Processing and Sorting of the Prohormone Convertase 2 Propeptide
2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês
10.1074/jbc.m003547200
ISSN1083-351X
AutoresLaurent Muller, Angus Cameron, Yolanda M. Fortenberry, Ekaterina V. Apletalina, Iris Lindberg,
Tópico(s)Biotin and Related Studies
ResumoThe prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion. The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion. prohormone convertase aminomethylcoumarin α-melanocyte-stimulating hormone endoplasmic reticulum trans-Golgi network phorbol 12-myristate 13-acetate radioimmunoassay human 7B2-(155–186)-carboxy-terminal peptide Dulbecco's modified Eagle's medium bovine serum albumin high pressure liquid chromatography polyacrylamide gel electrophoresis 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol methylcoumarinamide adrenocorticotropic hormone. CT peptide, carboxyl-terminal peptide of 7B2 Chinese hamster ovary A feature common to a great variety of secreted and membrane proteins, ranging from receptors to peptide hormones and neuropeptides, is initial synthesis as a precursor that must undergo endoproteolytic processing in order to acquire biological activity (for reviews see Refs. 1Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva A.A.J. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (313) Google Scholar and 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (241) Google Scholar). A family of mammalian serine proteases responsible for these proteolytic maturation events, termed the prohormone/proprotein convertases (PCs),1 has been identified in the last 10 years (for reviews see Refs. 2Seidah N.G. Chretien M. Curr. Opin. Biotechnol. 1997; 8: 602-607Crossref PubMed Scopus (241) Google Scholar, 3Creemers J.W. Jackson R.S. Hutton J.C. Semin. Cell Dev. Biol. 1998; 9: 3-10Crossref PubMed Scopus (57) Google Scholar, 4Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). The PCs are structurally homologous to subtilisin and kexin and belong to the enzymatic superfamily of subtilases. All of the PCs contain the following five domains: an amino-terminal signal peptide that directs them to the secretory pathway; a propeptide of approximately 80–100 residues; a catalytic domain and a P domain, which are well conserved between PCs; and a carboxyl-terminal domain (which is specific for each PC). The P domain, only present within the eukaryotic subtilisin-like enzymes, is apparently required for activation (5Gluschankof P. Fuller R.S. EMBO J. 1994; 13: 2280-2288Crossref PubMed Scopus (86) Google Scholar) and may be responsible for regulation of enzymatic activity (6Zhou A. Martin S. Lipkind G. LaMendola J. Steiner D.F. J. Biol. Chem. 1998; 273: 11107-11114Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The carboxyl-terminal domain is not required for activation but contains structural determinants necessary for the membrane attachment and the sorting of the PCs (7Molloy S.S. Thomas L. VanSlyke J.K. Stenberg P.E. Thomas G. EMBO J. 1994; 13: 18-33Crossref PubMed Scopus (420) Google Scholar, 8Creemers J.W. Usac E.F. Bright N.A. Van de Loo J.W. Jansen E. Van de Ven W.J. Hutton J.C. J. Biol. Chem. 1996; 271: 25284-25291Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9De 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, 10Van de Loo J.W. Creemers J.W. Bright N.A. Young B.D. Roebroek A.J. Van de Ven W.J. J. Biol. Chem. 1997; 272: 27116-27123Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Finally, the role of the propeptide remains to be determined precisely. This domain is specifically required for the activation of the PCs, and its proteolytic processing constitutes part of the mechanism of activation (7Molloy S.S. Thomas L. VanSlyke J.K. Stenberg P.E. Thomas G. EMBO J. 1994; 13: 18-33Crossref PubMed Scopus (420) Google Scholar, 11Rehemtulla A. Dorner A.J. