Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

Abstract

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359 Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

Activation of antioxidant defense during dehydration stress in the African clawed frog

“…Unlike mammalian GSTs, little is known about this family of proteins in amphibians. A few studies identified and characterized the different classes of GSTs present in X. laevis and mu, alpha, sigma, and pi classes have been found De Luca et al, 2002. However, the responses and regulation of these different classes of GSTs with respect to environmental stress in X. laevis has not previously been examined.…”

A novel amphibian Pi-class glutathione transferase isoenzyme from Xenopus laevis: importance of phenylalanine 111 in the H-site

“…For each sample, we used 100 units of Moloney murine leukaemia virus reverse transcriptase and gene-specific reverse primer, according the manufacturer's instructions. The reaction was carried out in 50 mM Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 5 mM dithiothreitol, 500 µM dNTP mix and 10 units of placental RNase inhibitor at 37 • C for 50 min, followed by 10 min at 90 • C. The following primers were used: Pirev (5 -TCACTTTTTGTGTTTGGGGG-3 ) for XlGSTP1-1; and Murev (5 -TTATTTTTTGTTGCCCCAGG-3 ) for XlGSTM1-1 [15].…”

“…RT-PCR was performed to investigate the presence of the XlGSTP1 transcript during embryo development as well as in different adult tissues. XlGSTM1-1 [15] was used as a positive control to evaluate the sensitivity of the method (results not shown). Figure 3 shows that a 420 bp product corresponding to XlGSTP1-1 was present in embryos, but not in the unfertilized eggs, liver, lung, heart, kidney and ovary.…”

“…We have also investigated the expression of GSTs in Xenopus laevis [14]. A new Mu-class GST has been cloned, expressed and characterized previously from this amphibian species [15]. Unlike B. bufo, X. laevis does not apparently express Pi-class GSTs [14]; however, analysis of the expressed sequence tag (EST) database suggests that a previously uncharacterized Pi-class GST isoform might be encoded by this amphibian species.…”

Cellular adaptation to dehydration stress : molecular adaptations for dehydration tolerance in the African clawed frog, Xenopus laevis

“…Unlike mammalian GSTs, little is known about this family of proteins in amphibians. A few studies identified and characterized the different classes of GSTs present in X. laevis and mu, alpha, sigma, and pi classes have been found De Luca et al, 2002;De Luca et al, 2003).…”