IDENTIFICATION OF HOLOCARBOXYLASE SYNTHETASE (HCS) PROTEINS IN HUMAN PLACENTA

Holocarboxylase synthetase (HCS) is a key enzyme in biotin utilization in eukaryotic cells. In a previous work from our laboratory, we descr ibed the cloning and sequencing of a full-length human HCS cDNA. Due t o the presence of three candidate sites for initiation of translation, the identification of full-length HCS proteins remains uncertain. Usi ng antibodies directed against human HCS sequences, we have identified , in human placenta, three cytosolic HCS proteins, of 86, 82 and 76 kD a. Similar results were observed in lysates of cells transfected with an HCS expression vector, as well as with human HCS cDNA transcribed a nd translated in a cell-free system. When anti-HCS antibodies were tes ted for their ability to inhibit HCS enzymatic activity, only the anti body directed against a region of HCS from Ile(128) to Pro(398), and n ot the antibodies against more proximal N-terminal regions inhibited H CS activity, suggesting that the sequence from Ile(128) to Pro(398) is essential for the catalytic activity of this enzyme. HCS synthesized in a cell-free system was not translocated into rat liver mitochondria . These results suggest that our human HCS cDNA encodes the cytosolic forms of the enzyme. These results also suggest that mRNA encoding cyt osolic HCS can be translated from all three translation initiation cod ons, Met(1), Met(7) and Met(58). (C) 1998 Elsevier Science B.V. All ri ghts reserved.