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8235-8239Crossref PubMed Scopus (72) Google Scholar, 12Creemers J.W. Vey M. Schafer W. Ayoubi T.A. Roebroek A.J. Klenk H.D. Garten W. Van de Ven W.J. J. Biol. Chem. 1995; 270: 2695-2702Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Propeptides have historically been involved in several different aspects of the post-translational modification and maturation of proproteins, both in the intracellular targeting, stabilization, and activation of proenzymes, as well as inhibition of their cognate enzymes. Propeptides are responsible for the targeting of cytosolic proteins (13Wagner I. van Dyck L. Savel'ev A.S. Neupert W. Langer T. EMBO J. 1997; 16: 7317-7325Crossref PubMed Scopus (37) Google Scholar) and secreted proteins, including proteases (14Valls L.A. Hunter C.P. Rothman J.H. Stevens T.H. Cell. 1987; 48: 887-897Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 15Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 2105-2116Crossref PubMed Scopus (190) Google Scholar, 16McIntyre G.F. Godbold G.D. Erickson A.H. J. Biol. Chem. 1994; 269: 567-572Abstract Full Text PDF PubMed Google Scholar) and peptide precursors such as prosomatostatin (17Sevarino K.A. Stork P. Ventimiglia R. Mandel G. Goodman R.H. Cell. 1989; 57: 11-19Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 18Stoller T.J. Shields D. J. Cell Biol. 1989; 108: 1647-1655Crossref PubMed Scopus (102) Google Scholar). Propeptides are also responsible for the stabilization of proenzymes, as in the case of cathepsins (19Mach L. Mort J.S. Glossl J. J. Biol. Chem. 1994; 269: 13036-13040Abstract Full Text PDF PubMed Google Scholar). Another major function of propeptides is their role in the activation of the mature protein they will form (20Gray A.M. Mason A.J. Science. 1990; 247: 1328-1330Crossref PubMed Scopus (221) Google Scholar, 21Suter U. Heymach Jr., J.V. Shooter E.M. EMBO J. 1991; 10: 2395-2400Crossref PubMed Scopus (129) Google Scholar). In the case of proteases, propeptides are involved in both the activation of the zymogen and the regulation of the activity of the mature enzyme (for review see Ref. 22Khan A.R. James M.N. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (381) Google Scholar). These features result both from the role of the propeptide as an intramolecular chaperone that controls zymogen folding and from the inhibitory potency of the propeptide. The intramolecular chaperone function of the propeptide was first demonstrated for prosubtilisin and pro-α-lytic protease and has since been extended to a number of proproteins (for reviews see Refs. 23Inouye M. Enzyme ( Basel ). 1991; 45: 314-321Crossref PubMed Scopus (92) Google Scholar, 24Baker D. Shiau A.K. Agard D.A. Curr. Opin. Cell Biol. 1993; 5: 966-970Crossref PubMed Scopus (152) Google Scholar, 25Siezen R.J. Leunissen J.A.M. Shinde U. Shinde U. Inouye M. in Intramolecular Chaperones and Protein Folding. R. G. Landes Co., Austin, TX1995: 233-256Google Scholar). Among these various functions, little is known concerning the role of the PC propeptides, because the activation mechanism of these zymogens has not yet been completely elucidated. The proposed model for the activation of the subtilases occurs through the following steps. 