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.
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
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Zhou
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Lindberg
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