Cell-surface Processing of Pro-ADAMTS9 by Furin
2006; Elsevier BV; Volume: 281; Issue: 18 Linguagem: Inglês
10.1074/jbc.m511083200
ISSN1083-351X
AutoresBon-Hun Koo, Jean-Michel Longpré, Robert Somerville, J. Preston Alexander, Richard Leduc, Suneel Apte,
Tópico(s)Cell Adhesion Molecules Research
ResumoProcessing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases. Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases. Zinc metalloendopeptidases, like most proteases, are synthesized as zymogens, and the N-terminal propeptide is usually excised. Propeptide excision usually leads to enzymatic activation, an important regulatory event, and it can occur intracellularly, at the cell surface, or extracellularly through a variety of proteolytic mechanisms. In one such mechanism, the propeptide is proteolytically excised by serine proteases of the mammalian subtilisin-like proprotein convertase (PC) 4The abbreviations used are: PC, proprotein convertase; MMP, matrix metalloprotease; TGN, trans-Golgi network; Pro-Cat, signal peptide, propeptide, and catalytic domain; cmk, chloromethyl ketone; PNGase F, peptide N-glycosidase F; siRNA, small interference RNA; CHO, Chinese hamster ovary cells; ADAM, a disintegrin and metalloprotease domain; ADAMTS, a disintegrin and metalloprotease domain with thrombospondin type 1 repeats. 4The abbreviations used are: PC, proprotein convertase; MMP, matrix metalloprotease; TGN, trans-Golgi network; Pro-Cat, signal peptide, propeptide, and catalytic domain; cmk, chloromethyl ketone; PNGase F, peptide N-glycosidase F; siRNA, small interference RNA; CHO, Chinese hamster ovary cells; ADAM, a disintegrin and metalloprotease domain; ADAMTS, a disintegrin and metalloprotease domain with thrombospondin type 1 repeats. family (1Thomas G. 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It is a type I transmembrane protein that is itself synthesized as a zymogen that undergoes autocatalytic intramolecular activation (16Leduc R. Molloy S.S. Thorne B.A. Thomas G. J. Biol. Chem. 1992; 267: 14304-14308Abstract Full Text PDF PubMed Google Scholar). Furin cleaves on the carboxyl side of a consensus recognition site that is rich in basic residues (e.g. Arg-Xaa-Arg/Lys-Arg↓) (2Bergeron F. Leduc R. Day R. J. Mol. Endocrinol. 2000; 24: 1-22Crossref PubMed Scopus (164) Google Scholar, 4Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar, 5Seidah N.G. Prat A. Essays Biochem. 2002; 38: 79-94Crossref PubMed Scopus (185) Google Scholar, 17Zheng M. Seidah N.G. Pintar J.E. Dev. Biol. 1997; 181: 268-283Crossref PubMed Scopus (41) Google Scholar). Most furin resides in the TGN, but some is present at the plasma membrane and shuttles between the cell surface and the TGN (18Mayer G. Boileau G. Bendayan M. J. 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Chem. 1993; 268: 26461-26465Abstract Full Text PDF PubMed Google Scholar). However, the physiological role of cell-surface or secreted furin in processing endogenous cellular products has remained elusive.The ADAMTS proteases are a family of 19 secreted enzymes, of which some have critical physiological functions and have been implicated in inherited human disorders, namely Ehlers-Danlos syndrome type VIIC (ADAMTS2), Weill-Marchesani syndrome (ADAMTS10), and inherited thrombocytopenic purpura (ADAMTS13) (23Apte S.S. Int. J. Biochem. Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (210) Google Scholar, 24Colige A. Sieron A.L. Li S.W. Schwarze U. Petty E. Wertelecki W. Wilcox W. Krakow D. Cohn D.H. Reardon W. Byers P.H. Lapiere C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; 65: 308-317Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 25Dagoneau N. Benoist-Lasselin C. Huber C. Faivre L. Megarbane A. Alswaid A. Dollfus H. Alembik Y. Munnich A. Legeai-Mallet L. Cormier-Daire V. Am. J. Hum. Genet. 2004; 75: 801-806Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 26Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemieniak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sabio H. Bouhassira E.E. Upshaw Jr., J.D. Ginsburg D. Tsai H.M. Nature. 2001; 413: 488-494Crossref PubMed Scopus (1419) Google Scholar). Overexpression of ADAMTS5 and ADAMTS4 is implicated in proteolytic loss of aggrecan, a major cartilage component, in arthritis (27Arner E.C. Curr. Opin. Pharmacol. 