1) Autocatalytic cleavage of the propeptide; 2) binding of the propeptide to the mature enzyme, which results in inhibition of catalytic activity; and 3) inactivation/degradation of the propeptide, which terminates the inhibition by the propeptide (26Shinde U. Inouye M. J. Mol. Biol. 1995; 247: 390-395Crossref PubMed Scopus (52) Google Scholar). A study of the activation of furin provided evidence that this model could also be applicable to the PCs (27Lesage G. Prat A. Lacombe J. Thomas D.Y. Seidah N.G. Boileau G. Mol. Biol. Cell. 2000; 11: 1947-1957Crossref PubMed Scopus (27) Google Scholar). Recently, the inhibitory potency of the PC1 and PC7 propeptides was also demonstrated (28Boudreault A. Gauthier D. Lazure C. J. Biol. Chem. 1998; 273: 31574-31580Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 29Zhong M. Munzer J.S. Basak A. Benjannet S. Mowla S.J. Decroly E. Chretien M. Seidah N.G. J. Biol. Chem. 1999; 274: 33913-33920Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Whereas the activation pathway of furin shares some characteristics with other PCs, the activation of PC2 is unique in the following respects (for review see Ref. 30Muller L. Lindberg I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 69-108Crossref PubMed Scopus (125) Google Scholar). (i) The PC2 propeptide is not removed in the endoplasmic reticulum (ER) within minutes after synthesis but rather in the acidic compartments of the trans-Golgi network (TGN)/secretory granules with a half-life in the hour range (31Guest P.C. Arden S.D. Bennett D.L. Clark A. Rutherford N.G. Hutton J.C. J. Biol. Chem. 1992; 267: 22401-22406Abstract Full Text PDF PubMed Google Scholar, 32Benjannet S. Rondeau N. Paquet L. Boudreault A. Lazure C. Chretien M. Seidah N.G. Biochem. J. 1993; 294: 735-743Crossref PubMed Scopus (172) Google Scholar, 33Shen F.S. Seidah N.G. Lindberg I. J. Biol. Chem. 1993; 268: 24910-24915Abstract Full Text PDF PubMed Google Scholar, 34Zhou A. Mains R.E. J. Biol. Chem. 1994; 269: 17440-17447Abstract Full Text PDF PubMed Google Scholar). (ii) Intermediate molecular forms, including a major 71-kDa protein with an amino-terminally truncated propeptide, have been described in addition to pro-PC2 and PC2 (31Guest P.C. Arden S.D. Bennett D.L. Clark A. Rutherford N.G. Hutton J.C. J. Biol. Chem. 1992; 267: 22401-22406Abstract Full Text PDF PubMed Google Scholar, 33Shen F.S. Seidah N.G. Lindberg I. J. Biol. Chem. 1993; 268: 24910-24915Abstract Full Text PDF PubMed Google Scholar). (iii) Pro-PC2 must interact with the neuroendocrine protein 7B2 in the secretory pathway in order to become competent for activation (35Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar); if 7B2 has not been encountered intracellularly, proteolytic removal of the propeptide leads to the formation of an inactive mature enzyme species, termed unproductive maturation (36Zhu X. Muller L. Mains R.E. Lindberg I. J. Biol. Chem. 1998; 273: 1158-1164Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 37Westphal C.H. Muller L. Zhou A. Zhu X. Bonner-Weir S. Schambelan M. Steiner D.F. Lindberg I. Leder P. Cell. 1999; 96: 689-700Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Pro-PC2 thus offers unique opportunities for delineating the role of the convertase propeptide in the activation mechanism of convertases. Indeed, pro-PC2 is the only PC that has been purified as an activable zymogen (38Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (86) Google Scholar), thus permitting in vitro analysis of propeptide maturation. In addition, because pro-PC2 is the only PC that exits the ER as a zymogen, it is possible to distinguish between the following two steps in the folding process of its zymogen: global folding required for exit from the ER, and local folding of the catalytic site required for activation. Accordingly, we have been able to identify mutations in PC2 catalytic domain that result in the generation of proenzymes that are processed, in the TGN/secretory granules, into totally inactive PC2s (36Zhu X. Muller L. Mains R.E. Lindberg I. J. Biol. Chem. 1998; 273: 1158-1164Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In this report, we have investigated