S-sulfonation of transthyretin is an important trigger step in the formation of transthyretin-related amyloid fibril.
Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluorescent microscopic properties of TTR incubated with and without cysteine-S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37 degrees. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine-S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent KD of 7.75x10(-6) M. On the other hand, the Bmax of cysteinyl TTR was 1.38, and its KD was 3.52x10(-6) M while the Bmax of reduced TTR was 0.86, and its KD was 2.86x10(-6) M. Moreover, we detected positive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluorescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, this study showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril.
Nakanishi
Toyofumi
T
Department of Clinical and Laboratory Medicine, 2-7 Daigaku-cho, Takatsuki-city, 569-8668, Osaka, Japan. nakanisi@poh.osaka-med.ac.jp
Yoshioka
Masanori
M
Moriuchi
Kazuyoshi
K
Yamamoto
Daisuke
D
Tsuji
Motomu
M
Takubo
Takayuki
T
D000682:Amyloid / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009206:Myocardium / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D011228:Prealbumin / Q000737:chemistry*; D013450:Sulfones / Q000737:chemistry*; D013997:Time Factors
D000682:Amyloid; D011228:Prealbumin; D013450:Sulfones; D003545:Cysteine; D010100:Oxygen