Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. Th e expression of human PACE4 in RPE.40 cells (furinnull mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild -type characteristics, including sensitivity to Sindbis virus and the abili ty to process the low-density-lipoprotein receptor-related protein. Express ion of PACE4 in these cells failed to restore wild-type sensitivity to Pseu domonas exotoxin A. Go-expression of human PACE4 in these cells with either a secreted form of the human insulin proreceptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co- expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 c ells express mRNA species for four PACE4 isoforms, suggesting that any endo genous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experimen ts in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes bet ween 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secret ed an endoprotease with the same Ca2+ dependence and temperature sensitivit y as that observed in membrane fractions of these cells assayed in vitro. T hese results, in conjunction with other published work, demonstrate that PA CE4 is an endoprotease with more stringent substrate specificity and more l imited operating parameters than furin.