Identification of Prodomain Determinants Involved in ADAMTS-1 Biosynthesis
2004; Elsevier BV; Volume: 279; Issue: 32 Linguagem: Inglês
10.1074/jbc.m313151200
ISSN1083-351X
AutoresJean‐Michel Longpré, Richard Leduc,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe metalloprotease ADAMTS-1 (adisintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR173 and RKKR235) revealed that Arg235 was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys106, Tyr108, Gly110, Cys125, and Cys181 and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis. The metalloprotease ADAMTS-1 (adisintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR173 and RKKR235) revealed that Arg235 was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys106, Tyr108, Gly110, Cys125, and Cys181 and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis. Proteolysis of extracellular substrates by the ADAMTS 1The abbreviations used are: ADAMTS, adisintegrin and metalloprotease with thrombospondin type I motif; PC, proprotein convertase; MMP, matrix metalloproteinases; CHO RPE.40, Chinese hamster ovary resistance to Pseudomonas exotoxin A. (adisintegrin and metalloprotease with thrombospondin type I motif) family, which consists of at least 19 members, is an important mechanism regulating events such as cartilage biosynthesis, angiogenesis, and cell motility and growth (1Iruela-Arispe M.L. Carpizo D. Luque A. Ann. N. Y. Acad. Sci. 2003; 995: 183-190Crossref PubMed Scopus (86) Google Scholar). The first member, ADAMTS-1 (peptidase M12.222, Merops data base), identified as a cachexia-associated gene expressed in colon tumor cells (2Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar), along with ADAMTS-4 and ADAMTS-5 (also called aggrecanases), degrades to different extents the cartilage proteoglycan aggrecan and lectican or aggrecan-like proteins such as brevican and versican. This suggested a significant participation of these enzymes in conditions such as arthritis (3Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (620) Google Scholar, 4Abbaszade I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 5Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. J. Biol. Chem. 2001; 276: 13372-13378Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 6Matthews R.T. Gary S.C. Zerillo C. Pratta M. Solomon K. Arner E.C. Hockfield S. J. Biol. Chem. 2000; 275: 22695-22703Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). ADAMTS-1 is also anti-angiogenic, a property possibly explained by the recent finding that it sequesters vascular endothelial growth factor-1 (7Luque A. Carpizo D.R. Iruela-Arispe M.L. J. Biol. Chem. 2003; 278: 23656-23665Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The phenotype of ADAMTS-1-/- mice revealed marked reduction in size with body weights of ∼70% of their wild-type or heterozygous littermates, and fertilization was impaired in females (8Shindo T. Kurihara H. Kuno K. Yokoyama H. Wada T. Kurihara Y. Imai T. Wang Y. Ogata M. Nishimatsu H. Moriyama N. Oh-hashi Y. Morita H. Ishikawa T. Nagai R. Yazaki Y. Matsushima K. J. Clin. Investig. 2000; 105: 1345-1352Crossref PubMed Scopus (272) Google Scholar). ADAMTS-2, ADAMTS-14, and ADAMTS-3 are procollagen N-proteinases that proteolytically remove amino peptides in the processing of type I and type II procollagens to collagens (9Colige 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 (302) Google Scholar, 10Colige A. Vandenberghe I. Thiry M. Lambert C.A. Van Beeumen J. Li S.W. Prockop D.J. Lapiere C.M. Nusgens B.V. J. Biol. Chem. 2002; 277: 5756-5766Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Fernandes 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 (197) Google Scholar). Deficiency of ADAMTS-2 led to an inherited connective tissue disorder called dermatosparaxis in animals and the Ehlers-Danlos syndrome (dermatosparactic-type) in humans. Reports have recently demonstrated that mutations in the ADAMTS-13 gene cause thrombotic thrombocytopenic purpura, a coagulation disorder, and that ADAMTS-13 is required for the processing of large von Willebrand factor multimers (12Levy 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 (1449) Google Scholar). The functions of most of the other ADAMTS members remain to be better