Elsevier

Gene

Volume 240, Issue 1, 15 November 1999, Pages 149-155
Gene

Genomic sequence of human glyoxalase-I: analysis of promoter activity and its regulation

https://doi.org/10.1016/S0378-1119(99)00420-5Get rights and content

Abstract

Glyoxalase-I is a glutathione-binding protein involved in the detoxification of methylglyoxal, a by-product of glycolysis. Aberrations in the expression of human glyoxalase in cancer and diabetes have been reported. To gain a better understanding of the glyoxalase-I regulation under normal physiological conditions and in disease processes, we have cloned 12 kb of genomic sequence, comprising five exons, separated by four introns. A fragment comprising 982 bp of 5′ flanking region was used in the pSEAP reporter system to identify the minimal promoter and to locate any cis-acting functional elements. This region contained a minimal promoter between −20 and −160 bp. Cells transfected with a construct containing the 5′ flanking sequence exhibited a 45-fold higher activity over vector transfected cells. A twofold reproducible increase in reporter activity was seen with insulin and ZnCl2 treatments, indicating a functionally operative insulin response element (IRE) and metal response element (MRE). Knowledge regarding the regulation of glyoxalase-I may provide insights into the importance of this enzyme in human diseases.

Introduction

Glyoxalase-I is an integral component of pathways leading to the detoxification of methylglyoxal and other α-oxoaldehydes [for a review, see Thornalley (1990)]. This ubiquitous enzyme catalyzes the conversion of the methylglyoxal-glutathione conjugate to S-D lactoylglutathione, which in turn is hydrolyzed by glyoxalase-II into D-lactate and glutathione (GSH). This cytosolic enzyme system is among the earliest expressed during embryogenesis and development and persists through maturation, adult life and senescence (McLellan and Thornalley, 1989). The widespread distribution of this enzyme system in prokaryotes and eukaryotes suggests a fundamental, evolutionarily conserved biological function in cells. The main physiological substrates for the glyoxalase system are methylglyoxal formed from the Embden–Meyerhof pathway and glyoxal formed from lipid peroxidation and glycation reactions (Thornalley, 1990, Thornalley, 1998). Methylglyoxal can react with arginine and lysine residues in proteins (Selwood and Thornalley, 1993) and, at high concentrations, inhibits glycolytic enzymes. Toxicity of methylglyoxal can eventually lead to growth inhibition (Ayoub et al., 1993, Halder et al., 1992, Leoncini et al., 1989). When mice were given 50–400 mg/kg doses of methylglyoxal, a dose-dependent decrease in liver detoxifying enzymes, superoxide dismutase (SOD), glutathione S-transferases (GST), catalase, glyoxalase-I and glyoxalase-II was observed. Methylglyoxal also enhanced lipid peroxidation and decreased the GSH content in the liver (Choudhary et al., 1997). Methylglyoxal also binds to DNA and RNA, induces cross-links in DNA resulting in enhanced levels of sister-chromatid exchange (Brambilla et al., 1985, Faggin et al., 1985). These observations emphasize the deleterious consequences of chronic exposure to methylglyoxal and the importance of regulating the glyoxalase system.

During the past several years, many investigators have reported abberant expression of the glyoxalase system in diseases including, diabetes mellitus, cancer and malaria. Concentrations of methylglyoxal, S-D lactoyl glutathione and D-lactate were found to be elevated in the blood samples of both insulin-dependent and -independent diabetic patients, compared to normal healthy controls (McLellan and Thornalley, 1992a, McLellan and Thornalley, 1992b, McLellan et al., 1993, Thornalley et al., 1989). Ayoub et al. (1993) have shown elevated levels of glyoxalase-I and decreased levels of glyoxalase-II in several tumor cell lines of urological origin compared to non-malignant counterparts. Another study by Di Ilio et al. (1995) measured glyoxalase-I and glyoxalase-II activities in urogenital tumor and non-tumor tissues and found decreased glyoxalase-I levels in 10 out of 15 kidney tumors compared to corresponding normal kidney tissue. Elevated levels of glyoxalase-I were also reported in human prostate cancer (Davidson et al., 1999). Studies from our laboratory have shown elevations in glyoxalase-I activities in 16 out of 21 colon tumors compared to corresponding normal colon (Ranganathan and Tew, 1993).

When red blood cells were infected with the malarial parasite, Plasmodium falsiparum, D-lactate production by the glyoxalase system increased ∼30-fold compared to uninfected red blood cells (Vander Jagt et al., 1990). The wide-ranging occurrence of abnormal expression of the glyoxalase system in disease processes led to the concept of synthesis of glyoxalase inhibitors as possible therapeutic drugs. Diethyl esters of glyoxalase inhibitors have shown antimalarial activity in infected red blood cells and antiproliferative activity in human leukemia cells (Lo and Thornally, 1992). Recently, the crystal structure of human glyoxalase-I was resolved (Cameron et al., 1997), and involvement of an active site Zn2+ ligand in the catalytic site was shown (Ridderstrom et al., 1998). Currently, little is known about factors pertinent to regulation of the glyoxalase system. To unravel the structural basis of glyoxalase-I regulation, we cloned and sequenced glyoxalase-I genomic DNA and explored the regulatory function of its 5′ flanking region.

Section snippets

Cloning of the human glyoxalase-I gene

Human placental genomic library packaged into lambda fix II vector was purchased from Stratagene (La Jolla, CA) and plated onto 150 mm agar plates at the appropriate titer. After overnight incubation at 37°C, the plates were chilled and overlayed with nitrocellulose membranes to transfer small amounts of DNA from plaques onto membranes, using standard protocols (Sambrook et al., 1989). A 623 bp human glyoxalase-I cDNA that was cloned and sequenced previously in our laboratory (Ranganathan et al.,

Results

The cloned and sequenced human glyoxalase-I gene was found to be ∼12.0 kb in length, comprising five exons, separated by four introns (Fig. 1). The coding region starts at 1017 bp of the first exon and ends at 10 381 bp of the fifth exon. The first exon was contiguous with 982 bp of 5′ non-coding region immediately upstream of the coding region. The transcription start site determined by using a methyl-cap-specific primer and gene-specific primer set was 56 bp upstream from the ATG site. DNA sequence

Discussion

The ubiquitous and abundant expression of glyoxalase-I is suggestive of an important physiological function for the enzyme. Treatment of cells in vitro with methylglyoxal resulted in G1 cell-cycle arrest and toxicity followed by apoptosis (Kang et al., 1996). In vivo studies involving treatment of mice with methylglyoxal result in decreases of antioxidant enzymes (Choudhary et al., 1997). Glyoxal and methylglyoxal are reactive dicarbonyl compounds formed by degradation of glycolytic

Acknowledgements

We thank Pat Kraus for typing the manuscript. This work was supported in part by National Institutes of Health grants #CA06927 and #RR05539; National Institutes of Health grant #CA53893 to K.D.T.; and by an appropriation from the Commonwealth of Pennsylvania.

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    Genbank Accession Nos. AF146017 and AF146651.

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    Present address: DuPont Pharmaceuticals, Stine-Haskell Research Center, Newark, Delaware, USA.

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