Molecular characterization, enzymatic analysis, and purification of murine proprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells.
A cDNA coding for the murine proprotein convertase-1 (mPC1 also known as mPC3 or mSPC3) was inserted into the Autographa californica nuclear polyhedrosis virus. Following infection of Spodoptera frugiperda cells, the recombinant N-glycosylated protein is secreted into the cell culture medium from which it can be purified to homogeneity as a fully enzymatically active enzyme. Two major secreted molecular forms of mPC1 with apparent molecular weights of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodetected in the medium. Automated NH2-terminal sequencing reveals that all three forms result from processing at the predicted zymogen activation site whereas both the 75- and the 71-kDa forms are truncated at their COOH-terminus. Labeling by an active-site titrant demonstrates that the 85-kDa form is optimally labeled at near neutral pH whereas the COOH-truncated forms are optimally labeled at acidic pH. Additionally it is shown that the 85-kDa mPC1 is transformed into the COOH-truncated forms following in vitro incubation at acidic pH levels and in presence of calcium. Concomitantly, the transformation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in enzymatic activity upon assaying at pH 6.0. The 71-kDa form can be recovered after purification at a level of 1 to 1.5 mg per liter of cell culture medium and is enzymatically stable only in the pH range from 5.0 to 6.5. Cells treated with tunicamycin show a drastically reduced secretion of the convertase in the medium but are not affected by swainsonine and deoxymannojirimycin. Finally, the 85-kDa secreted mPC1 is shown to be sulfated.
Boudreault
A
A
Protein Engineering Network of Centre of Excellence, Laboratory of Neuropeptides Structure and Metabolism, J. A. de Sève Laboratories of Molecular Neuroendocrinology, Institut de Recherches Cliniques de Montréal, Québec, Canada.
Gauthier
D
D
Rondeau
N
N
Savaria
D
D
Seidah
N G
NG
Chrétien
M
M
Lazure
C
C
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation / Q000187:drug effects; D016695:Glycosyltransferases / Q000037:antagonists & inhibitors; D006863:Hydrogen-Ion Concentration; D051379:Mice; D017924:Nucleopolyhedroviruses / Q000235:genetics; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013431:Sulfates / Q000378:metabolism; D017026:Swainsonine / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D013431:Sulfates; D014415:Tunicamycin; D017485:1-Deoxynojirimycin; D016695:Glycosyltransferases; D043484:Proprotein Convertases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases; D017026:Swainsonine; D002118:Calcium