Molecular Cloning, Characterization, and Expression in Escherichia coli of Full-length cDNAs of Three Human Glutathione S-Transferase Pi Gene Variants

doi:10.1074/jbc.272.15.10004

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*

From the ^‡ Section of Molecular Therapeutics, Department of Experimental Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and the
^¶ Department of Medicinal Chemistry and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677

^§ To whom correspondence should be addressed. Tel.: 713-0792-3495; Fax: 713-794-5514.

Abstract

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 K_m (CDNB) and a 3-4-fold higher K_cat/K_m 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.

Footnotes

↵* This work was supported by Grants CA55835 and CA55261 from NCI, National Institutes of Health and by a research grant award from the Kleberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U30897[GenBank] and U62589[GenBank].
↵¹ The abbreviations used are:

GST

glutathione S-transferase

GSH

glutathione

CDNB

1-chloro-2,4-dinitrobenzene

PCR

polymerase chain reaction

H-site

electrophile-binding active site

bp

base pair(s)

RT

reverse transcription

PAGE

polyacrylamide gel electrophoresis.
↵³ F. Ali-Osman and L. Pleasants, manuscript in preparation.
↵²F. Ali-Osman, J. M. Bruner, T. Cutluk, K. Hess, manuscript in preparation.
- Received June 20, 1996.
- Revision received December 5, 1996.