Comparative characterization of two forms of recombinant human SPC1 secreted from Schneider 2 cells.
SPC1 (furin/PACE), an enzyme belonging to the S8 group of serine endoproteases, is a type I integral membrane protein that catalyzes the processing of a multitude of precursor proteins. We report here the use of transfected Drosophila melanogaster Schneider 2 cells to produce milligram amounts of two forms of recombinant human SPC1. In order to investigate the role of the cysteine-rich region (CRR) of SPC1, we compared the biochemical and enzymatic properties of hSPC1/714 that has the C-terminal tail and transmembrane region of the native enzyme removed with that of hSPC1/585 which had, in addition, the CRR deleted. Two stable cell lines were established. The S2-hSPC1/714 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 the enzymes were glycosylated. Automated NH(2)-terminal sequencing revealed that all purified forms resulted from processing at the expected zymogen activation site. Removal of the CRR resulted in a broadening of the enzyme's pH range, a shift of K(0.5) for Ca(2+), and a shorter enzymatic half-life when compared 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 expression system will meet the demand for material necessary to perform biochemical and structural studies that are needed to further our understanding of this and other SPCs at the molecular level.
Denault
J B
JB
Laboratory of Neuropeptide Structure and Metabolism, Institut de Recherches Cliniques de Montréal, Québec, Canada.
Lazure
C
C
Day
R
R
Leduc
R
R
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004331:Drosophila melanogaster / Q000166:cytology; D004795:Enzyme Stability; D045683:Furin; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013381:Subtilisins; C468731:FURIN protein, human; D045683:Furin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003545:Cysteine; D002118:Calcium