title: Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain.
abstract: PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null 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 ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor 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 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments 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 between 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.
authors.affiliation: Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington VT 05405, USA. jsucic@flint.umich.edu
authors.forename: J F
authors.initials: JF
authors.lastname: Sucic
authors.forename: J M
authors.initials: JM
authors.lastname: Moehring
authors.forename: N M
authors.initials: NM
authors.lastname: Inocencio
authors.forename: J W
authors.initials: JW
authors.lastname: Luchini
authors.forename: T J
authors.initials: TJ
authors.lastname: Moehring
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004167:Diphtheria Toxin / Q000378:metabolism; D005098:Exotoxins / Q000378:metabolism; D045683:Furin; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012845:Sindbis Virus / Q000254:growth & development; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D037521:Virulence Factors; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002118:Calcium; D017077:Culture Media, Conditioned; D004167:Diphtheria Toxin; D005098:Exotoxins; D045683:Furin; D007527:Isoenzymes; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D043484:Proprotein Convertases; D011498:Protein Precursors; D012333:RNA, Messenger; D011972:Receptor, Insulin; D011971:Receptors, Immunologic; D012697:Serine Endopeptidases; D013381:Subtilisins; D037521:Virulence Factors; D014841:von Willebrand Factor; D000097668:Pseudomonas aeruginosa Exotoxin A; C030325:anthrax toxin; C051512:insulin proreceptor; C080654:PCSK6 protein, human