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• W2128548712 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. 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. In vitro characterization of the novel proprotein convertase PC7.Journal of Biological ChemistryVol. 272Issue 48PreviewDr. Hendy's name did not appear in the list of authors. He is affiliated with the Departments of Medicine, Physiology and Human Genetics, McGill University and the Royal Victorial Hospital, Montreal, QC H3A 1A1, Canada. Full-Text PDF Open Access 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 (1Seidah N.G. Chrétien M. Trends Endocrinol. Metab. 1992; 3: 133-140Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 2Rouillé Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (317) Google Scholar). These enzymes are related to bacterial subtilisins and to the yeast prohormone processing protease kexin. There are presently seven known members of this family that have been grouped under the generic name proprotein convertases (PCs). 1The abbreviations used are: PC, proprotein convertase(s); rPC7, rat PC7; hfurin, human furin; VV, vaccinia virus; BTMD, before the transmembrane domain; BCRD, before the cysteine-rich domain; ECL, enhanced chemiluminescence; MAP, multiple antigenic peptide; MCA, 4-methylcoumarin-7-amide; pro-EGF, pro-epidermal growth factor; pro-PTH, pro-parathyroid hormone; ykexin, yeast kexin; BFA, brefeldin A; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; RP-HPLC, reverse phase-high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid; pfu, plaque-forming units. These include furin (also known as PACE), PC1 (also known as PC3), PC2, PACE4, PC4, PC5 (also known as PC6) (for reviews, see Refs. 1Seidah N.G. Chrétien M. Trends Endocrinol. Metab. 1992; 3: 133-140Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 2Rouillé Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (317) Google Scholar, 3Seidah N.G. Chrétien M. Day R. Biochimie ( Paris ). 1994; 76: 197-209Crossref PubMed Scopus (383) Google Scholar, 4Van de Ven W.J.M. Roebroek A. Crit. Rev. Oncog. 1993; 4: 115-136PubMed Google Scholar), and, most recently, PC7 (5Seidah N.G. Hamelin J. Mamarbachi M. Dong W. Tadros H. Mbikay M. Chrétien M. Day R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar) (also known as LPC (6Meerabux J. Yaspo M.-L. Roebroek A.J. Van de Ven W.J.M. Lister T.A. Young B.D. Cancer Res. 1996; 56: 448-451PubMed Google Scholar), PC8 (7Bruzzaniti A. Goodge K. Jay P. Taviaux S.A. Lam M.H.C. Berta P. Martin T.J. Moseley J.M. Gillespie M.T. Biochem. J. 1996; 314: 727-731Crossref PubMed Scopus (88) Google Scholar), or SPC7 (8Constam D.B. Calfon M. Robertson E.J. J. Cell Biol. 1996; 134: 181-191Crossref PubMed Scopus (90) Google Scholar)). Tissue distribution analyses of these enzymes indicate that PC1 and PC2 are expressed mainly in neural and endocrine tissues, PC4 exclusively in reproductive germ cells, and PC5 and PACE4 to varying degrees in many tissue types (1Seidah N.G. Chrétien M. Trends Endocrinol. Metab. 1992; 3: 133-140Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 2Rouillé Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (317) Google Scholar, 3Seidah N.G. Chrétien M. Day R. Biochimie ( Paris ). 1994; 76: 197-209Crossref PubMed Scopus (383) Google Scholar). Similar to furin (3Seidah N.G. Chrétien M. Day R. Biochimie ( Paris ). 1994; 76: 197-209Crossref PubMed Scopus (383) Google Scholar, 4Van de Ven W.J.M. Roebroek A. Crit. Rev. Oncog. 