Molecular characterization, enzymatic analysis, and purification of murineproprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells

A cDNA coding for the murine proprotein convertase-l (mPC1 also known as mP C3 or mSPC3) was inserted into the Autographa californica nuclear polyhedro sis virus. Following infection of Spodoptera frugiperda cells, the recombin ant N-glycosylated protein is secreted into the cell culture medium from wh ich it can be purified to homogeneity as a fully enzymatically active enzym e. Two major secreted molecular forms of mPC1 with apparent molecular weigh ts of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodete cted in the medium. Automated NH2-terminal sequencing reveals that all thre e forms result from processing at the predicted zymogen activation site whe reas 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 o ptimally labeled at near neutral pH whereas the COOH-truncated forms are op timally 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 transfo rmation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in en zymatic 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 medi um 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 conver tase in the medium but are not affected by swainsonine and deoxymannojirimy cin, Finally, the 85-kDa secreted mPC1 is shown to be sulfated. (C) 1998 Ac ademic Press.