clarified. The structure of all of the ADAMTS members includes a signal peptide for access to the secretory pathway, a prodomain, a catalytic metalloprotease domain, a disintegrin-like domain, and a carboxyl-terminal ancillary domain having a conserved modular structure but containing a variable number of thrombospondin, type I repeat-like domains (2Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). Unlike the ADAM family, the ADAMTS members do not contain a transmembrane domain yet they may be located in the vicinity of the cell via binding to cell surface molecules or the pericellular matrix (13Somerville 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 (274) Google Scholar). Thus ADAMTSs are secreted proteins anchored to the cell surface or to the extracellular matrix (14Kuno K. Terashima Y. Matsushima K. J. Biol. Chem. 1999; 274: 18821-18826Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). ADAMTS proteases, similar to ADAM and MMPs, are synthesized as zymogens, which require activation via the proteolytic removal of a prodomain. The size of the prodomain varies, because it is in part determined by the location of the carboxylterminal most furin-processing site; however, in all of the ADAMTSs with the exception of ADAMTS-13, which has an overall atypical primary structure, the size is ∼200 residues. At least one sequence, but usually multiple furin recognition sequences, occurs in the prodomain of most members of the ADAMTS family. ADAMs, MMPs, and ADAMTS-1, ADAMTS-2, ADAMTS-4, ADAMTS-5, ADAMTS-9, and ADAMTS-12 (3Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (620) Google Scholar, 4Abbaszade I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 13Somerville 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 (274) Google Scholar, 15Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (208) Google Scholar, 16Cal S. Arguelles J.M. Fernandez P.L. Lopez-Otin C. J. Biol. Chem. 2001; 276: 17932-17940Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Wang W.M. Lee S. Steiglitz B.M. Scott I.C. Lebares C.C. Allen M.L. Brenner M.C. Takahara K. Greenspan D.S. J. Biol. Chem. 2003; 278: 19549-19557Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) have been shown to be processed by furin-like proprotein convertases. The convertase family is comprised of seven calcium-dependent serine proteases (18Bergeron F. Leduc R. Day R. J. Mol. Endocrinol. 2000; 24: 1-22Crossref PubMed Scopus (165) Google Scholar) that recognize various sequence motifs containing permutations of basic residues in the P1, P2, P4, and P6 positions (19Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4762) Google Scholar). One of the most studied convertases, furin, is concentrated in the trans-Golgi network and cycles between this compartment and the cell surface through the exocytic/endocytic pathway (20Molloy S.S. Thomas L. Kamibayashi C. Mumby M.C. Thomas G. J. Cell Biol. 1998; 142: 1399-1411Crossref PubMed Scopus (83) Google Scholar). The autoactivation and intracellular trafficking of furin are well characterized events (21Thomas G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 753-766Crossref PubMed Scopus (940) Google Scholar). Because zymogen activation is a critical post-translational regulatory step, we have addressed in this study, the critical determinant affecting this process. Although convertases like PACE4 and PC6B may process proADAMTS-1, we demonstrate that furin is the most efficient convertase at cleaving the proADAMTS-1 precursor intracellularly at Arg235 of the furin recognition sequence RKKR235. Moreover, we also identify different conserved residues within the prodomains of ADAMTS family members that could be involved in the maturation of proADAMTS-1. These observations could be of broad significance to understand the regulation of the ADAMTS proteases. Cell Culture and Transfection—QBI-293A cells (293A, Quantum Biotechnologies, Montréal, QC, Canada) derived from the human embryonic kidney-293 cell line were grown in complete Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. CHO RPE.40 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Subconfluent cells were transfected with 2 μl of FuGENE 6 reagent (Roche Diagnostics, Laval, QC, Canada) per microgram of DNA 