Enzymatic properties of carboxyl-terminally truncated prohormone convertase 1 (PC1/SPC3) and evidence for autocatalytic conversion.
Previous studies have shown that the prohormone convertase 1 (PC1, or SPC3), a member of the new eukaryotic subtilisin-like proteinase family, undergoes a series of proteolytic processing events during its biosynthesis. The first cleavage, of the amino-terminal prosegment, is probably involved in enzyme activation, while the secondary cleavages at the carboxyl terminus are of unknown significance and occur mainly in cells possessing a regulated secretory pathway. In this work, we found that 87-kDa PC1, a homogeneous recombinant protein, could spontaneously convert to 74- and 66-kDa forms in vitro. Limited digestion of 87-kDa PC1 using chymotrypsin and trypsin could also generate 74- and 66-kDa-like PC1s, which were enzymatically active against the fluorogenic peptide carbobenzoxy-Arg-Tyr-Lys-Arg-aminomethylcoumarin. The 74/66-kDa PC1 generated by spontaneous conversion was purified away from the 87-kDa form and enzymatically characterized. Compared to the 87-kDa form, 74/66-kDa PC1 was more active but less stable. In addition, 74/66-kDa PC1 exhibited a narrower pH optimum (between 5.0 and 5.5) and was activated by higher concentrations of calcium. Carboxyl-terminally truncated PC1 also appeared to be more sensitive to certain protease inhibitors than 87-kDa PC1. Taken together, our results suggest that autocatalysis could be involved in carboxyl-terminal cleavages of PC1. These carboxyl-terminal cleavages of PC1 result in alterations in certain PC1 properties and may therefore possess potential significance with respect to prohormone processing.
Zhou
Y
Y
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Lindberg
I
I
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells; D002918:Chymotrypsin / Q000378:metabolism; D006224:Cricetinae; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D043484:Proprotein Convertases; D002918:Chymotrypsin; D014357:Trypsin; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases