A Comprehensive Analysis of Selenium-Binding Proteins in the Brain Using Its Reactive Metabolite.

The intracellular metabolism of selenium in the brain currently remains unknown, although the antioxidant activity of this element is widely acknowledged to be important in maintaining brain functions. In this study, a comprehensive method for identifying the selenium-binding proteins using PenSSeSPen as a model of the selenium metabolite, selenotrisulfide (RSSeSR, STS), was applied to a complex cell lysate generated from the rat brain. Most of the selenium from L-penicillamine selenotrisulfide (PenSSeSPen) was captured by the cytosolic protein thiols in the form of STS through the thiol-exchange reaction (R-SH+PenSSeSPen→R-SSeSPen+PenSH). The cytosolic protein species, which reacted with the PenSSeSPen mainly had a molecular mass of less than 20 kDa. A thiol-containing protein at m/z 15155 in the brain cell lysate was identified as the cystatin-12 precursor (CST12) from a rat protein database search and a tryptic fragmentation experiment. CST12 belongs to the cysteine proteinase inhibitors of the cystatin superfamily that are of interest in mechanisms regulating the protein turnover and polypeptide production in the central nervous system and other tissues. Consequently, CST12 is suggested to be one of the cytosolic proteins responsible for the selenium metabolism in the brain.

An effective method for profiling the selenium-binding proteins using its reactive metabolic intermediate.

Currently, the intracellular reduction and/or transport of selenium still remain unknown. Certain reduced forms of selenium species are thought to be reactive with various endogenous molecules, particularly thiol-containing proteins. In this study, a profiling method for identifying the selenium-binding proteins using L-penicillamine selenotrisulfide (PenSSeSPen) as a model of the selenium metabolic intermediate was applied to the cell lysate generated from the rat liver. Several proteins with cysteine thiol were found to be reactive with PenSSeSPen through the thiol-exchange reaction by MALDI TOF-MS analysis. The most distinctive cysteine-containing protein at m/z 14,313 in the liver cell lysate was identified as the liver fatty acid-binding protein based on a rat protein database search and a tryptic fragmentation experiment. This methodology could be used for determining the selenium-binding proteins and/or selenium-interactive species and provide a better understanding of the selenium metabolism and utilization in biological systems.