1993; 4: 115-136PubMed Google Scholar), PC7 has a nearly ubiquitous tissue distribution as assessed by mRNA expression (5Seidah N.G. Hamelin J. Mamarbachi M. Dong W. Tadros H. Mbikay M. Chrétien M. Day R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar,7Bruzzaniti A. Goodge K. Jay P. Taviaux S.A. Lam M.H.C. Berta P. Martin T.J. Moseley J.M. Gillespie M.T. Biochem. J. 1996; 314: 727-731Crossref PubMed Scopus (88) Google Scholar, 8Constam D.B. Calfon M. Robertson E.J. J. Cell Biol. 1996; 134: 181-191Crossref PubMed Scopus (90) Google Scholar), suggesting that it could, like furin (4Van de Ven W.J.M. Roebroek A. Crit. Rev. Oncog. 1993; 4: 115-136PubMed Google Scholar), be involved in the processing of precursors within the constitutive secretory pathway. Interestingly, in lymphoid-associated tissues such as thymus, spleen, and lymph nodes, the expression of PC7 appears to be especially high (5Seidah N.G. Hamelin J. Mamarbachi M. Dong W. Tadros H. Mbikay M. Chrétien M. Day R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar). The sites where PCs cleave peptide precursors of various hormones, growth factors, and viral envelope glycoproteins have generally been defined as (Arg/Lys)-(X)n-(Arg/Lys)↓ wheren = 0, 2, 4, or 6 residues (9Seidah N.G. Shinde U. Inouye M. Intramolecular Chaperones and Protein Folding. R. G. Landes, Austin, TX1995: 181-203Google Scholar, 10Nakayama K. Watanabe T. Nakagawa T. Kim W.S. Nagahama M. Hosaka M. Hatsuzawa K. Kondoh-Hashiba K. Murakami K. J. Biol. Chem. 1992; 267: 16335-16340Abstract Full Text PDF PubMed Google Scholar, 11Molloy S.S. Bresnahan P.A. Leppla S.H. Klimpel K.R. Thomas G. J. Biol. Chem. 1992; 267: 16396-16402Abstract Full Text PDF PubMed Google Scholar). Due to this wide-ranging specificity and the overlapping expression of various convertases in different tissues, it is often difficult to assign cleavage of a given precursor to a particular enzyme. Although it is clear that PC1, PC2 (12Benjannet S. Rondeau N. Day R. Chrétien M. Seidah N.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3564-3568Crossref PubMed Scopus (539) Google Scholar, 13Thomas L. Leduc R. Thorne B.A. Smeekens S.P. Steiner D.F. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5297-5301Crossref PubMed Scopus (276) Google Scholar, 14Benjannet S. Reudelhuber T. Mercure C. Rondeau N. Chrétien M. Seidah N.G. J. Biol. Chem. 1992; 267: 11417-11423Abstract Full Text PDF PubMed Google Scholar), and PC5-A (15de Bie I. Marcinkiewicz M. Malide D. Lazure C. Nakayama K. Bendayan M. Seidah N.G. J. Cell Biol. 1996; 135: 1261-1275Crossref PubMed Scopus (141) Google Scholar) are the enzymes most likely to be active in secretory vesicles (9Seidah N.G. Shinde U. Inouye M. Intramolecular Chaperones and Protein Folding. R. G. Landes, Austin, TX1995: 181-203Google Scholar), processing within the compartments of the early secretory pathway may be best performed by furin, PACE4, PC5-B (16Vollenweider F. Benjannet S. Decroly E. Savaria D. Lazure C. Thomas G. Chrétien M. Seidah N.G. Biochem. J. 1996; 314: 521-532Crossref PubMed Scopus (82) Google Scholar, 17Seidah N.G. Benjannet S. Pareek S. Savaria D. Hamelin J. Goulet B. Laliberté J. Lazure C. Chrétien M. Murphy R. Biochem. J. 1996; 314: 951-960Crossref PubMed Scopus (243) Google Scholar), and, most likely, PC7. This paper characterizes the in vitro properties of full-length and soluble rat PC7 (rPC7) overexpressed in mammalian cells infected with the corresponding vaccinia virus (VV) recombinants. A polyclonal rPC7 antiserum has been produced and used to identify the biosynthetic forms, likely cellular location, and site of zymogen cleavage of rPC7 in these cells. In addition, processing of several synthetic fluorogenic and peptidyl substrates is examined to elucidate the cleavage specificity of this newest member of