Characterization of STARD4 and STARD6 Proteins in Human Ovarian Tissue and Human Granulosa Cells and Cloning of Human STARD4 Transcripts
Abstract
Progesterone is essential hormone for pregnancy, which is produced by the human corpus luteum in early pregnancy until the placenta assumes this function. Transport of the cholesterol from the outer to the inner mitochondrial membrane is the rate limiting step for the de novo synthesis of pregnenolone (the precursor to progesterone), a process mediated by STARD1. STARD1 contains a C-terminal lipid binding domain which binds cholesterol and an N-terminal domain targeting STARD1 to the mitochondrial membrane. Unlike STARD1, STARD4 and STARD6 proteins lack a mitochondrial targeting sequence; however, they can bind cholesterol and increase steroidogenesis in model systems. In this study, we found both STARD4 and STARD6 mRNA expression in all steroidogenic compartments of human ovarian tissue. Western blots of human ovarian tissue demonstrated STARD4 and STARD6 immunoreactive protein at approximate molecular mass of 30 kDa and 43 kDa, respectively. Microscopically, STARD4 and STARD6 proteins are localized to steroidogenic cells of ovarian follicles (in granulosa and theca cells) and luteal cells. In order to determine the actual protein mass for ovarian STARD4, mRNA from human luteinized granulosa cells (hLGC), we aimed to clone all major transcripts of STARD4 from a cDNA library we generated from granulosa cell poly (A) RNA. Two major transcripts were isolated for STARD4. First one possessing all six exons with a small variation of 6 nucleotides in the untranslated exon 1 was predicted to yield a full-length protein of 205 aa. The second transcript possessed an exon 4 deletion, which introduced a premature stop codon and is predicted to yield a truncated protein with sizes of either 55 amino acids or 107 amino acids. Both cDNAs were placed in expression vectors. The full-length recombinant STARD4 protein appeared on Western blot at an approximate size of 24 kDa. Using the COS cell F2-steroidogenic assay, transfected full-length and exon 4-deleted transcript protein functions were compared. Full-length STARD4 protein was able to increase the pregnenolone production significantly compared to empty vector, whereas truncated (exon 4 deleted) STARD4 was not. Although full-length STARD4 has the potential to increase steroidogenesis, it is unlikely that the truncated protein has similar ability.