Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells.
Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.
Jean
F
F
J.A. de Sève Laboratory of Molecular, Clinical Research Institute of Montréal, Québec, Canada.
Basak
A
A
Rondeau
N
N
Benjannet
S
S
Hendy
G N
GN
Seidah
N G
NG
Chrétien
M
M
Lazure
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010907:Pituitary Hormones / Q000378:metabolism; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics
D009842:Oligopeptides; D010907:Pituitary Hormones; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1