the following four aspects of the function of the propeptide in the activation of pro-PC2: 1) the processing of the propeptide in vitro, using purified recombinant pro-PC2; 2) the processing of the PC2 propeptide in vivo, using mutated pro-PC2s stably transfected in neuroendocrine cells; 3) the inhibitory potency of propeptide fragments using synthetic peptides; and 4) the fate of the propeptide in the TGN/secretory granules. Unless specifically stated, all PC and peptide antisera used were polyclonal antisera raised in rabbits. The antiserum against PC2 (LSU18) was directed against a carboxyl-terminal peptide of mature PC2 (35Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar). The antiserum against the PC2 propeptide (LSU26) was directed against residues His58–Asp80 (Fig. 1, hatched gray box) of mouse pro-PC2 conjugated to keyhole limpet hemocyanin (Pierce) using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide; the peptide contained an amino-terminal tyrosine to enable iodination. The antiserum against α-melanocyte-stimulating hormone (α-MSH) was obtained from Chemicon (Temecula, CA) and was raised in sheep (39Elias C.F. Saper C.B. Maratos-Flier E. Tritos N.A. Lee C. Kelly J. Tatro J.B. Hoffman G.E. Ollmann M.M. Barsh G.S. Sakurai T. Yanagisawa M. Elmquist J.K. J. Comp. Neurol. 1998; 402: 442-459Crossref PubMed Scopus (771) Google Scholar). AtT-20 cells were cultivated at 37 °C in 5% CO2. AtT-20 cells were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% Nuserum IV (Becton Dickinson, Franklin Lakes, NJ), and 2.5% fetal bovine serum (Irvine Scientific, Santa Ana, CA). Transfected cell lines were cultivated with either one or both of the following selection agents: 200 μg/ml active G418 (Life Technologies, Inc.) and/or 100 μg/ml hygromycin (Sigma). The AtT-20/PC2 cell line was kindly provided by Dr. R. E. Mains (The Johns Hopkins School of Medicine, Baltimore, MD) (34Zhou A. Mains R.E. J. Biol. Chem. 1994; 269: 17440-17447Abstract Full Text PDF PubMed Google Scholar). We have already characterized the AtT-20/PC2/7B2 and the Rin/Proenkephalin/7B2 cell lines (40Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Crossref PubMed Scopus (76) Google Scholar). In order to obtain cell lines co-expressing 7B2 and mutated pro-PC2s, AtT-20 cells were first transfected with a pCEP4-derived plasmid (Invitrogen, Carlsbad, CA) encoding rat 27-kDa 7B2 (35Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar). A high expressing clone was selected using immunoblotting and was supertransfected with pcDNA3 (Invitrogen) encoding mutated mouse pro-PC2 cDNAs. The mutations were obtained by the polymerase chain reaction-mediated primer overlap method as described previously (35Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar). For this purpose aHindIII-KpnI fragment of mouse pro-PC2 was amplified and reintroduced into the HindIII vector site and the pro-PC2 KpnI site inHindIII/KpnI-cut pcDNA3/pro-PC2. The mutagenesis primers used were as follows: 5′-GCCAAGCACAAGCACAGCCTACACCATAAGCGG-3′ and 5′-GCTGTGCTTGTGCTTGGCCTTTGCAAGCCCATT-3′ for the M53-56 mutant; 5′-GACAAGCACAAGCACGGGTACAGGGACATCAAT-3′ and 5′-CCCGTGCTTGTGCTTGTCAAATCCTTCTTGTTG-3′ for the M81-84 mutant; 5′-GTCCCTGTACCCGTGCTTTTTACGGTCAAATCC-3′ and 5′-AAGCACGGGTACAGGGACATCAAT-3′ for the R84H mutant; 5′-AAGGCAGGGTACAGGGACATCAAT-3′ and 5′-CCCTGCCTTTTTACGGTCAAATCC-3′ for the R84A mutant; and 5′-AAACACAGAGGGTACAGGGACATC-3′ and 5′-CCTGTACCCTCTGTGTTTACGGTCAAATCCTTC-3′ for the K83H mutant. All cDNA segments generated by polymerase chain reactions were verified by DNA sequencing. Cells were transfected using Lipofectin (Life Technologies, Inc.) (35Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar). Three independent clones were analyzed for each mutation. 