Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

Abstract

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…Numerous studies have shown that physiological concentrations of HNE can stimulate cellular proliferation (187)(188)(189), differentiation (190,191), and cytoprotective response (181,(192)(193)(194)(195)(196)(197), through affecting multiple signaling pathways. As low as 0.5 μM, HNE can induce transcription of glutamate cysteine ligase, the first enzyme of GSH synthesis, through activation of the Nrf2-EpRE signaling pathways (198).…”

Regulation of Neuronal Glutathione Synthesis

“…Methionine is an important amino acid for producing cysteine via the trans-sulfuration pathway, which supplies approximately 50% of the cysteine needed for GSH synthesis in the liver (45). However, the brain trans-sulfuration pathway is thought to be negligible because the pathway-related enzymatic activity and RNA expression were found to be much lower than those in the liver (46). A primary culture experiment demonstrated incubation with cysteine or NAC, but not methionine, to increase the neuronal GSH level (47).…”

Abnormal Lipid Metabolism in Cystathionine β-Synthase-deficient Mice, an Animal Model for Hyperhomocysteinemia

“…Although liver is the major organ for the Cbs/CBS expression (Fig. 1) and the transsulfuration pathway (18), and hepatic steatosis is the common pathology observed in severe HHCY patients (2) and CBS Ϫ/Ϫ mice (15), the underlying mechanism has not been elucidated. CBS Ϫ/Ϫ mice suffer from severe growth retardation and some of them start to die after 2 weeks of age (15).…”

“…After the separated RNA was transferred to a Hybond-XL nylon membrane (Amersham Biosciences), the hybridization was performed as described previously (18). The entire open reading frame of rat CBS cDNA was used as a specific probe for the mouse Cbs transcript.…”