24 h prior to each experiment. Site-directed Mutagenesis—Human ADAMTS-1 cDNA in pcDNA-3.1MycHis has been previously described (22Rodriguez-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 (134) Google Scholar). The construction of ADAMTS-1 prodomain mutants was performed using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA) with the following oligonucleotides: R173A (5′-CACCTCCTGCGGCGGAATGCTCAGGGCGACGTAGGCGGC-3′, mutated codon is underlined); R235A (5′-ATAAGAAAGAAGGCTTTTGTGTCCAGTC-3′); C106A (5′-GACCTGGCGCACGCTTTCTACTCCGGC-3′); Y108A (5′-GCGCACTGCTTCGCTTCCGGCACCGTG-3′); G110A (5′-TGCTTCTACTCCGCTACCGTGAATGGC-3′); T111A (5′-CTTCTACTCCGGCGCTGTGAATGGCGATC-3′); V112A (5′-CTACTCCGGCACCGCTAATGGCGATCCC-3′); P116A (5′-GTGAATGGCGATGCTAGCTCGGCTGCC-3′); C125A (5′-GCCCTCAGCCTCGCTGAGGGCGTGCGC-3′); C181A (5′-GTAGGCGGCACGGCTGGGGTCGTGGAC-3′); and 8A (amino acids 137-144 mutated to alanine) (5′-GCCTTCTACCTGCTGGGGGCTGCTGCTGCTGCTGCTGCTGCTCCCGCCGCCAGCGAGCGC-3′). The different convertases were expressed using the following constructions. The human furin cDNA in pCI-Neo was described elsewhere (23Denault J. Bissonnette L. Longpre J. Charest G. Lavigne P. Leduc R. FEBS Lett. 2002; 527: 309-314Crossref PubMed Scopus (25) Google Scholar). The cDNA encoding mouse PC6B was subcloned in NotI of pRcCMV (a gift from K. Nakayama, Tsukuba, Japan). The human PC7 (a gift from R. Day, Université de Sherbrooke, Sherbrooke, Canada) and PACE4 (a gift from R. E. Mains, The University of Connecticut Health Center, Farmington, CT) cDNA were inserted in EcoRI/XbaI-linearized pcDNA3. All of the constructs were confirmed by DNA sequencing. Production of Anti-human ADAMTS-1 and Anti-PC7 Antibodies—The human ADAMTS-1 polyclonal antibodies were obtained from rabbits injected with a peptide (Ile-His-Asp-Glu-Gln-Lys-Gly-ProGlu-Val-Thr-Ser-Cys) coupled to keyhole limpet hemocyanin (Pierce). The peptide encompasses residues 295-306 of the human ADAMTS-1 catalytic domain. The hPC7 polyclonal antibodies were obtained from rabbits injected with a peptide (Asp-Gly-ProHis-Gln-Leu-Gly-Lys-Ala-Ala-Leu-Gln-His-Cys) coupled to keyhole limpet hemocyanin. The peptide encompasses residues 298-310 of the human PC7 catalytic domain. Rabbits were injected with antigen emulsified in an equal volume of TiterMax adjuvant (Sigma-Aldrich). Metabolic Labeling and Immunoprecipitation—Metabolic-labeling experiments were performed 24 h post-transfection. Cells were washed with warm phosphate-buffered saline and incubated in Met/Cys-free medium (modified Eagle's medium Select-Amine kit, Invitrogen) supplemented with 10% dialyzed fetal calf serum, 1 mml-glutamine, and 50 μCi of Expre35S35S (PerkinElmer Life Science Products) for the indicated period of time (pulse). Chase was done in complete medium. After recovering the medium, the cell layer was washed with phosphate-buffered saline and the cells were lysed with 1 ml of radioimmunoprecipitation assay buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 4 mm EDTA, and 0.1% SDS) containing protease inhibitors (1 μm aprotinin, 10 μm pepstatin, 10 μm leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Samples were centrifuged to remove insoluble material. Human ADAMTS-1 antiserum, anti-furin (24Denault J.B. Lazure C. Day R. Leduc R. Protein Expression Purif. 2000; 19: 113-124Crossref PubMed Scopus (21) Google Scholar), anti-PACE4 (Alexis Biochemicals), anti-PC6B (Alexis Biochemicals); or anti-PC7 was added, and samples were incubated overnight at 4 °C. Protein A/G Plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) beads were added and incubated for 1 h at 4 °C. Beads were washed three times with 1 ml of radioimmunoprecipitation assay buffer, and labeled proteins were resolved by SDS-PAGE. Gels were treated with ENHANCE reagent (PerkinElmer Life Science), dried, and exposed for fluorography. Brefeldin A at 36 μm (Sigma) or monensin at 3.6 or 36 μm (Sigma) was added to the labeling mixture when indicated. Western Blot—Transfected CHO RPE.40 cells were lysed in 1 ml of radioimmunoprecipitation assay buffer. 