Cloning and characterization of a novel oxidoreductase KDRF from a human bone marrow-derived stromal cell line KM-102.
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Koishi R, Kawashima I, Yoshimura C, Sugawara M, Serizawa N
Cloning and characterization of a novel oxidoreductase KDRF from a human bone marrow-derived stromal cell line KM-102.
J Biol Chem. 1997 Jan 24;272(4):2570-7.
- PubMed ID
- 8999974 [ View in PubMed]
- Abstract
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.