Cloning and expression of a novel mammalian thioredoxin.
We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.
Spyrou
G
G
Department of Biosciences at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden. Giannis.Spyrou@mbb.ki.Se
Enmark
E
E
Miranda-Vizuete
A
A
Gustafsson
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013879:Thioredoxins