2002; 2: 322-329Crossref PubMed Scopus (134) Google Scholar). ADAMTS proteases (except ADAMTS13, the von Willebrand factor-processing protease, whose propeptide contains 41 amino acid residues) are synthesized with propeptides of ∼220-240 amino acids, which are larger than the MMP and ADAM propeptides. Almost all ADAMTS propeptides have consensus sites for the attachment of N-linked oligosaccharide (Asn-Xaa-Thr/Ser, where Xaa is any amino acid except Pro). Consensus recognition sequences for PCs are present at the junction of the propeptide and catalytic domain, and ADAMTS proteases may have additional, more N-terminal PC-processing sites within their propeptides. Previous studies of ADAMTS1, ADAMTS4, and ADAMTS7 suggested that, like ADAMs and the furin-processed MMPs, ADAMTS proteases were processed in the TGN by PCs, although some cell-surface processing of ADAMTS7 was also observed (11Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 12Kuno K. Terashima Y. Matsushima K. J. Biol. Chem. 1999; 274: 18821-18826Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 13Longpre J.M. Leduc R. J. Biol. Chem. 2004; 279: 33237-33245Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Wang P. Tortorella M. England K. Malfait A.M. Thomas G. Arner E.C. Pei D. J. Biol. Chem. 2004; 279: 15434-15440Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 28Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang L.W. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).ADAMTS9 is one of only two ADAMTS proteases that is highly conserved during evolution (29Llamazares M. Cal S. Quesada V. Lopez-Otin C. J. Biol. Chem. 2003; 278: 13382-13389Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 31Huxley-Jones J. Apte S.S. Robertson D.L. Boot-Handford R.P. Int. J. Biochem. Cell Biol. 2005; 37: 1838-1845Crossref PubMed Scopus (42) Google Scholar). Its mRNA is widely expressed (29Llamazares M. Cal S. Quesada V. Lopez-Otin C. J. Biol. Chem. 2003; 278: 13382-13389Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), especially during embryonic development (32Jungers K.A. Le Goff C. Somerville R.P. Apte S.S. Gene Expr. Patterns. 2005; 5: 609-617Crossref PubMed Scopus (74) Google Scholar). ADAMTS9 is up-regulated by inflammatory cytokines in chondrocytes (33Demircan K. Hirohata S. Nishida K. Hatipoglu O.F. Oohashi T. Yonezawa T. Apte S.S. Ninomiya Y. Arthritis Rheum. 2005; 52: 1451-1460Crossref PubMed Scopus (87) Google Scholar). It may have a role in atherosclerosis and arthritis, because it can proteolytically process the proteoglycans versican and aggrecan (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), which are important constituents of the vessel wall, and cartilage, respectively. This potential biological significance of ADAMTS9 prompted an in-depth analysis of the processing of its propeptide. In the initial characterization of ADAMTS9, we demonstrated that it could be processed at more than one site (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In continuing these studies, we have made the unexpected observation that ADAMTS9 processing is exclusively extracellular and occurs at the cell surface in cells that express high levels of furin. These results establish a precedent for the cell-surface activation of precursor endogenous proteins by furin and are possibly of broad biological relevance.EXPERIMENTAL PROCEDURESExpression Plasmids and Site-directed Mutagenesis—Plasmids for expression of full-length ADAMTS9, or a truncated form containing the signal peptide, propeptide, and catalytic domain (Pro-Cat) with C-terminal Myc and His tags were described previously (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Due to low expression levels of full-length ADAMTS9, we excised its open reading frame with the Myc-His tags as a NotI/PmeI fragment and then recloned this into pCEP4 (Invitrogen) digested with KpnI and blunt-ended followed by NotI digestion. To insert a FLAG tag between Thr276 and Arg277 in the propeptide, site-directed mutagenesis (QuikChange kit, Stratagene) was performed using the forward primer 5′-AATAAGACGGACAACACAGACTACAAGACGATGACGACAAGAGAGAAAAGAGGACCCAC-3′ (with FLAG encoding site underlined) and the reverse primer 5′-GTGGGTCCTCTTTTCTCTCTTGTCGTCATCGTCTTTGTAGTCTGTGTTGTCCGTCTTATT-3′ (FLAG encoding site underlined). Plasmids for expression of full-length ADAMTS1 or the propeptide and catalytic domain of ADAMTS7 (ADAMTS7-Pro-Cat) were previously described (11Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 28Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang L.W. