HOMOCYSTEINYLATED TRANSTHYRETIN IN HUMAN PLASMA BY ELECTROSPRAY IONIZATION MS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。血浆总同型半胱氨酸水平升高是心血管疾病的重要独立危险因素，也是阿尔茨海默病和S病的新兴危险因素。尽管超过70%的Hcys是与血浆蛋白相连的二硫键，但对载体蛋白的识别以及同型半胱氨酸化对蛋白质功能的影响(S)，人们知之甚少。最近，我们报道了淀粉样变性蛋白转甲状腺蛋白(TTR，前白蛋白)通过Cys10处的二硫键携带Hcys。从人血浆中提纯的~(35)S-L-同型半胱氨酸标记的TTRSDSPAGE分析显示有两条带，分别对应于二聚体(27.5 kDa)和四聚体(55 KDa)。在进行SDS-PAGE分析之前，用b-巯基乙醇处理放射性标记样品，从TTR中去除放射性标记，证实存在二硫键。对人血浆(未经DTT治疗)免疫沉淀物的ESI MS分析提供了明确的证据，证明TTR是人类免疫缺陷病毒的携带者。同型半胱氨酸化的TTR-Cys10-S-S-Hcys(同型半胱氨酸化)在健康人和终末期肾病和同型半胱氨酸尿症患者的血浆中很容易检测到。体外同型半胱氨酸同型半胱氨酸化的体外研究表明，血浆中同型半胱氨酸化的TTR值与L-HCys(0-500M)的添加量成正比。在正常和高同型半胱氨酸血症的血浆中，Hcys的总浓度和同型半胱氨酸化的Ttr之间也发现了类似的相关性。因此，同型半胱氨酸化的TTR似乎是HCys负荷的一个敏感指标。研究结果发表在《J·生物》杂志上。化学。在上一个报告年度内。基于这项工作的诊断测试专利申请正在缓慢地通过该系统。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Elevation in the level of total plasma homocysteine (Hcys) is a strong independent risk factor for cardiovascular disease and an emerging risk factor for Alzheimer¿s disease. Although over 70% of total Hcys is disulfide linked to plasma proteins, little is known about the identity of the carrier proteins the effect(s) of homocysteinylation on protein function. Recently, we reported that the amyloidogenic protein transthyretin (TTR, prealbumin) carries Hcys by disulfide linkage at Cys10. SDS-PAGE analysis of 35S-L-homocysteine radiolabeled TTR purified from human plasma revealed two bands corresponding to dimeric (27.5 kDa) and tetrameric (55 kDa) TTR. Treatment of the radiolabeled sample with b-mercaptoethanol prior to SDS-PAGE analysis removed the radiolabel from TTR, confirming the presence of a disulfide linkage. ESI MS analysis of the TTR immunoprecipitated from human plasma (without DTT treatment) provides unambiguous evidence that TTR is a carrier for Hcys. TTR-Cys10-S-S-Hcys (homocysteinylated) is readily detectable in plasma from healthy individuals and patients suffering from end-stage renal disease and homocystinuria. In vitro studies on dose-dependent homocysteinylation of TTR showed that the level of homocysteinylated TTR in plasma is directly proportional to the amount of L-Hcys (0-500 ¿M) added. A similar correlation was found between total Hcys concentration and homocysteinylated TTR in normal and hyperhomocysteinemic plasma. Thus, homocysteinylated TTR appears to be a sensitive indicator of Hcys burden. The results were published in J. Biol. Chem. during the last reporting year. The patent application for a diagnostic test based on this work is slowly progressing through the system.