the PC family. The full-length cDNA of rPC7 (5Seidah N.G. Hamelin J. Mamarbachi M. Dong W. Tadros H. Mbikay M. Chrétien M. Day R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar) that had been inserted into the pBluescript vector (Stratagene) was digested with (5′) HindIII and (3′) BbsI to remove a pair of extra 5′ ATG codons (5Seidah N.G. Hamelin J. Mamarbachi M. Dong W. Tadros H. Mbikay M. Chrétien M. Day R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar). Following the addition of a linker to close the sequence, the insert (5′) HindIII/(3′)XbaI was ligated into the HindIII/NheI sites of the VV transfer vector PMJ602 (18Davison A.J. Moss B. Nucleic Acids Res. 1990; 18: 4285-4286Crossref PubMed Scopus (99) Google Scholar). This produced the rPC7 full-length recombinant virus (VV:rPC7). The native initiator sequence of this construct, CTGATGC, was subsequently modified by polymerase chain reaction to GTGATG Gto generate a favorable Kozak consensus sequence for the initiation of protein translation (19Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar). Three soluble forms of rPC7 were constructed from this full-length VV:rPC7K clone by digestion with restriction enzymes followed by addition of the appropriate linkers. Thus, digestion withBsu36I produced a form called BTMD- (before thetransmembrane domain) rPC7K, which ends at Gly-Tyr-Ser622. Similarly, digestion with HgaI generated two shorter forms of rPC7, called BTMD-S-rPC7, ending at Tyr-Gly-Ser578 or at Val-Asp-Ile585. Vaccinia virus recombinants of the three constructs were then isolated as described previously (12Benjannet S. Rondeau N. Day R. Chrétien M. Seidah N.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3564-3568Crossref PubMed Scopus (539) Google Scholar, 14Benjannet S. Reudelhuber T. Mercure C. Rondeau N. Chrétien M. Seidah N.G. J. Biol. Chem. 1992; 267: 11417-11423Abstract Full Text PDF PubMed Google Scholar). Human BCRD- (before the cysteine-richdomain) furin (BCRD-hfurin), ending at Ser-Ser-Gly586, has been described previously (20Paquet L. Bergeron F. Boudreault A. Seidah N.G. Chretien M. Mbikay M. Lazure C. J. Biol. Chem. 1994; 269: 19279-19285Abstract Full Text PDF PubMed Google Scholar). The yeast recombinant VV:ykexin was kindly provided by Dr. D. Thomas (Biotechnology Research Institute, Montreal, Canada). BSC40 cells, an African green monkey kidney epithelial cell line, were grown to confluence in 10% CO2 at 37 °C on 15-cm tissue culture dishes using minimum essential medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 28 μg/ml gentamycin. Following a brief rinse in phosphate-buffered saline, cells were infected with recombinant viruses at 2–4 plaque forming units (pfu)/cell in 5 ml of phosphate-buffered saline containing 0.01% (v/v) bovine serum albumin for 30 min at room temperature. They were then returned to minimum essential medium plus fetal bovine serum and incubated overnight at 37 °C. Membrane preparations of rPC7K− and yeast kexin (ykexin)-infected cells were made according to the procedure of Bresnahan et al. (21Bresnahan P.A. Leduc R. Thomas L. Thorner J. Gibson H.L. Brake A.J. Barr P.J. Thomas G. J. Cell Biol. 1990; 111: 2851-2859Crossref PubMed Scopus (288) Google Scholar) with several modifications: 1) cells were harvested at 18 h post-infection by scraping in 20 mm Tris acetate, pH 7.0; 2) suspensions from the Dounce homogenizer were centrifuged at 16,000 × gfor 15 min at 4 °C in a microcentrifuge; 3) the supernatants, representing soluble cellular material, were removed and stored at −20 °C for enzymatic activity and Western blot analyses; 4) the membrane pellets, resuspended in the