Characterization of cysteine and homocysteine bound to human serum transthyretin

Abstract

Background Cysteine-bound transthyretin (TTR) in serum is believed to be associated with deposition of amyloid in familial amyloidotic polyneuropathy (FAP). We examined the binding of cysteine and homocysteine with TTR, and the effect of such binding on TTR structure. Methods The concentration of serum cysteine and homocysteine was measured using HPLC and fluorescence detection. TTR was analyzed using isoelectric focusing (IEF), SDS-polyacrylamide gel electrophoresis (PAGE) and mass spectrometry. Results Incubation of serum at 4 °C resulted in an approximate doubling of the amount of both cysteine and homocysteine bound to TTR. Incubation of serum TTR with cysteine–HCl at 37 °C markedly altered the IEF and SDS-PAGE profiles of TTR. The main ion peaks of TTR were observed at m/ z 13,761 and 13,881, and assigned as free TTR and TTR + cysteine respectively. Incubation of purified TTR with dithiothreitol followed by cysteine–HCl resulted in loss of the latter peak and an increase in the m/ z 13,761 peak, while incubation with cysteine–HCl alone did not cause such a change. Conclusion Cysteine- and homocysteine-bound TTR was more easily denatured by cysteine–HCl than free TTR. Binding of cysteine and homocysteine may alter the structure and characteristics of serum TTR, and may facilitate TTR denaturation and deposition.