Cloning, expression, and characterization of a class-mu glutathione transferase from human muscle, the product of the GST4 locus.

Abstract

A class-mu glutathione transferase cDNA clone, GTHMUS, was isolated from human myoblasts and its sequence was determined. The sequence predicts a protein of molecular weight 25,599 whose 24 amino-terminal residues are identical to those of the class-mu isoenzyme expressed from the GST4 locus. The GTHMUS cDNA shares 93.7% nucleotide sequence identity with a human liver cDNA clone, GTH411, that is encoded at the GSTJ locus. Comparison of the liver and muscle cDNA sequences shows two regions of remarkable sequence conservation: a 140-nucleotide region in the 5' coding portion of the molecule that has a single silent nucleotide substitution, and a 550-nucleotide region, including the entire 3' noncoding region, that has only three nucleotide substitutions or deletions. This sequence conservation suggests that gene conversion has occurred between the human GSTI and GST4 glutathione transferase gene loci. The human muscle and liver glutathione transferase clones GTHMUS and GTH411 have been expressed in Escherichia coli. The kinetic mechanism of the muscle enzyme was examined in product inhibition studies. The inhibition patterns are best modeled by a steadystate ordered bi-bi reaction mechanism. Glutathione is the first substrate bound and chloride ion is the first product released. Chloride ion inhibits the muscle enzyme.The glutathione transferases (RX: glutathione R-transferase, EC 2.5.1.18) are a family of proteins that detoxify chemical carcinogens, either by conjugating glutathione to reactive electrophilic sites or by direct binding (1,2). The soluble glutathione transferase enzymes have been grouped into three classes according to protein sequence similarity and antibody crossreactivity-alpha, mu, and pi (2). Members of the same class share 70-95% protein sequence identity, while interclass alignments show 25-30% sequence identity. These enzymes are abundant in mammalian liver. Class-alpha and -pi isoenzymes have been characterized from human, mouse, and rat liver. Genetic loci for six human glutathione transferase enzymes have been described: GSTJ-GST6 (3)(4)(5).

Genetic monitoring of human polymorphic cancer susceptibility genes by polymerase chain reaction: application to glutathione transferase mu.

“…The exon and intervening intron regions of the GST1 gene that were amplified by PCR show nearly 100% homology with other functional and psuedo-GST p. loci (13)(14)(15). In our hands, the PCR primer sequences of Comstock et al (8) for exons 4-5 often produced spurious PCR fragments with lengths of approximately 100 bp or approximately 265 bp, which appeared in samples from individuals with no GT p, activity.…”

“…In our hands, the PCR primer sequences of Comstock et al (8) for exons 4-5 often produced spurious PCR fragments with lengths of approximately 100 bp or approximately 265 bp, which appeared in samples from individuals with no GT p, activity. These primers may not adequately discriminate between the GST1 locus and the highly homologous human muscle-specific GST4 locus or the testis-specific GT p. locus, neither of which is polymorphic (13,15). Because of this problem, primers for exons 6 and 7 were designed to detect the GST1 locus specifically.…”

GSTM1 (Glutathione S-transferase M1)

“…The end points of the polymorphic GSTM1 deletion are: the left repeated region 5 kb downstream from the 3?-end of the GSTM2 gene and 5 kb upstream from the beginning of the GSTM1 gene; the right repeated region 5 kb downstream from the 3?-end of the GSTM1 and 10 kb upstream from the 5?-end of the GSTM5 gene (Xu et al, 1998). The cDNAs encoded by GSTM1 and GSTM2 share a remarkable 99% sequence identity (Vorachek et al, 1991). The fact that GSTM1 and GSTM2 are physically linked suggests that the frequent deletion of the GSTM1 locus is caused by unequal crossing-over (Pearson et al, 1993).…”

Review

“…To date, five GSTmu subunits have been identified in different tissues: GSTM1 -1 (two alleles) is expressed in liver and peripheral blood (Seidegard etal., 1987); GSTM2-2 is expressed in muscle (Vorachek et al, 1991); GSTM3-3 (Campbell etal., 1990) and GSTM4-4 are expressed in testis (Ross and Board, 1993) and GSTM5-5 is expressed in the brain (Takahashi ef al., 1993). GSTM1-1 is virtually absent in 50% of the human population.…”