Abstract
Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.
Highlights
Mammalian prohormone convertases comprise a family of serine proteinases whose function is the cleavage of peptide precursor molecules at distinct single or pairs of basic residues (1, 2)
The 17-amino acid peptide sequence indicated within the P-domain was used as an immunogen to obtain a polyclonal rat PC7 (rPC7) antiserum, permitting the identification and characterization of the various forms of rPC7 obtained via overexpression in BSC40 cells
Following overnight incubation of cells infected with vaccinia virus (VV) recombinants expressing either rPC7K or BTMD-rPC7K, the cell extracts, and media were analyzed by Western blotting
Summary
Vaccinia Virus Constructs—The full-length cDNA of rPC7 (5) that had been inserted into the pBluescript vector (Stratagene) was digested with (59) HindIII and (39) BbsI to remove a pair of extra 59 ATG codons (5). RP-HPLC Analysis of Synthetic Peptide Substrate Digests—The proPTH tridecapeptide (KSVKKRSVSEIQL), the pro-PC4 heptadecapeptide (YETLRRRVKRSLVVPTD), and the pro-EGF hexadecapeptide (HLREDDHHYSVRNSDS) were reacted at room temperature with enzymes in the reaction mixture as described above for enzymatic activity determinations. The digestion products were analyzed using RP-HPLC separation (Varian, model 9010) on a Beckman 5-mm Ultrasphere C18 column (0.2 3 25 cm) as described above for peptide purification except that the buffer system contained 0.01% triethylamine in both the aqueous and organic phases, the flow rate was 1 ml/min, the linear gradient of acetonitrile was 5–30% over 45 min, and monitoring was carried out at a wavelength of 210 nm. The Km determinations were based on the amounts of the C-terminal cleavage product of each substrate peptide, and the resulting data were analyzed as described above for the pERTKR-MCA peptide
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