30 μl of each sample were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot analysis was performed with monoclonal mouse anti-actin antibody (Chemicon). The antibodies were visualized with the horseradish peroxidase-coupled sheep anti-mouse immunoglobulin (Amersham Biosciences) using the Western Lightning Chemiluminescence Reagent Plus according to the manufacturer's instructions (PerkinElmer Life Sciences). Deglycosylation—Deglycosylation of immunoprecipitated proteins from cell lysate and conditioned medium of ADAMTS-1 transfected cells was performed following immunoprecipitation using 10 units of N-glycosidase F (Roche Applied Science) for 3 h at 37 °C in 150 mm sodium phosphate, pH 7.4, 50 mm EDTA, 0.1% SDS, 1% 2-mercapthoethanol, and 0.5% Triton X-100 followed by SDS-PAGE and fluorography. Biosynthesis of ADAMTS-1—Previous work (22Rodriguez-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 (134) Google Scholar) describe how processing of proADAMTS-1 is required for its activation. To better characterize how the proregion of ADAMTS-1 is involved in the biosynthesis of the enzyme, we transfected human kidney 293 cells with an expression vector containing the complete human ADAMTS-1 cDNA. Pulse-chase experiments were initially carried out to analyze the onset of ADAMTS-1 synthesis. ADAMTS-1 proteins were immunoprecipitated with a polyclonal anti-ADAMTS-1 recognizing the IHDEQKGPEVTS sequence present in the metalloproteinase domain of ADAMTS-1. As seen in Fig. 1B, we detected in cell lysates after a 15-min pulse a 110-kDa form that corresponds well to the theoretical molecular mass (100 kDa, 916 residues) of unprocessed proADAMTS-1 (Fig. 1A). Following a 15-min chase period, a 87-kDa form corresponding to the proteolytically active ADAMTS-1 appears in the cells with a complete processing of proADAMTS-1 after 60 min of chase. The mature 87-kDa ADAMTS-1 is also detected in the media after 30 min. A 2-hour chase period enabled us to detect a doublet at 65 kDa in the media (Fig. 1C), a result of C-terminal processing by a metal-loproteinase (22Rodriguez-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 (134) Google Scholar). Deglycosylation of ADAMTS-1—Longer pulse labeling and chase times enabled us to detect a doublet at 87 and 65 kDa. Because ADAMTS-1 possesses three N-glycosylation sites, we investigated the glycosylated state of the doublets. Deglycosylation with N-glycosidase F after metabolic labeling reveals that the upper band of the doublet at 87 kDa represents an N-glycosylated form as can be seen by the significant shift in electrophoretic mobility to an apparent molecular mass of 82 kDa, more in line with the calculated molecular mass of 79 kDa, which would correspond to mature ADAMTS-1 (residues 236-950, Fig. 1C). Moreover, the two forms at 65 kDa also appear to be N-glycosylated because both forms are shifted to lower molecular masses following N-glycosidase F treatment. Processing of ProADAMTS-1 by Convertases—Although furin is the better characterized proprotein convertase, PACE4, PC6B, and PC7 also cleave precursor proteins within the constitutive secretory pathway (25Zhou 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) but their precise function is still unclear. To investigate whether these convertases process proADAMTS-1, we co-transfected ADAMTS-1 cDNA with the cDNA of these different convertases in the furin-deficient CHO RPE.40 cells (26Spence M.J. Sucic J.F. Foley B.T. Moehring T.J. Somat. Cell Mol. Genet. 1995; 21: 1-18Crossref PubMed Scopus (30) Google Scholar). As can be seen in Fig. 2A, proADAMTS-1 is processed to 87 kDa in CHO RPE.40 cells by endogenous proteases but less effectively than in 293 cells as demonstrated by the presence of the 110-kDa zymogen form in the medium (compare Fig. 2A with Fig. 1 where the 110-kDa form is detected in the medium). Co-transfection of furin with ADAMTS-1 led to the complete processing of the 110-kDa form into the 87-kDa band and the 65-kDa doublet. PACE4 and PC6B also cleaved proADAMTS-1 as demonstrated by the reduced intensity of the 110-kDa zymogen form in both cells and media but not as efficiently as furin. Interestingly, PC7-dependent processing of proADAMTS-1 was very weak. Expression levels of the different convertases were verified by immunoprecipitating each convertase when co-transfected with ADAMTS-1 (Fig. 2B). Fig. 2B shows that mature furin (apparent molecular mass of 100 kDa) (27Bissonnette L. Charest G. Longpre J.M. Lavigne P. Leduc R. Biochem. J. 2004; 379: 757-763Crossref PubMed Scopus (15) Google Scholar), PACE4 (103 kDa) (28Taniguchi T. Kuroda R. Sakurai K. Nagahama M. Wada I. Tsuji A. Matsuda Y. Biochem. Biophys. Res. Commun. 