A NOVEL HUMAN PACE4 ISOFORM, PACE4E IS AN ACTIVE PROCESSING PROTEASE CONTAINING A HYDROPHOBIC CLUSTER AT THE CARBOXY-TERMINUS

PACE4 is a processing protease which processes the precursor protein t o the mature protein, Currently, four PACE4 isoforms have been reporte d [Tsuji, A. et al, (1994) Biochem. Biophys, Res, Commun, 200, 943-950 ], In this study, me have cloned cDNA encoding a novel isoform, PACE4E , by screening the human brain cerebellum cDNA library and reverse tra nscriptase-polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells, The PACE4E cDNA encoded an amino acid sequence o f 975 residues, The sequence from the amino terminus to Arg(900) Of PA CE4E was identical to the corresponding sequence of PACE4A, but the ca rboxy terminal sequence (75 residues) was unique and contained a hydro phobic cluster (LeU(952)-Gly(968)). PACE4E cDNA was transiently transf ected in COS-1 cells, and the expressed proteins were a 112-kDa precur sor form and a 105-kDa mature form, They were secreted into the cultur e medium, but their secretion was retarded compared with that of PACE4 A, The expression of a mutant of PACE4E truncated up to the hydrophobi c cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intrac ellularly due to interaction with the membrane through the hydrophobic cluster, On the contrary, the transient expression experiment of PACE 4C showed that only 68-kDa protein (precursor form) was detected in th e cell and not secreted into the medium, In addition, coexpression exp eriment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.