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Cytochrome P450 CYP3A in marsupials: Cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus)


Adaweyah El-Merhibi a, Suong N.T. Ngo b,⁎,1, Tamara A. Crittenden c, Ceilidh L. Marchant c, Ieva Stupans d, Ross A. McKinnon e,⁎⁎


a Women's and Children's Health Research Institute, Women's and Children's Hospital, North Adelaide, SA 5006, Australia
b School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA 5371, Australia
c School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia
d School of Science and Technology, University of New England, Armidale, NSW 2351, Australia
e School of Medicine, Flinders Centre for Innovation in Cancer, Flinders University, Adelaide, SA 5001, Australia

 

ABSTRACT

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics
and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene
subfamilies from marsupials and demonstrated important species-differences in both activity and tissue
expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned
and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala
(Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin Ndemethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant
differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas,
with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (pb0.01). No
gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising
anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and
female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences
detected across test marsupials. A 1610 bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned
by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid
sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino
acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and
displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat,
rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala
hepatic CYP3A78 gene and enables further functional analyses of CYP3A enzymes in marsupials. Given the
significant role that CYP3A enzymes play in the metabolism of both endogenous and exogenous compounds, the
clone provides an important step in elucidating the metabolic capacity of marsupials.

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