Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.
Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.
Leduc
R
R
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Molloy
S S
SS
Thorne
B A
BA
Thomas
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D045683:Furin; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D010450:Endopeptidases; D013381:Subtilisins; D045683:Furin