Cloning and expression of a cDNA for human thioredoxin.
Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity. We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes). Screening of this library with synthetic oligonucleotide probes, constructed from the NH2-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin. The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine). This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34). These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin. Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates. It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase. Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.
Wollman
E E
EE
Fédération Centre National de la Recherche Scientifique, UA 1156/Institut National de la Santé et de la Recherche Médicale/Institut Gustave Roussy, Villejuif, France.
d'Auriol
L
L
Rimsky
L
L
Shaw
A
A
Jacquot
J P
JP
Wingfield
P
P
Graber
P
P
Dessarps
F
F
Robin
P
P
Galibert
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D007075:Immunoglobulin M; D008291:Malate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics*
D001426:Bacterial Proteins; D007075:Immunoglobulin M; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D004247:DNA; D008291:Malate Dehydrogenase; D004229:Dithiothreitol