Cloning and sequencing of mammalian glutathione reductase cDNA

Abstract

The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full-length cDNA to the mRNA of the human enzyme is described. An initial cDNA clone designated AGRM-B11 was isolated by plaque-screening of an induced mouse cDNA expression library in the l g t l l vector with a rabbit antibody probe to human glutathione reductase. 2sIodine-labelled whole anti-rabbit immunoglobulin was used as second antibody. EcoRI digestion of the AGRM-B11 clone released a 720-bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques. (a) Escherichia coli Y1089 lysogenized with 1.GRM-B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti-(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting. (b) The GRM-B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM-B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318.The insert of the pGRM-B11 plasmid was used as a bonajide nucleic acid probe to screen mouse and human cDNA libraries prepared in the i g t l l or in the AgtlO vector. The first full-length cDNA clone (AGRH-Mev10) was identified in a human cDNA library based on RNA of human placental cells. Its insert was composed of three EcoRI fragments of 720, 613 and 336 bp. The three fragments were recloned in the Bluescript vector and sequenced. The largest fragment (pGRH-B) is colinear with the mouse sequence cloned in the pGRM-B1 1 plasmid. The fragment of intermediate size (pGRH-CT) comprises the 3' end of the mRNA and the poly(A) tail while the short fragment (pGRH-NT) corresponds to the 5' region of the mRNA. The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.Glutathione reductase, a homodimeric flavoprotein of 2 x 52 kDa, catalyzes the production of glutathione (GSH) from glutathione disulfide (GSSG) at the expense of NADPH :As part of the glutathione redox cycle, the enzyme plays a role in the detoxification of reactive oxygen species [l]. In addition, by adjusting the [GSH]/[GSSG] ratio, it is involved in a number of cellular functions including the activation of dormant cells [2] and the regulation of the cell cycle [3].Most structural information is available for the enzyme from human erythrocytes. This includes the amino acid sequence [4], the three-dimensional geometry at 0.15-nm resolution [5], the binding mode of the substrates [6-81 and the

Molecular characterization and expression of Onchocerca volvulus glutathione reductase

“…The amino acid sequence has 53 % identity with HsGR. This is much higher than the similarity to the putative GR of the free living nematode C. elegans, with only 30 % sequence identity [19,31,32]. As shown in Table 2, 8 out of 19 residues involved in GSSG binding are different in ' CeGR ' compared with the HsGR, whereas the O GR deduced amino acid sequence only shows one conservative replacement within these residues.…”

Cloning, Sequencing, and Regulation of the Glutathione Reductase Gene from the Cyanobacterium Anabaena PCC 7120

“…2 shows a single protein band with an apparent subunit molecular mass of about 50 kDa after the last purification step. Unexpectedly, the enzyme failed to bind to 2Ј,5Ј-ADP Sepharose, commonly used for affinity chromatography purification of the enzyme from other organisms, such as human erythocytes (27), E. coli (18), and pea (28).…”

“…GR cDNA has been obtained from two plants, pea (16) and Arabidopsis thaliana (17), as well as from mouse and human cells (18). However, the regulation of gor in response to oxidative stress has been reported only for E. coli and Salmonella typhimurium (19,20), in which OxyR (a transcriptional activator) regulates the overexpression of nine proteins, including GR.…”

Low-level Nitric Oxide Blunts Oxidant Injury via Up-regulating Glutathione Synthes.

“…Mouse GR cDNA was a kind gift from Prof. Dieter Werner, German Cancer Research Center. 25) Mouse γ-GCS and GAPDH cDNAs were synthesized by RT-PCR (Titan, Boehringer Mannheim, Mannheim) from mouse liver total RNA using oligo DNA primers for γ-GCS (5′-CACAT-CTACCAC-GCAGTCA-3′ and 5′-TTCGCTTTT -CTA-AATCCTGA-3′) and GAPDH (5′-TGAAG-GTCGGTGTGAACGGA-TTTGGC-3′ and 5′-CA-TGTAGGCCATGAGGCCACCAC-3′). cDNA was amplified (35 cycles, 94°C, 1 min; 55°C, 1 min; 72°C, 1 min) and PCR products were sub-cloned into the pGEM-T vector (Promega, Madison, WI, U.S.A.) for amplification.…”