Processing and sorting of the prohormone convertase 2 propeptide.
The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.
Muller
L
L
Department of Biochemistry and Molecular Biology, LSU Health Sciences Center, New Orleans, Louisiana 70112, USA.
Cameron
A
A
Fortenberry
Y
Y
Apletalina
E V
EV
Lindberg
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D020134:Catalytic Domain; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry*; D017354:Point Mutation; D011233:Precipitin Tests; D045706:Proprotein Convertase 2; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D010455:Peptides; D011994:Recombinant Proteins; D013381:Subtilisins; D045706:Proprotein Convertase 2