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).Antibodies and Immunoblotting—The peptides RP1 and RP4 representing different regions of the ADAMTS9 propeptide were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and conjugated to KLH. New Zealand White male rabbits (7-8 pounds) were immunized with the conjugates at biweekly intervals for 8 weeks. After an initial injection in Freund's complete adjuvant, subsequent injections were given in incomplete adjuvant. Antibody titer was measured by enzyme-linked immunosorbent assay using free peptides. Affinity-purified antibodies were prepared using the respective immobilized antigens. Anti-penta-His monoclonal antibody, anti-Myc monoclonal antibody 9E10, and anti-FLAG M2 monoclonal antibody were obtained from commercial sources (Invitrogen and Sigma-Aldrich). Anti-furin monoclonal antibody was purchased from Alexis Biochemicals (San Diego, CA). Immunoblotting was done using denaturing SDS-PAGE and electroblotting to polyvinylidene fluoride membrane followed by detection of the bound antibody using enhanced chemiluminescence (Amersham Biosciences).Cell Culture, Transfection, and Cell Treatments—HEK293F cells stably transfected with ADAMTS9 or Pro-Cat, COS-1 cells, LoVo cells (ATCC no. CCL-299), CHO-K1 cells, CHO RPE.40 (35Spence M.J. Sucic J.F. Foley B.T. Moehring T.J. Somat. Cell Mol. Genet. 1995; 21: 1-18Crossref PubMed Scopus (30) Google Scholar), and rat chondrosarcoma RCS-LTC cells (36Fernandes R.J. Hirohata S. Engle J.M. Colige A. Cohn D.H. Eyre D.R. Apte S.S. J. Biol. Chem. 2001; 276: 31502-31509Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) were maintained as described previously (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) or as per vendors' instructions. Transient transfections were done using FuGENE6 (Roche Diagnostics, Indianapolis, IN) as per the manufacturer's recommendations. For inhibition of PCs, cells were treated with the PC inhibitory peptide dec-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk, Calbiochem) at different concentrations as indicated or with the polypeptide α1-PDX (Portland variant of α 1-antitrypsin) (37Dufour E.K. Denault J.B. Hopkins P.C. Leduc R. FEBS Lett. 1998; 426: 41-46Crossref PubMed Scopus (33) Google Scholar).Detection of Cell-surface Processing—Biotinylation of cells was done on ice to prevent labeling of intracellular proteins, essentially as previously described (28Somerville R.P. Longpre J.M. Apel E.D. Lewis R.M. Wang L.W. Sanes J.R. Leduc R. Apte S.S. J. Biol. Chem. 2004; 279: 35159-35175Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). As a control to eliminate cell-surface proteins, cells were treated with 0.05% trypsin/0.53 mm EDTA for 20 min on ice, and trypsin was subsequently inactivated with 10% fetal bovine serum-supplemented medium.To examine the fate of cell-surface biotinylated protein, biotinylated cells were transferred to serum-free medium, and cells and media were collected at sequential time points. Biotinylated proteins were captured from samples using streptavidin-agarose (Sigma-Aldrich) and eluted by boiling in Laemmli sample buffer followed by SDS-PAGE and Western blotting with appropriate antibodies.Pro-Cat was purified from lysates of stably transfected cells using Pro-Bond resin (Invitrogen). For determination of processing of exogenously added Pro-Cat by cells, untransfected HEK293F cells were cultured to confluence on 6-well plates and then in 293 SFM-II medium (Invitrogen). Purified Pro-Cat (50 μg/well) was added to untransfected HEK293F cells, and the cells were cultured further over an 8-h period. The medium was taken for analysis at successive time points. In parallel experiments purified Pro-Cat was incubated with cell-free conditioned medium from HEK293F cells to investigate whether the processing was cell-mediated or not. The samples were analyzed by Western blotting with anti-Myc.Enzymatic Deglycosylation—Removal of N-linked carbohydrate with peptide N-glycosidase F (PNGase F, New England Biolabs, Beverly, MA) was performed as previously described (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar).Co-transfection of Pro-Cat and Proprotein Convertases—Furin-deficient CHO RPE.40 cells (35Spence M.J. Sucic J.F. Foley B.T. Moehring T.J. Somat. Cell Mol. Genet. 