Proprotein convertase PC1/3-related peptides are potent slow tight-binding inhibitors of murine PC1/3 and Hfurin

The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH2-terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with Ki in the low nanomolar range. Additional cleavages at Trp residues yielded fragment9-71, which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH2-terminal fragment1-98 is cleaved at two sites, as revealed through Western blotting using NH2-terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/31-98 peptide, albeit at a much lesser extent with a micromolar Ki and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.