NAKANISHI Toyofumi Osaka Medical College, Faculty of Medicine, Assistant professor, 医学部, 助教授 (10247843)
MIYAZAKI Ayako Osaka Medical College, Faculty of Medicine, Lecturer, 医学部, 講師 (20298772)
|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2005 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 2004 : ¥2,200,000 (Direct Cost : ¥2,200,000)
During the process of transthyretin (TTR) analysis, we found unique modified forms of TTR ; those are S-sulfo-TTR and TTR with the substitution of cysteine at position 10 by glycine. To clarify the generation process of these unique derivatives in vitro, we analyzed commercially purchased TTR and synthetic peptide with the same sequence as the cysteine-containing part of TTR, i.e., SKCPLMVK, after incubation at pH 8.3. Changes in the molecular weights and amino acid sequences of peptides were analyzed by LC/ESI-MS. Both experiments showed that various derivatives were generated, which revealed substitutions of the cysteine residue to glycine, dehydroalanine, S-thiocysteine, and S-sulfocysteine residues, which were confirmed by molecular mass and collision induced dissociation spectra. The existence of the TTR derivatives suggests that transformation starts from a β-elimination of a disulfide linkage to dehydroalanine and S-thiocysteine. The TTR and peptides were hydrolysed with HCl and analysed by amino acid analysis. We failed to confirm the increase of glycine and generation of new amino acids such as lanthionine.
Commercially purchased TTR and serum TTR were denatured and reduced. These were analyzed by SDS-PAGE and MALDI-TOFMS. Both showed bands corresponding to a monomer, dimer, 60kDa, 45kDa, 28kDa, and 20kDa polymers by SDS-PAGE. In addition to these, serum TTR showed a 55kDa component. The content of polymers was more than 10% in purified TTR and serum TTR by SDS-PAGE. However, by MALDI-TOFMS, no polymer components were observed. The solvent for MALDI-TOFMS was a strong acid but did it not contain SDS. TTR might form non-covalent polymers in reagents for denaturation such as SDS. Further studies are necessary to elucidate the possible relationships among dehydroalanine generation, non-covalent polymerization, and amyloido-fiber formation of TTR molecules.