5 × 105 AtT-20 cells were used 40–48 h after seeding in 6-well plates. They were labeled with either 500 μCi/ml [35S]methionine and -cysteine (Pro-Mix, Amersham Pharmacia Biotech) for 20 min or 500 μCi/ml [35S]methionine (Amersham Pharmacia Biotech) for 8 h. Cells were then directly extracted or chased in DMEM containing 2% fetal bovine serum. For the analysis of the maturation of mutant pro-PC2s, proteins were extracted under denaturing conditions, and immunoprecipitation was conducted at 4 °C as described previously (35Zhu X. Lindberg I. J. Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (144) Google Scholar). Briefly, extracts were first incubated for 1 h in the presence of protein A coupled to Sepharose CL-4B (Amersham Pharmacia Biotech). The pre-cleared supernatants were then incubated for 4 h in the presence of PC2 antiserum LSU18. The immune complexes were precipitated with protein A coupled to Sepharose CL-4B and washed once with the immunoprecipitation buffer, once with 0.5 m NaCl in phosphate-buffered saline, and twice with Dulbecco's modified Eagle's medium. They were then boiled in Laemmli sample buffer prior to separation by SDS-PAGE on 8.8% acrylamide gels. Gels were either treated for fluorography with Amplify (Amersham Pharmacia Biotech) or directly exposed to a phosphoscreen and analyzed using either a PhosphorImager (Storm, Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (Molecular Dynamics) or on an InstantImager (Packard Instrument Co.). For the in vitro analysis of pro-PC2 processing, cells were chased in DMEM containing 2% fetal bovine serum for 2, 4, or 12 h in the presence of 1 μm bafilomycin A1 (Kamiya Biomedical Company, Seattle, WA). Proteins were then extracted under native conditions as described previously (41Muller L. Zhu X. Lindberg I. J. Cell Biol. 1997; 139: 625-638Crossref PubMed Scopus (87) Google Scholar), and PC2 forms were immunoprecipitated as described above. The beads were then resuspended in 100 mm Bis-Tris, 100 mm sodium acetate at the indicated pH and incubated at 37 °C for 30 min. The reaction was stopped by adding Laemmli sample buffer and boiling for 5 min. Proteins were analyzed as described above. Pro-PC2 was purified from the medium of CHO cells expressing high levels of pro-PC2 and 7B2 as described previously (42Johanning K. Juliano M.A. Juliano L. Lazure C. Lamango N.S. Steiner D.F. Lindberg I. J. Biol. Chem. 1998; 273: 22672-22680Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Pro-PC2 (∼5–15 μg) was incubated in 100 mm Bis-Tris, 100 mm sodium acetate at the indicated pH at 37 °C. In some experiments the incubations were performed in the presence of 1 mm EDTA or 10 μm of the carboxyl-terminal peptide of 7B2 (CT peptide), a PC2-specific inhibitor (43Lindberg I. van den Hurk W.H. Bui C. Batie C.J. Biochemistry. 1995; 34: 5486-5493Crossref PubMed Scopus (76) Google Scholar). An aliquot was boiled in Laemmli sample buffer for Western blotting. Another aliquot was stopped by adding 4 volumes of 8 m urea and boiled for 5 min for the HPLC analysis of the fragments generated. Samples were fractionated on a C4 column (Vydac, Hesperia, CA) at a 1 ml/min flow rate using a three-step gradient consisting of 0–40% buffer B for 57 min, 40–70% B for 37 min, 70–100% B for 27 min, with buffer B being 80% acetonitrile with 0.06% trifluoroacetic acid and buffer A being 0.052% trifluoroacetic acid. 100-μl duplicate aliquots of each fraction were dried in the presence of carrier bovine serum albumin (BSA) and analyzed for PC2 propeptide content using a novel radioimmunoassay (RIA) using the LSU26 antiserum (see below). Amino-terminal peptide sequencing of the immunoreactive fractions was performed by the Protein and Carbohydrate Structure Facility (University of Michigan, Ann Arbor, MI), and matrix-assisted laser desorption ionization/time of flight-mass spectroscopy was performed by the LSU Core Labs (LSU Health Sciences Center, New Orleans, LA). PC2 activity was measured using 0.2 mm pGlu-Arg-Thr-Lys-Arg-MCA (Peptides International, Lexington, KY) (final concentration) as a substrate as described previously (39Elias C.F. Saper C.B. Maratos-Flier E. Tritos N.A. Lee C. Kelly J. Tatro J.B. Hoffman G.E. Ollmann M.M. Barsh G.S. Sakurai T. Yanagisawa M. Elmquist J.K. J. Comp. Neurol. 