1995; 21: 1-18Crossref PubMed Scopus (30) Google Scholar) and LoVo cells were transfected with Pro-Cat alone or together with plasmids encoding the proprotein convertases furin, PACE4, and PC5A (kindly provided by Dr. Nabil Seidah). 48 h after transfection, cells were incubated in 293 SFM-II medium for 24 h, and Western blotting of the cell lysate and medium was performed with anti-Myc.Cell-surface Cross-linking and Immunoprecipitation—48 h after transfection with the Pro-FLAG-Cat plasmid, HEK293F cells were washed four times with phosphate-buffered saline and treated with the thiolcleavable, membrane-nonpermeable cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) (Pierce Endogen, Rockford, IL) to cross-link molecules at the cell surface. The cells were lysed in lysis buffer (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, protease inhibitor mixture, Roche Diagnostics) for 1 h at 4 °C and centrifuged. The soluble portion of the lysate was transferred to a fresh tube and incubated overnight with anti-FLAG-agarose (Sigma-Aldrich) at 4 °C with rotation. Anti-FLAG-agarose was pelleted by centrifugation at 1000 rpm in a microcentrifuge and washed six times in lysis buffer. The supernatant was discarded, and the bound protein was eluted with 0.1 m glycine/HCl (pH 3.5) from the resin. The eluted samples were analyzed by Western blotting with anti-furin monoclonal antibody (Alexis Biochemicals) or RP4 antibody.Metabolic Labeling and Pulse-Chase Analysis—HEK-293F cells stably expressing Pro-Cat were grown to confluence and incubated in Dulbecco's modified Eagle's medium without cysteine and methionine (Sigma-Aldrich). After 24 h, the medium was replaced with cysteinemethionine-free medium containing radioactive cysteine/methionine (50 μCi/well in 6-well plates, Pulse medium) for 15 min using EXPRE35S35S (PerkinElmer Life Sciences). For pulse-chase analysis, the pulse medium was removed, and the cells were washed three times on ice with phosphate-buffered saline containing 0.5 mm MgCl2 and 0.5 mm CaCl2, followed by incubation for 0, 15, 30, 60, or 120 min in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Chase medium) at 37 °C. At the end of the chase period, cells were placed on ice, the medium was collected, and the cells were washed four times with phosphate-buffered saline and extracted with lysis buffer. In parallel, samples were processed for removal of cell-surface proteins in these assays by treatment with trypsin/EDTA for 20 min on ice, and trypsin was subsequently inactivated with 10% fetal bovine serum supplemented medium. The media and cell lysates were processed for immunoprecipitation with anti-Myc followed by fluorography as described previously (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar).RNA Interference—HEK293F cells stably expressing Pro-Cat were transfected with 20 pmol of Furin ShortCut siRNA Mix (New England Biolabs) using Lipofectamine 2000 (Invitrogen). After 48 h of incubation without antibiotics, the medium was changed to 293 SFM-II medium (Invitrogen), and cells were incubated for a further 24 h. The conditioned medium and cell lysate were analyzed by Western blotting with anti-Myc and anti-RP4. Cell-surface biotinylation was done as above.RESULTSThe Proteolytically Processed ADAMTS9 Propeptide Is Present in the Conditioned Medium but Not in the Cell Lysate—The ADAMTS9 propeptide (Fig. 1, A and B) has five consensus furin-recognition sequences (Arg-Xaa-Xaa-Arg). Arg-Lys-Asp-Arg33 and Arg-Thr-Arg-Arg74 are near the signal peptidase processing site, whereas the remaining three sites (Arg-Glu-Lys-Arg280, Arg-Thr-His-Arg283, and Arg-Thr-Lys-Arg287) overlap and are clustered near the junction of the propeptide and catalytic domain. We previously showed that mutation of Arg74 and Arg287 abrogated processing at these sites, whereas mutation of Arg280, which compromised two putative sites, was without effect (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). The propeptide also contains three sequence motifs for attachment of N-linked oligosaccharides (Fig. 1B), whereas the catalytic domain has none.Two nonoverlapping synthetic peptides from the propeptide (RP1 and RP4; sequences are not provided for proprietary reasons of Triple Point Biologics) within the propeptide were used for the production of polyclonal antisera. In HEK293F cells stably expressing full-length ADAMTS9 (Fig. 2A), anti-RP4 recognized the expected ∼180-kDa zymogen in the cell lysate. Smaller polypeptides (37 kDa and a ∼20-22-kDa doublet) were recognized