Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala (Phascolarctos cinereus)
Ngo, SNT, McKinnon, RA & Stupans, I 2003, Gene, vol. 309, no. 1, pp. 91-99.
Reverse transcription polymerase chain reaction was used to amplify two sections of koala liver acyl CoA oxidase (AOX) cDNA named AOX1 and AOX2. The two cDNAs were 2039 bp each, encoding for proteins of 662 amino acids in length. Transfection of these cDNAs into Cos-7 cells resulted in cells with palmitoyl-CoA oxidase activity. Apparent Km values (an inverse measure of affinity) were the same order of magnitude as rat and human AOX enzymes. Northern analysis found a more intense AOX mRNA band for koala liver than human and rat, and genomic DNA southern blot analysis found a single <14 kb AOX gene fragment in all three species. It is therefore likely that the lack of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in koala hepatic tissue is due to enzymes downstream of AOX or deficiencies in mitochondrial β-oxidation enzymes.
The two AOX cDNAs were isolated using polymerase chain reaction (PCR) then sequenced, with differences occurring between nucleotides 269 and 429. Both rats and humans have an AOX gene that utilises differential splicing to give different mRNAs, and these had high a nucleotide similarity to the koala cDNA. The cDNAs contain conserved motifs seen in other AOX sequences, and yeast has previously been found to also contain two AOX cDNAs. Total palmitoyl-CoA oxidase activity was determined to be twelve- and five- fold less than rat and human liver homogenates, respectively, demonstrating a significantly lower functional protein level. Transfection of the cDNAs into Cos-7 cells resulted in cells with AOX1 protein expressed at a much higher rate and AOX2 at a lower rate than rat AOX2. Kinetic analysis of the cDNA gave Km values of 28 and 38 μM for AOX1 and AOX2 respectively, compared to previously recorded values of ~10 μM for rat and human purified AOX1 enzymes. The human liver homogenate Km was 70 μM, consistent with other studies. Northern analysis of mRNA samples showed a single band at 5 kb for each individual koala, compared to rat and human mRNA showing lower intensity bands at 4 and 3 kb, respectively. The higher intensity band on the koala mRNA western blot indicates higher expression levels of AOX mRNA in koalas than rats and humans. The rat and human band sizes and relative intensities were comparable to previous studies’ values. Southern blot analysis used EcoRI-digested genomic DNA from livers of male koalas, rats, wallabies, possums and bandicoots, and showed one AOX gene fragment <14 kb for the koala, wallaby and rat samples, suggesting one AOX gene.
This study improved current knowledge of koala genetics and metabolism pathways, and the interrelation between them. Better information on koala liver function has many benefits, including improving our understandings of the marsupial’s diet and food consumption habits.
Summarised by Laura Wait
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