1998; 402: 442-459Crossref PubMed Scopus (771) Google Scholar). The assay was performed at 37 °C in 100 mm sodium acetate, pH 5, containing 5 mmCaCl2 and 0.4% octyl glucoside, in the presence of a protease inhibitor mixture composed of 1 μm pepstatin, 0.28 mm tosylphenylalanine chloromethyl ketone, 1 μm trans-epoxysuccinic acid, and 0.14 mm tosyl-lysyl chloromethyl ketone (this inhibitor mixture was omitted when purified recombinant PC2 was used). In order to determine the enzymatic activity of the wild type and mutant PC2s, intracellular pro-PC2/PC2 was immunopurified from cell extracts (obtained by nondenaturing lysis from triplicate plates) using antiserum LSU18 bound to protein A-coupled Sepharose CL-4B. An aliquot of these same cell extracts was saved for protein determination using the BCA protein assay (Pierce), and similar amounts of cellular protein were subjected to Western blotting using antiserum against PC2 (LSU18). The activity of immunopurified enzymes prepared from the various cell lines was assessed under standard conditions for a 2-h reaction time and was normalized to the total intracellular content of PC2 forms expressed in that cell line. The entire experiment was repeated independently with similar results, and the results were also confirmed using overnight conditioned media from each cell line. For analysis of inhibition by propeptide-related peptides, duplicate reactions were used, consisting of 7 nm mouse recombinant pro-PC2, 0 to 0.1 mm of the peptide to be tested, and 0.2 mm fluorogenic substrate. Prior to addition to the reaction mixture, purified recombinant mouse pro-PC2 was diluted in the assay buffer and preactivated by incubating for 30 min at 37 °C to obtain the 66-kDa form. Peptides corresponding to residues 57–84, 57–80 (containing an S57Y substitution), and 35–56 of mouse pro-PC2 were synthesized by LSU Health Sciences Center Core Labs. After preactivated PC2 was mixed with the peptide, the reaction mixtures were incubated at room temperature for 30 min, and the reaction was initiated by adding substrate followed by incubation at 37 °C for 30 min. The amount of released aminomethylcoumarin (AMC) was measured with a microtiter plate fluorometer (Ascent, Labsystems) using excitation and emission wavelengths of 380 and 460 nm, respectively. The amount of released product was calculated by reference to the fluorescence of free AMC standard. IC50 values were calculated by applying nonlinear regression to the data using the GraphPad Prism program. The values were determined from four independent experiments using two different PC2 preparations. Purified pro-PC2 was activated at pH 5.0 for 30 min at room temperature prior to addition of 0.2 mm Glu(P)-Arg-Thr-Lys-Arg-MCA and varying (0–1 μm) quantities of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Alexis Biochemicals, San Diego, CA) under conditions such thatET ≫ Kiapp. Reactions were incubated at 37 °C. Least square regression was used to calculateET from the resulting quasi-linear plots as described by the Morrison equation (44Williams J.W. Morrison J.F. Methods Enzymol. 