Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA).
Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A-type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N-terminal part of the polypeptide. Co-expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione-S-transferase-hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N-acetyl methionine sulfoxide.
Kuschel
L
L
Arbeitsgruppe Molekulare und zelluläre Biophysik, Klinikum der Friedrich-Schiller-Universität Jena, Germany.
Hansel
A
A
Schönherr
R
R
Weissbach
H
H
Brot
N
N
Hoshi
T
T
Heinemann
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line / Q000201:enzymology; D002531:Cerebellum / Q000201:enzymology; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005260:Female; D005333:Fetus / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007668:Kidney / Q000201:enzymology / Q000254:growth & development; D007938:Leukemia / Q000201:enzymology; D008099:Liver / Q000196:embryology / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008223:Lymphoma / Q000201:enzymology; D051150:Methionine Sulfoxide Reductases; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009865:Oocytes / Q000201:enzymology; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase