Isolation and transient expression of a cDNA encoding l-gulono-γ-lactone oxidase, a key enzyme for l-ascorbic acid biosynthesis, from the tiger shark Scyliorhinustorazame

Abstract

We present the first description of a cDNA encoding the l-gulono-γ-lactone oxidase (GLO) from a fish, a cartilaginous shark species, Scyliorhinus torazame. An expressed sequence tag (EST) from the shark kidney, which showed high similarity with a rat GLO gene, was isolated and its full-length sequence (1752 bp) was determined. The putative shark GLO (sGLO) cDNA sequence contained 98 bp of 5′-untranslated region, an open reading frame consisting 440 amino acids, and 334 bp of 3′-untranslated region including the poly(A+) tail. The deduced amino acid sequence was 63% identical to the rat GLO sequence and showed high conservation in the flavine adenine dinucleotide (FAD)-binding region. In addition, the calculated molecular mass (50,976 Da), theoretical p I (7.17) and hydropathy profile were similar to those of the rat GLO. Using both reverse transcription-PCR (RT-PCR) assays and the sGLO cDNA as a probe in Northern hybridisation experiments, expression was demonstrated in the shark kidney but not in any other tissues (brain, intestine, liver, muscle, pituitary and spleen). Biologically functional GLO activity was demonstrated by direct delivery of an expression vector containing the sGLO cDNA into kidney of far eastern catfish ( Silurus asotus), which lacks endogenous GLO activity. Transient expression of GLO activity was dependent on the amount of plasmid injected (up to 120 μg of DNA), and persisted for 12 days post injection, as demonstrated by RT-PCR and biochemical assays.