above buffer, were diluted 1:1 (v/v) with glycerol and aliquoted for storage at −20 °C. Under these conditions, enzymatic activity was stable for at least 6 months. For the soluble VV:BTMD-rPC7K construct, cells at 18 h postinfection were washed three times with serum-free minimum essential medium and then incubated in the same medium for an additional 6 h. Following centrifugation at 1,500 × g for 10 min to remove cell debris, the media were concentrated approximately 20-fold on Centriprep-30s (Amicon), diluted 1:1 with glycerol, and stored at −20 °C. BCRD-hfurin, produced in the same manner, was further purified on DEAE-Bio-Gel A agarose (Bio-Rad) as reported (22Jean F. Basak A. Rondeau N. Benjannet S. Hendy G.N. Seidah N.G. Chrétien M. Lazure C. Biochem. J. 1993; 292: 891-900Crossref PubMed Scopus (88) Google Scholar). A similar further purification of BTMD-rPC7 consistently resulted in very poor recoveries of activity, most likely due to oxidative damage. Nonetheless, using the small amount of active BTMD-rPC7 recovered from this purification, we were able to ascertain that the properties (pH optimum and K m (app) using a pentapeptide fluorogenic substrate (see below)) of the DEAE-purified BTMD-rPC7 were essentially identical to those of the enzyme obtained from concentrated media (results not shown). Media and membrane extracts from rPC7-infected BSC40 cells were run on a 7.5% SDS-PAGE and the separated proteins electroeluted onto polyvinylidene difluoride membranes (Schleicher and Schuell). Protein bands were visualized by enhanced chemiluminescence (ECL) (Boehringer Mannheim) using a primary rabbit antiserum raised against a synthetic multiple antigenic peptide (MAP) (23Basak A. Boudreault A. Chen A. Chrétien M. Seidah N.G. Lazure C. J. Pept. Sci. 1995; 1: 385-395Crossref PubMed Scopus (41) Google Scholar) based on the amino acid sequence (Ala449-Ser-Tyr-Val-Ser-Pro-Met-Leu-Lys-Glu-Asn-Lys-Ala-Val-Pro-Arg-Ser465) located in the P-domain of rPC7 (5, Fig. 1). The specificity of the antiserum was verified by demonstrating that the bands visualized in the rPC7-infected cells could be blocked by preincubation with 200 μg/ml linear synthetic peptide of the MAP that had been prepared separately using normal, solid phase Fast-Moc chemistry (23Basak A. Boudreault A. Chen A. Chrétien M. Seidah N.G. Lazure C. J. Pept. Sci. 1995; 1: 385-395Crossref PubMed Scopus (41) Google Scholar). In addition, the antiserum did not cross-react when used on membrane pellets of BSC40 cells infected with vaccinia virus recombinants of other known membrane-associated convertases such as hfurin, mPC5/6-B, or ykexin. For quantitative analyses, densitometric readings of ECL films were carried out using the Gel Plotting macro of a Macintosh image analysis software (a public domain National Institutes of Health Image program). BSC40 cells were infected with the VV:rPC7 construct at 4 pfu/cell. Seventeen hours postinfection, the cells were pulse- or pulse-chase labeled withl-[35S]Met as described (12Benjannet S. Rondeau N. Day R. Chrétien M. Seidah N.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3564-3568Crossref PubMed Scopus (539) Google Scholar, 16Vollenweider F. Benjannet S. Decroly E. Savaria D. Lazure C. Thomas G. Chrétien M. Seidah N.G. Biochem. J. 1996; 314: 521-532Crossref PubMed Scopus (82) Google Scholar). When present during the preincubation as well as the pulse periods, the concentrations of tunicamycin and brefeldin A (BFA) were 5 and 2.5 μg/ml, respectively. As described previously (24Benjannet S. Rondeau N. Paquet L. Boudreault A. Lazure C. Chrétien M. Seidah N.G. Biochem. J. 1993; 294: 735-743Crossref PubMed