Analysis of methionine/selenomethionine oxidation and methionine sulfoxide reductase function using methionine-rich proteins and antibodies against their oxidized forms.
Methionine (Met) residues are present in most proteins. However, this sulfur-containing amino acid is highly susceptible to oxidation. In cells, the resulting Met sulfoxides are reduced back to Met by stereospecific reductases MsrA and MsrB. Reversible Met oxidation occurs even in the absence of stress, is elevated during aging and disease, but is notoriously difficult to monitor. In this work, we computationally identified natural Met-rich proteins (MRPs) and characterized three such proteins containing 21-33% Met residues. Oxidation of multiple Met residues in MRPs with H(2)O(2) and reduction of Met sulfoxides with MsrA/MsrB dramatically influenced the mobility of these proteins on polyacrylamide gels and could be monitored by simple SDS-PAGE. We further prepared antibodies enriched for reduced and Met sulfoxide forms of these proteins and used them to monitor Met oxidation and reduction by immunoblot assays. We describe applications of these reagents for the analysis of MsrA and MsrB functions, as well as the development of the assay for high-throughput analysis of their activities. We also show that all Met sulfoxide residues in an MRP can be reduced by MsrA and MsrB. Furthermore, we prepared a selenomethionine form of an MRP and found that selenomethionine selenoxide residues can be efficiently reduced nonenzymatically by glutathione and other thiol compounds. Selenomethionine selenoxide residues were not recognized by antibodies specific for the Met sulfoxide form of an MRP. These findings, reagents, assays, and approaches should facilitate research and applications in the area of Met sulfoxide reduction, oxidative stress, and aging.
Le
Dung Tien
DT
Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, USA.
Liang
Xinwen
X
Fomenko
Dmitri E
DE
Raza
Ashraf S
AS
Chong
Chom-Kyu
CK
Carlson
Bradley A
BA
Hatfield
Dolph L
DL
Gladyshev
Vadim N
VN
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D000079923:Copper Transporter 1; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism*; D051150:Methionine Sulfoxide Reductases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*
D000906:Antibodies; C490078:CTR1 protein, S cerevisiae; D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D008840:Microfilament Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D012645:Selenomethionine; D008715:Methionine; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C520510:Msrb2 protein, mouse; C027219:methionine sulfoxide reductase; C013111:methionine sulfoxide