Cloning and Characterization of a Novel Oxidoreductase KDRF from a Human Bone Marrow-derived Stromal Cell Line KM-102

• Published in: The Journal of biological chemistry Vol. 272; no. 4; pp. 2570 - 2577
• Main Authors: Koishi, Ryuta, Kawashima, Ichiro, Yoshimura, Chigusa, Sugawara, Mie, Serizawa, Nobufusa
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.01.1997; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Base Sequence; Blotting, Northern; Blotting, Western; Bone Marrow - enzymology; Bone Marrow Cells; Cell Line; Cloning, Molecular; DNA, Complementary - chemistry; Glutathione Reductase - chemistry; Glutathione Reductase - genetics; Humans; Molecular Sequence Data; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Stromal Cells - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.4.2570

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 ∼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-derivedreductase-like factor), and KDRF may play a role in scavenging reactive oxygen intermediates, which are possibly toxic to cells, in response to inflammatory stimuli.