Scopus (173) Google Scholar), cells were lysed, and the proteins therein, along with those in the culture media, were immunoprecipitated by incubation with either the rPC7 antiserum (see above) plus protein A-agarose or with lentil lectin-Sepharose (25Zhong M. Benjannet S. Lazure C. Munzer S. Seidah N.G. FEBS Lett. 1996; 396: 31-36Crossref PubMed Scopus (28) Google Scholar). Following SDS-PAGE, the bands were visualized by exposure to x-ray film. For microsequence analyses, BSC40 cells infected with the VV:rPC7 construct were pulse-labeled for 4 h in the presence of [3H]Leu or [3H]Ile and processed as above. The labeled proteins were then run on an SDS-PAGE, after which the appropriate bands were eluted and analyzed on an Applied Biosystem gas-phase sequenator (model 470A) as described previously (12Benjannet S. Rondeau N. Day R. Chrétien M. Seidah N.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3564-3568Crossref PubMed Scopus (539) Google Scholar, 25Zhong M. Benjannet S. Lazure C. Munzer S. Seidah N.G. FEBS Lett. 1996; 396: 31-36Crossref PubMed Scopus (28) Google Scholar). Enzymatic activity was assessed using the fluorogenic synthetic peptide substrate pERTKR-MCA (Peptides International) (26Jean F. Boudreault A. Basak A. Seidah N.G. Lazure C. J. Biol. Chem. 1995; 270: 19225-19231Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) as follows. The enzyme preparation (usually 5 μl) was added to the assay reaction mixture consisting of (final concentration) 50 mm Tris acetate (pH 6.5), 1 mm CaCl2, 1% Triton X-100 (v/v) in the case of membrane-bound enzyme preparations, and 50 μm2-mercaptoethanol in the case of BCRD-hfurin. The reaction (in a total volume of 100 μl) was initiated at room temperature in a microtiter plate by the addition of pERTKR-MCA at a final concentration of 0.1 mm. Fluorescence was monitored at 0, 30, 60, and 90 min using a Perkin-Elmer spectrofluorometer (model LS 50 B) set to an excitation wavelength of 370 nm and an emission wavelength of 460 nm. In all cases, the rate of substrate hydrolysis was constant for at least 60 min. For pH sensitivity experiments, enzymatic activity was evaluated as above using a ternary buffer system containing 17.3 mm sodium acetate, 17.3 mm MES, and 17.3 mm Tris acetate adjusted to the appropriate pH with acetic acid or NaOH. To determine the Ca2+ activation requirement of PC7, increasing concentrations of EDTA were added to the assay mixture described above for enzymatic activity measurements but lacking CaCl2. Based on these results, the assay mixture was then supplemented with 1 mm EDTA and varying concentrations of CaCl2. For the inhibitor profiles, selected compounds were added to the fluorogenic assay medium 30 min before the addition of pERTKR-MCA. In the case of EDTA and EGTA, the indicated concentrations of the chelator were added to the assay mixture without CaCl2. Fluorogenic assays were carried out substituting various MCA substrates for pERTKR-MCA. For K m determinations, increasing amounts of pERTKR-MCA were added to the reaction mixture. The data, plotted as hydrolysis activityversus [pERTKR-MCA], were subject to nonlinear regression analysis (KaleidaGraph) to determine the K m andV max values. For the representative assays depicted in the figures, the data were expressed as a percentage of the calculated V max and replotted. The pro-PC4 and pro-epidermal growth factor (pro-EGF) peptides were synthesized on an automated solid phase peptide synthesizer (Applied Biosystems, model 431A) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)-based FastMoc chemistry (22Jean F. Basak A. Rondeau N. Benjannet S. Hendy G.N. Seidah N.G. Chrétien