1979; 63: 437-467Crossref PubMed Scopus (659) Google Scholar). Brains from wild type mice or from PC2 knockout mice (45Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Crossref PubMed Scopus (353) Google Scholar) were directly homogenized on ice in 0.1m HCl. Extracts were frozen, thawed, and centrifuged before lyophilization. Dried samples were then made up to 1.5 ml in 0.1% trifluoroacetic acid and centrifuged, and 500 μl of the clear supernatant was fractionated by HPLC. The propeptide was assayed in duplicate aliquots of each fraction by RIA, as described below. AtT-20 cells, expressing PC2 alone or PC2 and 7B2, or Rin5f cells, expressing proenkephalin alone or with 7B2, were plated in 35-mm wells. Confluent cells were first incubated for 2 h under basal conditions. The medium was replaced for 3 more h, with fresh medium containing 100 nm phorbol 12-myristate 13-acetate (PMA). Collected media were spun to remove floating cells and directly assayed for propeptide immunoreactive by RIA. Cells were homogenized in 1m acetic acid, 20 mm HCl, and 0.1% (v/v) β-mercaptoethanol. The extracts were frozen, thawed, centrifuged, and lyophilized before being subjected to the propeptide and α-MSH/ACTH-(1–13)-NH2 or Met-enkephalin RIAs. In some experiments, the cell extracts or the stimulated medium were separated by HPLC as described above. Media were first concentrated using SepPak C18 columns (Waters, Milford, MA) eluted with 60% isopropyl alcohol, 0.1% trifluoroacetic acid and diluted in buffer A prior to application to the HPLC column. Aliquots of the HPLC fractions were dried together with 50 μl of 0.05% carrier BSA, and the propeptide content was measured by RIA. Propeptide RIAs were carried out using 100 μl of a 1:40,000 dilution of antiserum LSU26, 100 μl of sample or standard, and 100 μl of iodinated peptide containing 10,000 cpm in RIA buffer consisting of 0.1 msodium phosphate, pH 7.4, 0.1% heat-treated BSA, 0.1% sodium azide, and 50 mm sodium chloride. In order to enable iodination, the peptide to be radiolabeled (pro-PC2 58–80) was synthesized with the addition of an amino-terminal Tyr. This Tyr57-substituted peptide was also used to generate the standard curve. The IC50 of the assay was 60 fmol, and the range of the standard curve was from 1 to 500 fmol. Other details of the RIA procedure were as described previously (46Lindberg I. J. Biol. Chem. 1986; 261: 16317-16322Abstract Full Text PDF PubMed Google Scholar). Samples were subjected to carboxypeptidase B digestion prior to propeptide assay (42Johanning K. Juliano M.A. Juliano L. Lazure C. Lamango N.S. Steiner D.F. Lindberg I. J. Biol. Chem. 1998; 273: 22672-22680Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The Met-enkephalin RIA has already been described (46Lindberg I. J. Biol. Chem. 1986; 261: 16317-16322Abstract Full Text PDF PubMed Google Scholar).The α-MSH/ACTH-(1–13)-NH2 RIA was accomplished using a 1:120,000 final dilution of antiserum (raised in sheep against α-MSH conjugated to thyroglobulin; Chemicon AB5087) and a standard curve of 1–250 fmol. Due to the specificity of the antibody for the amide moiety, cross-reaction was negligible for either intact ACTH, α-MSH-free acid, or ACTH-(1–10) (39Elias C.F. Saper C.B. Maratos-Flier E. Tritos N.A. Lee C. Kelly J. Tatro J.B. Hoffman G.E. Ollmann M.M. Barsh G.S. Sakurai T. Yanagisawa M. Elmquist J.K. J. Comp. Neurol. 1998; 402: 442-459Crossref PubMed Scopus (771) Google Scholar). 2L. Muller, A. Cameron, Y. Fortenberry, E. V. Apletalina, and I. Lindberg, unpublished results. Overall sequence conservation between the PC propeptides is quite low, as shown in Fig. 1,panel A. However, all propeptides are cleaved at a common RXKR locus, referred to as the primary site (Fig. 1, panel B), generating the mature PCs. In addition, all of the PCs except PC7 contain a secon
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