Cloning and characterization of a novel oxidoreductase KDRF from a human bone marrow-derived stromal cell line KM-102.
A cDNA clone coding for a novel oxidoreductase was cloned from a human bone marrow-derived stromal cell line KM-102. We screened a cDNA library constructed from the mRNA of KM-102 cells stimulated with phorbol 12-myristate 13-acetate and calcium ionophore A23187 using a 32P-labeled 15-mer synthetic oligonucleotide (5'-TAAATAAATAAATAA-3') probe. This probe was designed as a complementary sequence to the three reiterated AUUUA sequences, which are contained in the 3'-untranslated regions of cytokine and some proto-oncogene mRNAs and correlate with rapid mRNA turnover. Then, we obtained one cDNA clone, and further sequence analysis revealed that it coded for a new protein exhibiting 30 to approximately 40% homology with glutathione reductase. By fusion protein analysis, this protein showed reducing activities on 2, 6-dichlorophenol-indophenol and 5,5'-dithio-bis(2-nitrobenzoic acid) but only a weak reducing activity on oxidized glutathione. Although it lacked a stretch of hydrophobic amino acids in its N terminus, it was secreted by monkey kidney-derived COS-1 cells when we introduced the expression plasmid into them and also secreted by a human lung carcinoma cell line A549. Northern blot analysis revealed that the mRNA turnover of this protein was regulated by inflammatory stimuli in KM-102 cells. These results show that this protein may have scavenging enzyme properties and has its mRNA expression regulated in a similar fashion to cytokine genes or proto-oncogenes. Thus, we named it KDRF (KM-102-derived reductase-like factor), and KDRF may play a role in scavenging reactive oxygen intermediates, which are possibly toxic to cells, in response to inflammatory stimuli.
Koishi
R
R
Biomedical Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan.
Kawashima
I
I
Yoshimura
C
C
Sugawara
M
M
Serizawa
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001853:Bone Marrow / Q000201:enzymology*; D001854:Bone Marrow Cells; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D017154:Stromal Cells / Q000201:enzymology
D018076:DNA, Complementary; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; C103914:KDRF oxidoreductase; D005980:Glutathione Reductase