In vitro and in vivo interactions of homocysteine with human plasma transthyretin.
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 (Cys10) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35S-L-homocysteine followed by SDS-PAGE and PhosphorImaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with beta-mercaptoethanol prior to SDS-PAGE removed the disulfide-bound homocysteine. Transthyretin-Cys10-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-Cys10-S-S-homocysteine and transthyretin-Cys10-S-S-sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations, whereas the ratio of transthyretin-Cys10-S-S-cysteine to that of unmodified transthyretin decreased. The hyperhomocysteinemic burden is thus reflected in the plasma levels of transthyretin-Cys10-S-S-homocysteine, which in turn may contribute to the pathological consequences of amyloid disease.
D000682:Amyloid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006710:Homocysteine / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D013058:Mass Spectrometry; D008623:Mercaptoethanol / Q000737:chemistry; D008956:Models, Chemical; D011228:Prealbumin / Q000737:chemistry* / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000737:chemistry
D000682:Amyloid; D004220:Disulfides; D011228:Prealbumin; D001613:beta 2-Microglobulin; D006710:Homocysteine; D008623:Mercaptoethanol; D003545:Cysteine