M. Lazure C. Biochem. J. 1993; 292: 891-900Crossref PubMed Scopus (88) Google Scholar, 23Basak A. Boudreault A. Chen A. Chrétien M. Seidah N.G. Lazure C. J. Pept. Sci. 1995; 1: 385-395Crossref PubMed Scopus (41) Google Scholar). The following side chain protecting groups were used:tert-butyloxycarbonyl for Lys; tert-butyl for Ser, Thr, Tyr, Asp, and Glu; and 2,2,5,7,8-pentamethylchroman 6-sulfonyl for Arg. At the end of the synthesis, the peptides were cleaved from the resin and fully deprotected by treating the resin with reagent K (22Jean F. Basak A. Rondeau N. Benjannet S. Hendy G.N. Seidah N.G. Chrétien M. Lazure C. Biochem. J. 1993; 292: 891-900Crossref PubMed Scopus (88) Google Scholar, 23Basak A. Boudreault A. Chen A. Chrétien M. Seidah N.G. Lazure C. J. Pept. Sci. 1995; 1: 385-395Crossref PubMed Scopus (41) Google Scholar) for 5 h, followed by lyophilization and repeated washings with ether. The peptides were purified by RP-HPLC (Beckman, model 5500) on a semi-preparative Vydac column 218TP510G8 (1.0 × 25 cm) (Chromatographic Specialty Corp., Canada). The buffer system consisted of an aqueous phase containing 0.1% trifluoroacetic acid (v/v) and an organic phase of acetonitrile also containing 0.1% trifluoroacetic acid (v/v). The elution was carried out at a flow rate of 2 ml/min beginning with a 5-min isocratic step of 5% organic phase followed by a linear gradient of 5–60% organic phase over 60 min. Eluted materials were monitored at a wavelength of 225 nm. Both peptides were fully characterized by FAB mass spectrometry as well as by amino acid analysis (23Basak A. Boudreault A. Chen A. Chrétien M. Seidah N.G. Lazure C. J. Pept. Sci. 1995; 1: 385-395Crossref PubMed Scopus (41) Google Scholar). The synthetic pro-parathyroid hormone (pro-PTH) tridecapeptide was a gift from Dr. G. N. Hendy, Calcium Research Laboratory, Royal Victoria Hospital, McGill University, and has been described previously (27Hendy G.N. Bennett H.P.J. Gibbs B.F. Lazure C. Day R. Seidah N.G. J. Biol. Chem. 1995; 270: 9517-9525Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The pro-PTH 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. Time courses of substrate digestion by BCRD-hfurin, rPC7, and BTMD-rPC7 were first carried out to optimize the digestion conditions and to define the period during which the reaction proceeded at a constant rate (not shown). Thus, the reaction times chosen for the K m determinations were from 10 to 20 min. The digestion products were analyzed using RP-HPLC separation (Varian, model 9010) on a Beckman 5-μm Ultrasphere C18 column (0.2 × 25 cm) as described above for peptide purification except that the buffer system also 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 collected peptides (two product peaks along with that of the undigested peptide, not shown) were identified and quantitated by amino acid composition as described (27Hendy G.N. Bennett H.P.J. Gibbs B.F. Lazure C. Day R. Seidah N.G. J. Biol. Chem. 1995; 270: 9517-9525Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The K m 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. A schematic representation of the sequence of the full-length rPC7 and three truncated versions lacking the putative transmembrane domain and cytosolic tail (5Seidah N.G. Hamelin J. Mamarbachi M. Dong W. Tadros H. Mbikay M. Chrétien M. Day R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3388-3393Crossref PubMed Scopus (225) Google Scholar), denoted BTMD-rPC7 and C-terminally shortened versions called BTMD-S-rPC7, is shown in Fig.1. These molecular structures are compared with those of ykexin, the full-length hfurin, and its truncated