Analysis of modified structures of transthyretin, elucidation of disease etiology of amyloidosis and application to clinical laboratory tests

在转甲状腺素（TTR）分析过程中，我们发现了独特的TTR修饰形式；它们是s -磺基TTR和在10号位置用甘氨酸取代半胱氨酸的TTR。为了明确这些独特衍生物的体外生成过程，我们在pH 8.3孵育后，分析了市售TTR和与TTR含半胱氨酸部分相同序列的合成肽，即SKCPLMVK。采用LC/ESI-MS分析多肽分子量和氨基酸序列的变化。这两个实验都显示了不同的衍生物，这些衍生物显示了半胱氨酸残基被甘氨酸、去氢丙氨酸、s -硫代半胱氨酸和s -硫代半胱氨酸残基取代，并通过分子质量和碰撞诱导解离光谱证实了这一点。TTR衍生物的存在表明，转化从二硫键的β-消除到脱氢丙氨酸和s -硫半胱氨酸开始。用盐酸水解TTR和多肽，进行氨基酸分析。我们未能证实甘氨酸的增加和新氨基酸如硫代氨酸的产生。市售TTR和血清TTR变性和降低。采用SDS-PAGE和MALDI-TOFMS进行分析。SDS-PAGE均显示单体、二聚体、60kDa、45kDa、28kDa和20kDa聚合物对应的条带。此外，血清TTR含量为55kDa。SDS-PAGE法纯化的TTR和血清TTR中聚合物含量均大于10%。然而，通过MALDI-TOFMS，未观察到聚合物成分。MALDI-TOFMS的溶剂为强酸，但不含SDS。TTR可能在变性试剂（如SDS）中形成非共价聚合物。需要进一步的研究来阐明脱氢丙氨酸生成、非共价聚合和TTR分子淀粉样纤维形成之间的可能关系。

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.