Comparative characterization of two forms of recombinant human SPC1 secreted from Schneider 2 cells

SPC1 (furin/PACE), an enzyme belonging to the 58 group of serine endoprotea ses, is a type I integral membrane protein that catalyzes the processing of a multitude of precursor proteins. We report here the use of transfected D rosophila melanogaster Schneider 2 cells to produce milligram amounts of tw o forms of recombinant human SPC1, In order to investigate the role of the cysteine-rich region (CRR) of SPC1, we compared the biochemical and enzymat ic properties of hSPC1/714 that has the C-terminal tail and transmembrane r egion of the native enzyme removed with that of hSPC1/585 which had, in add ition, the CRR deleted. Two stable cell lines were established. The S2-hSPC 1/714 line secreted a major form of apparent molecular weight of 83 kDa and a minor form of 80 kDa whereas the S2-hSPC1/585 line secreted a single 59- kDa protein. PNGase F treatment of the different forms demonstrated that th e enzymes were glycosylated. Automated NH2-terminal sequencing revealed tha t all purified forms resulted from processing at the expected zymogen activ ation site. Removal of the CRR resulted in a broadening of the enzyme's pH range, a shift of K-0.5 for Ca2+, and a shorter enzymatic half-life when co mpared to the longer form, which suggest that the CRR of hSPC1 may help in stabilizing the enzyme's proteolytic activity. The use of this high-level e xpression system will meet the demand for material necessary to perform bio chemical and structural studies that are needed to further our understandin g of this and other SPCs at the molecular level. (C) 2000 Academic Press.