki
M
Nakamura
Hisayasu
H
Terazaki
Taro
T
Yamashita
Hirofumi
H
Kai
Katsuki
K
Haraoka
Akira
A
Imasato
Kazuo
K
Takechi
Kazuko
K
Nakagawa
Hiroaki
H
Okabe
Takashi
T
Ishizaki
D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D052160:Benzothiazoles; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008297:Male; D008854:Microscopy, Electron; D018384:Oxidative Stress; D010269:Paraquat; D011228:Prealbumin / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013844:Thiazoles
D000682:Amyloid; D052160:Benzothiazoles; D011228:Prealbumin; D013844:Thiazoles; C009462:thioflavin T; D010269:Paraquat