SFEBES2013 Poster Presentations Steroids (37 abstracts)
Identification of a novel CYP11B1 isoform in human adrenocortical cells
Samantha Alvarez-Madrazo 1 , Scott MacKenzie 1 , Alette Brinth 1 , Niall Fraser 1 , Rita Bernhardt 1, , John Connell 1, & Eleanor Davies 1
1BHF Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK; 2Department of Biochemistry, Saarland University, Saarbrücken, Germany; 3Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Dundee, UK.
The final reaction for cortisol production in the adrenal gland is catalysed by the 11β-hydroxylase enzyme, encoded by the CYP11B1 gene. Variants in this gene have been associated with alterations in cortisol levels, which increase blood pressure. This gene is traditionally thought to consist of 9 exons. However, recent evidence has predicted the existence of at least one alternatively spliced form.
The presence of novel CYP11B1 mRNA species in the H295R human adrenocortical cell line and non-diseased human adrenal tissue was investigated using RT-PCR, sequencing and western blotting.
Following RT-PCR, a larger band corresponding in size to an alternative form (ALT1) of CYP11B1 mRNA was observed on agarose gels, in addition to the wild-type (WT) form. Sequencing of the ALT1 band confirmed the presence of an additional exon between exons 2 and 3. In silico analysis of the 26 in-frame amino acids encoded by this exon predicts an insertion between alpha helices B’ and C of the enzyme. Western blotting using a custom antibody targeted at this insertion produced a band of the predicted size in total H295R cell protein. Control cells expressing only the WT form of CYP11B1 did not yield this band.
Further in vitro studies are required to investigate the effect of this alternative transcript on protein structure and cortisol production. The identification of a novel CYP11B1 isoform will broaden our understanding of adrenal physiology and its contribution to cortisol synthesis.
Declaration of funding: This work was supported by the TENOVUS Scotland Grant no. S11/11.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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