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Deduced amino acid sequence, gene structure and chromosomal location of a novel human class Mu glutathione S-transferase, GSTM4.

ABSTRACT: The Mu-Class glutathione S-transferases (GSTs) are subject to marked inter-individual variation in man, owing to the fact that 40-50% of the population fail to express M1 subunits. Mu-Class GST from two lymphoblastoid cell lines (expressing M1 subunits and the other 'nulled' for M1) have been studied. Both cell lines were found to express a Mu-Class GST that has not been described previously. The cDNA encoding this novel transferase, designated 'GSTM4' has been isolated and the enzyme shown to be comprised of 218 amino acids (including the initiator methionine residue) with an M(r) of approx. 25.5 kDa. Molecular cloning demonstrated that the lymphoblastoid cell line which expressed GSTM1 possessed the b allelic variant (i.e. that with an asparagine residue at position 173). The genes for GSTM4 and GSTM1b have been cloned and found to contain seven introns and eight exons. The coding region of the GSTM4 gene, including the seven introns, encompasses 5.0 kb, whereas the same region of GSTM1b is 5.5 kb; the difference in the size of the two genes is due to the length of intron 7. DNA sequencing allowed a GSTM4-gene-specific oligo-primer to be designed which has been utilized in a PCR-based assay to determine that the GSTM4 gene is located on chromosome 1.

SUBMITTER: Zhong S

PROVIDER: S-EPMC1132478 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Project description:We have isolated and characterized a cDNA and partial gene encoding a murine subfamily 1 Theta class glutathione transferase (GST). The cDNA derived from mouse GSTT1 has an open reading frame of 720 bp encoding a peptide of 240 amino acids with a calculated molecular mass of 27356 Da. The encoded protein shares only 51% deduced amino acid sequence identity with mouse GSTT2, but greater than 80% deduced amino acid sequence identity with rat GSTT1 and human GSTT1. Mouse GSTT1-1 was expressed in Escherichia coli as an N-terminal 6x histidine-tagged protein and purified using immobilized-metal affinity chromatography on nickel-agarose. The yield of the purified recombinant protein from E. coli cultures was approx. 14 mg/l. Recombinant mouse GSTT1-1 was catalytically active towards 1, 2-epoxy-3-(p-nitrophenoxy)propane, 4-nitrobenzyl chloride and dichloromethane. Low activity towards 1-menaphthyl sulphate and 1-chloro-2,4-dinitrobenzene was detected, whereas mouse GSTT1-1 was inactive towards ethacrynic acid. Recombinant mouse GSTT1-1 exhibited glutathione peroxidase activity towards cumene hydroperoxide and t-butyl hydroperoxide, but was inactive towards a range of secondary lipid-peroxidation products, such as the trans-alk-2-enals and trans,trans-alka-2,4-dienals. Mouse GSTT1 mRNA is most abundant in mouse liver and kidney, with some expression in intestinal mucosa. Mouse GSTT1 mRNA is induced in liver by phenobarbital, but not by butylated hydroxyanisole, beta-napthoflavone or isosafrole. The structure of mouse GSTT1 is conserved with that of the subfamily 2 Theta class GST genes mouse GSTT2 and rat GSTT2, comprising five exons interrupted by four introns. The mouse GSTT1 gene was found, by in situ hybridization, to be clustered with mouse GSTT2 on chromosome 10 at bands B5-C1. This region is syntenic with the location of the human Theta class GSTs clustered on chromosome 22q11.2. Similarity searches of a mouse-expressed sequence tag database suggest that there may be two additional members of the Theta class that share 70% and 88% protein sequence identity with mouse GSTT1, but less than 55% sequence identity with mouse GSTT2.

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Deduced amino acid sequence, gene structure and chromosomal location of a novel human class Mu glutathione S-transferase, GSTM4.

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