Loss of selenium from selenoproteins: Conversion of selenocysteine to dehydroalanine in vitro

Abstract

Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-␤-elimination of selenenic acid during sample processing.

Irreversible Inactivation of Glutathione Peroxidase 1 and Reversible Inactivation of Peroxiredoxin II by H2O2in Red Blood Cells

“…However, DHA is not frequently found in positions corresponding to Cys residues. This difference is likely attributable to the greater strength of the C-S bond (272 kJ=mol) compared with that of the C-Se bond (234 kJ=mol) (25). These multiple sources of DHA are consistent with our observation that direct blot analysis of crude RBC extracts yielded many positive bands (not shown).…”

“…Tryptic peptides prepared from selenoproteins have previously been observed to have lost Sec residues as a result of their conversion to DHA during protein purification or digestion (25,30). To determine whether GPx1 might lose selenium under oxidative stress, we incubated human GPx1 with 1 mM H 2 O 2 for 1 h at 378C.…”

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

“…An in depth investigation of the Se-incorporation in protein by 2D GE -LA-ICPMS, demonstrated that in this L. reuteri strain all the Se present in the proteins is incorporated as selenocysteine (SeCys), whereas methionine/selenomethionine substitution, which is typical for other microorganisms, such as yeast (25), did not occur. Note also that, in contrast to literature (26), the method developed here allows the preservation of the SeCys moiety during analysis Indeed, no dehydroalanine residues were detected.…”

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

“…Ma et al (30) observed the conversion of one or more selenocysteine residues of rat selenoprotein P to dehydroalanine, probably through the mechanism of oxidation of selenocysteine followed by a syn-␤-elimination of selenenic acid. The calculated mass of a PrxU variant containing dehydroalanine instead of selenocysteine would be 23,555 Da.…”