HOMOCYSTEINYLATED TRANSTHYRETIN IN HUMAN PLASMA BY ELECTROSPRAY IONIZATION MS

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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.