Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins.
We report the isolation of three full-length cDNAs corresponding to the mRNAs of closely related glutathione S-transferase (GST) Pi genes, designated hGSTP1*A, hGSTP1*B, and hGSTP1*C, expressed in normal cells and malignant gliomas. The variant cDNAs result from A --> G and C --> T transitions at nucleotides +313 and +341, respectively. The transitions changed codon 104 from ATC (Ile) in hGSTP1*A to GTC (Val) in hGSTP1*B and hGSTP1*C and changed codon 113 from GCG (Ala) to GTG (Val) in hGSTP1*C. Both amino changes are in the electrophile-binding active site of the GST Pi peptide. Computer modeling of the deduced crystal structures of the encoded peptides showed significant deviations in the interatomic distances of critical electrophile-binding active site amino acids as a consequence of the amino acid changes. The encoded proteins expressed in Escherichia coli and purified by GSH affinity chromatography showed a 3-fold lower Km (CDNB) and a 3-4-fold higher Kcat/Km for the hGSTP1*A encoded protein than the proteins encoded by hGSTP1*B and hGSTP1*C. Analysis of 75 cases showed the relative frequency of hGSTP1*C to be 4-fold higher in malignant gliomas than in normal tissues. These data provide conclusive molecular evidence of allelopolymorphism of the human GST Pi gene locus, resulting in active, functionally different GST Pi proteins, and should facilitate studies of the role of this gene in xenobiotic metabolism, cancer, and other human diseases.
Ali-Osman
F
F
Section of Molecular Therapeutics, Department of Experimental Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Akande
O
O
Antoun
G
G
Mao
J X
JX
Buolamwini
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D014644:Genetic Variation; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase