Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 CYP4A15
Ngo, SNT, McKinnon, RA & Stupans, I 2006, Gene, vol. 376, p. 123-132.
Digestion of eucalyptus oil compounds, or monoterpenes, is thought to be done by liver enzymes called cytochromes P450 (CYP). In the past it has been reported that koala liver has increased hydroxylation of lauric acid, a fatty acid, which is indicative of CYP4A enzyme activity. The molecular and enzymatic characteristics of CYP4A in koala liver are reported in this study.
Liver tissue was obtained from a koala and processed to extract a gene sequence for CYP4A, annotated as CYP4A15. The gene is 1544 base pairs long and is similar size to most animal CYP4A genes, with highest similarity to human CYP4A11 (69% similarity). The gene codes for a 500 amino acid protein. CYP4A protein has high similarity to CYP4A amino acid sequences of rat, rabbit, mouse and human; however, the koala sequence has two additional amino acids. When CYP4A15 gene was cloned into cells in a petri dish, its lauric acid hydroxylase activity was detected to be 4.79 ± 1.91 nmol/min/nmol CYP and this value is lower when compared to the same experiment in rats. The total protein activity for koala CYP4115 cloned gene was 0.45 ± 0.18 nmol/min/mg protein, and activity was lower than in non-cloned koala or rat samples, perhaps due to poor gene synthesis. Total amount of CYP in koala liver cells was 0.094 ± 0.001 nmol/mg protein. When liver cells were analysed for presence of CYP4A protein using an antibody, a protein 52 kDa in size was identified. It appears CYP4A may exist in greater amounts in koalas when compared to rat, human or wallaby samples, perhaps to aid in digestion of monoterpenes. Another protein of 48 kDA size was detected with the same antibody and this may be a modified version of CYP4A or another similar protein. When the messenger RNA (mRNA) of CYP4A15 gene was allowed to bind total liver mRNA, CYP4A15 mRNA bound a slightly larger segment of 2.6 kilobases (kb) in marsupial (koala and possum) samples than in human and rat samples, a 2.4 kb segment. An additional analysis of Southern Blotting, when flagged DNA is allowed bind other DNA on a blot, showed that CYP4A15 gene size varies across species: koala, rat, wallaby, possum and bandicoot. The koala diet is restricted to eucalyptus leaves which contain monoterpenes, highly toxic essential oil compounds. Their digestion may be aided by CYP4A enzymes which are possibly present in higher amounts in the koala for that purpose.
This study provides important molecular data about CYP4A enzyme in koala liver and raises the possibility of higher expression of the CYP4A15 gene in the koala liver than that in other species. These findings contribute to our growing understanding of the koala’s unique metabolic mechanisms that allow it to sustain its high toxin diet.
Summarised by Alexandra Selivanova
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