version lacking the cysteine-rich domain, called BCRD-hfurin. The 17-amino acid peptide sequence indicated within the P-domain was used as an immunogen to obtain a polyclonal rPC7 antiserum, permitting the identification and characterization of the various forms of rPC7 obtained via overexpression in BSC40 cells. Thus, following overnight incubation of cells infected with VV recombinants expressing either rPC7K or BTMD-rPC7K, the cell extracts, and media were analyzed by Western blotting. As shown in Fig.2, an rPC7-specific doublet (101 and 89 kDa, at a ratio of approximately 1:3 as determined by densitometric analysis of the ECL film) is visible only in the membrane fraction of cell lysates obtained from VV:rPC7K-infected cells. (The construct VV:rPC7K produced 2–3 times more enzyme than that containing the native initiator sequence, results not shown.) These two proteins were resistant to solubilization with 0.1m Na2CO3 and even low (<5%) concentrations of Triton X-100 (not shown), indicating that rPC7 is an integral membrane protein. These findings were further supported by enzymatic activity assays of crude cellular fractions (TableI), which showed that 93% of the total rPC7-specific pERTKR-MCA hydrolytic activity was present in cell membranes. Analysis of BSC40 cells infected with VV:ykexin revealed that 21% of its activity is shed into the medium during a 7-h collection (subsequent to an overnight incubation), whereas 66% remains membrane-associated (Table I). Similarly, infections of BSC40 cells with VV:hfurin (full-length) resulted in the secretion of active, soluble furin (28Rehemtulla A. Kaufman R.J. Blood. 1992; 79: 2349-2355Crossref PubMed Google Scholar, 29Vidricaire G. Denault J.B. Leduc R. Biochem. Biophys. Res. Commun. 1993; 195: 1011-1018Crossref PubMed Scopus (77) Google Scholar). These data thus confirm that rPC7, unlike hfurin and ykexin, remains as a membrane-associated protein which is not significantly shed into the medium.Figure 2Immunodetection of rPC7 and BTMD-rPC7.Samples from VV:wild type-, rPC7-, and BTMD-rPC7-infected BSC40 cells were processed as described under “Experimental Procedures” and run on 7.5% SDS reducing gels. Following electrotransfer to polyvinylidene difluoride membranes, protein bands were visualized via ECL detection using a primary rabbit antiserum raised against the synthetic rPC7 MAP (indicated in Fig. 1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IComparative distribution of selected convertase enzymatic activitiesEnzymeCulture mediumCell lysate supernatant1-aSupernatant from microcentrifuge spin at 16,000 × g of cells disrupted by Dounce homogenizer (see “Experimental Procedures”).Cell lysate membranesnmol/h 1-bEDTA inhibitable pERTKR-MCA hydrolysis. (% of total activity) 1-cRepresents percentage of the sum of the total activities from the various cell fractions collected from a P-15 tissue culture dish.WT1-dInfection at 2 pfu per cell.1.20.80.0(61)(39)(0)ykexin1-dInfection at 2 pfu per cell.6.03.618.6(21)(13)(66)rPC71-eInfection at 4 pfu per cell.0.40.918.2(2)(5)(93)BTMD-rPC71-eInfection at 4 pfu per cell.80.64.55.5(89)(5)(6)BCRD-hfurin1-dInfection at 2 pfu per cell.43.70.00.0(100)(0)(0)1-a Supernatant from microcentrifuge spin at 16,000 × g of cells disrupted by Dounce homogenizer (see “Experimental Procedures”" @default.
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• W2128548712 title "In Vitro Characterization of the Novel Proprotein Convertase PC7" @default.
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• W2128548712 doi "https://doi.org/10.1074/jbc.272.32.19672" @default.
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