In Vitro and in Vivo Interactions of Homocysteine with Human Plasma Transthyretin

Abstract

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimer's disease. Greater than 70% of the homocysteine in plasma is disulfide-bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging. The amyloidogenic protein transthyretin (prealbumin), as we now report, undergoes homocysteinylation at its single cysteine residue (Cys 10 ) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35 S-L-homocysteine followed by SDS-PAGE and PhosphorImaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with ␤-mercaptoethanol prior to SDS-PAGE removed the disulfide-bound homocysteine. Transthyretin-Cys 10 -S-S-homocysteine was then identified in vivo in plasma from normal donors, patients with end-stage renal disease, and homocystinurics by immunoprecipitation and high performance liquid chromatography/electrospray mass spectrometry. The ratios of transthyretin-Cys 10 -S-S-homocysteine and transthyretin-Cys 10 -S-S-sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations, whereas the ratio of transthyretin-Cys 10 -S-S-cysteine to that of unmodified transthyretin decreased. The hyperhomocysteinemic burden is thus reflected in the plasma levels of transthyretin-Cys 10

Factors Affecting S-Homocysteinylation of LDL Apoprotein B

“…Although increased protein S-thiolation is considered an in vivo marker of oxidative injury caused by noxious agents (21,22 ), the importance of this index in clinical studies has not been well documented. Thiols in plasma are mainly linked to albumin, but interactions between Hcy and ceruloplasmin, fibrin, and transthyretin have also been reported (23)(24)(25). These Hcy-mediated posttranslational modifications may have important functional consequences.…”

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

“…Due to the very high stability of the native wild-type TTR tetramer, such analyses became possible only with the recent development of fluorescence optics for the ana- lytical ultracentrifuge (30), which are several orders of magnitude more sensitive than the existing commercial optical systems. For our studies, rTTR was produced and modified at Cys 10 by S-sulfonation and S-cysteinylation to generate the two most predominant adducts in human serum (17)(18)(19)(20). These species, as well as unmodified rTTR, were covalently bound to the amine-reactive fluorescein derivative, FITC.…”

“…It is not immediately obvious how structural instability could be conferred to wild-type TTR, but post-translational modifications are a possibility. In human serum, TTR is extensively modified at its lone cysteine residue (Cys 10 ) through mixed disulfide bonds with a number of different compounds (17)(18)(19)(20). Of these, adducts of sulfonate (S-sulfonation) and cysteine (S-cysteinylation) are generally the most common.…”

Homocysteine levels are associated with MTHFR A1298C polymorphism in Indian population

“…It is believed that elevated levels of homocysteine might result in the accumulation of reactive oxygen species (Starkebaum and Harlan 1986) which has been implicated as a major factor in the pathogenesis of several diseases. It has also been proposed that homocysteine can potentially disrupt critical protein-disulfide bonds, thereby altering the structure and/or function of proteins (Sengupta et al 2001;Majors et al 2002;Lim et al 2003). Furthermore, an increase in the concentration of homocysteine leads to elevated levels of Sadenosyl homocysteine-an inhibitor for many methyl transferases-resulting in altered methylation of genes that subsequently leads to modulation of gene expression (Ping et al 2000).…”