Endocrine Abstracts (2001) 2 OC14

Characterisation of two splice variants of a novel serine protease expressed in steroidogenic tissues

S Omer, K Lomthiasong, PJ Lowry & AB Bicknell

School of animal and microbial sciences, Reading University, Reading, UK.

During the search for the serine protease that cleaves pro-gamma-melatropin to stimulate adrenal growth (Bicknell et al, Cell, 2001, 105: 903), we identified another novel protease which we called AmP. In situ hybridisation detected AmP transcripts in steroidogenic tissues such as the brain particularly in the PVN and the dentate gyrus, in leydig cells of the testis, in underdeveloped follicles in the ovary as well as in the adrenal cortex. Full length cloning identified two splice variants differing by a 222 nucleotide insertion in the 5' end of the short variant. The shorter variant codes for a 371 amino acid protein of 40.7kDa and computer analysis predicts it to be targeted to the cytosol while the longer 445 amino acid protein of 48.4 kDa is mitochondrial. Cellular targeting was confirmed by tagging with GFP. Using RT-PCR, the short variant was detected in the brain, adrenal, ovary, but not in the testis or placenta while the long variant was expressed in all of these tissues. Moreover, AmP-long was highly expressed in the brain in comparison with AmP-short. In situ hybridisation showed the expression of both variants across the whole adrenal cortex but not the medulla or the outer capsule. Due to the mitochondrial localisation of the long variant of the protease and its expression in steroidogenic tissues, it may be expected to be involved in the steroidogenic pathway, possibly by cleaving StAR or its homolouge MLN64. We investigated this by co-transfecting the two variants with StAR or MLN64 and F2 plasmid (Harikrishna et al, DNA and Cell Biol, 1993,12:371) into Cos-1 cells and measuring the effect on pregnenolone production. The results suggest that neither AmP variant affects steroidogenesis nor cleaves StAR as shown by western immunoblot.

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