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

在转甲状腺素分析过程中，我们发现了独特的转甲状腺素受体修饰形式：S-磺基-甲状腺激素受体和甘氨酸取代10位半胱氨酸的甲状腺激素受体。为了阐明这些独特的衍生物的体外生成过程，我们分析了商业购买的TTR和与TTR半胱氨酸部分序列相同的合成肽，即SKCPLMVK，在pH 8.3下孵育。用LC/ESI-MS分析多肽的相对分子质量和氨基酸序列的变化。这两个实验都表明生成了不同的衍生物，发现半胱氨酸残基取代了甘氨酸、脱氢丙氨酸、S-硫代半胱氨酸和S-磺基半胱氨酸残基，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，脱氢丙氨酸和S-硫代半胱氨酸的转化始于二硫键的β消除。用盐酸对TTR和多肽进行水解，并进行氨基酸分析。我们没有证实甘氨酸的增加和新氨基酸的产生，如羊毛硫氨酸。商业购买的TTR值和血清TTR值被变性和减少。用SDS-PAGE和MALDI-TOFMS对其进行了分析。经SDS-PAGE分析，均显示与单体、二聚体、60 kDa、45 kDa、28 kDa和20 kDa聚合物相对应的条带。此外，血清TTR还显示一个55 kDa的成分。经SDS-PAGE分析，提纯的TTR和血清TTR中聚合物含量均在10%以上。然而，MALDI-TOFMS没有观察到任何聚合物组分。MALDI-TOFMS的溶剂为强酸，但不含十二烷基硫酸钠。在变性试剂如十二烷基硫酸钠中，TTR可能形成非共价聚合物。需要进一步的研究来阐明脱氢丙氨酸的产生、非共价聚合和TTR分子的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.