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 (bMsr A), With dot blot analyses based on RNA from human tissues, expression of h MsrA was found in all tissues tested, with highest mRNA levels in adult kid ney and cerebellum, followed by liver, heart ventricles, bone marrow and hi ppocampus. In fetal tissue, expression was highest in the liver. No express ion of hmsr A was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation tim e 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-te rminal part of the polypeptide, Go-expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned en zyme 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 [H-3]N-acetyl methionine sulfoxide, (C) 1999 Federation of European Biochemical Societies.