in the conditioned medium. These bands were not seen in untransfected HEK293F cells (Fig. 2A). When the medium was deglycosylated using PNGase F, the 37-kDa band and the ∼22-kDa doublet increased in mobility and migrated at 28 and 15 kDa, respectively, demonstrating the presence of N-linked carbohydrate (Fig. 2B), characteristic of propeptide fragments. Notably, the ∼22-kDa doublet converted to a ∼15-kDa doublet, indicating two protein species rather than glycoforms of the same fragment. When these experiments were conducted using an ADAMTS9 active site mutant (ADAMTS9 Glu381 → Ala, a widely used metalloprotease inactivating mutation (12Kuno K. Terashima Y. Matsushima K. J. Biol. Chem. 1999; 274: 18821-18826Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 38Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjument-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar)), no differences were seen (Fig. 2B); thus, the anti-RP4-reative fragments were not autoproteolytic in origin. In Western blotting with anti-RP1 the medium showed a weakly immunoreactive 37-kDa band but not the ∼22-kDa doublet (Fig. 2C), whereas PNGase F treatment clearly resolved the 28-kDa band seen with RP4 and a 15-kDa doublet also seen with RP4. The comparable results with RP4 and RP1 as well as the presence of glycosylation identified these fragments in the medium as originating from the propeptide. The RP1 peptide is adjacent to an N-linkage site, and the carbohydrate may mask reactivity of the ∼22-kDa fragments without prior PNGase F treatment. Because anti-RP4 provided more robust immunoreactivity, it was used in subsequent experiments.FIGURE 2Characterization of propeptide antibodies and absence of intracellular processing in cells transfected with various ADAMTS9 plasmids. The zymogen (Z), processed propeptide fragments (P), and processed catalytic domain (C) are indicated. A-C, Western blot analysis of transfected HEK293F cell and conditioned medium (CM) expressing full-length ADAMTS9 or ADAMTS9-Glu381 → Ala (E>A). Antibodies used are shown under each immunoblot. D, Western blot analysis of HEK293F cells and conditioned medium (CM) expressing ADAMTS9 Pro-Cat using anti-RP4 (left panel) or anti-Myc (right panel). The structure of Pro-Cat is illustrated above the gels. E, effect of PNGase F on migration of anti-RP4-reactive bands from CM. F, Western blot analysis of CM, treated, or untreated with PNGase F from HEK293F cells expressing Pro-FLAG-Cat. The structure of this construct is shown above the gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because the large size and extensive glycosylation of full-length ADAMTS9 (30Somerville R.P. Longpre J.M. Jungers K.A. Engle J.M. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) precludes accurate resolution in gels, the above studies were repeated using an expression plasmid encoding the ADAMTS9 signal peptide, propeptide, and catalytic domain (Pro-Cat) with a Myc-His tag at the C terminus (Fig. 2D). The results were identical, i.e. anti-RP4 detected the intact Pro-Cat zymogen (55 kDa) only in the cell lysate, and 37- and 20-22-kDa fragments were found only in the conditioned medium (Fig. 2D). Similarly, anti-Myc detected only the unprocessed 55-kDa Pro-Cat zymogen in the cell lysate, and only the processed catalytic domain (29 kDa) in conditioned medium (Fig. 2D). Incubation of conditioned medium with PNGase F enhanced the mobility of propeptide fragments to the same extent as with full-length ADAMTS9 (Fig. 2E). That these fragments originated from the propeptide was further verified by insertion of a FLAG epitope tag between Thr276 and Arg277, i.e. upstream of the furin processing site at Arg287. Western blotting with anti-FLAG provided similar results to that with anti-RP4, with differences (Fig. 2F) reflecting distinct locations of the FLAG and RP4 epitopes within the propeptide. These results demonstrate that the propeptide is part of the intact zymogen in HEK293F cells, but that it is present as a distinct, proteolytically derived entity in their conditioned medium.We conclude that the 37-kDa band seen with all propeptide antibodies corresponds to the complete propeptide. When deglycosylated, its size (28 kDa), is compatible with processing at the most N- and C-terminal PC processing sites, i.e. Leu34-Arg287 (28 kDa). The site at which the processing of the pro
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