2002; 290: 878-884Crossref PubMed Scopus (9) Google Scholar), PC6B (theoretical molecular mass of 195 kDa), and PC7 (92 kDa) (29van 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) are all expressed at similar levels. Sample loading was verified by Western blot using anti-actin (Fig. 2C). These results suggest that proADAMTS-1 is efficiently converted to the active 87- and 65-kDa forms by furin and, to a lesser extent, by PACE4, PC6B, and PC7. Because furin in the most ubiquitously expressed of all of the convertases, it is very likely to be the bona fide physiological zymogen convertase of the ADAMTS family. However, it is conceivable that other proteases of this family may also play a role in zymogen activation under certain conditions. Arg235 Is Required for ProADAMTS-1 Maturation—Many ADAMTS family members possess more than one putative furin recognition sequence, RXXR, within their prodomain. There are two putative cleavage sites (RRNR173 and RKKR235) within the ADAMTS-1 prodomain. To assess the effect of abolishing either one of these sites on ADAMTS-1 biosynthesis, we performed site-directed mutagenesis on the P1 residue of each cleavage sequence by replacing Arg with Ala (ADAMTS-1/R173A and ADAMTS-1/R235A in Fig. 3A). Fig. 3B shows the result of metabolic labeling of 293A cells previously transfected either with ADAMTS-1/R173A or ADAMTS-1/R235A cDNA followed by immunoprecipitation with anti-ADAMTS-1 antiserum. We observed that the biosynthetic profile of the ADAMTS-1/R173A mutant did not differ from that associated with wild-type ADAMTS-1. However, cells transfected with the ADAMTS-1/R235A cDNA did not process the 110-kDa form, which is also found intact in the media, indicating the necessity of Arg at position 235 for cleavage. No other bands were detected, suggesting that the alternative furin recognition site at position 173 was not used to produce other maturation fragments. Moreover, no additional processing was observed such as the C-terminal processing of the 87-kDa form into the C-terminally cleaved forms, indicating that these latter entities require the preliminary cleavage of the zymogen form. Thus, it is possible that the C-terminally processed forms observed at 65 kDa arise from the autocatalytic action of the proteolytically active forms of ADAMTS-1. Because we had observed some processing of proADAMTS-1 in CHO RPE.40 cells, we investigated whether cleavage in these cells was also dependent on the furin recognition sites. Fig. 3C shows that proteolysis also required Arg235 for proper processing, because only the zymogen form (110 kDa) of ADAMTS-1/R235A was present in the cell extracts and in the media. Supplementing these cells with furin did not promote processing into the mature forms, suggesting the absolute requirement of Arg235 in the maturation process. Intracellular Localization of ADAMTS-1 Prodomain Cleavage—To further define the intracellular compartment where proADAMTS-1 activation occurs, we treated cells expressing ADAMTS-1 with the Golgi-disturbing agents Brefeldin A and monensin (Fig. 4). Pulse-labeling analysis revealed that Brefeldin A, which inhibits protein transport between the endoplasmic reticulum and the Golgi apparatus, abolished the processing of the 110-kDa proADAMTS-1 form into the 87- and 65-kDa mature forms. The cells were also treated with monensin, a known inhibitor of post-Golgi transport (30Dinter A. Berger E.G. Histochem. Cell Biol. 1998; 109: 571-590Crossref PubMed Scopus (319) Google Scholar). At 3.6 μm, monensin interfered with but did not completely abolish the production of the mature 87-kDa form that was detected in the media, although this secretion was blocked at higher doses (36 μm). Taken together, these results identify the Golgi network as the major site of proADAMTS-1 processing. Mutagenesis of Conserved ADAMTS-1 Prodomain Residues—To determine whether residues other than the furin recognition sequences are important in the maturation and secretion of ADAMTS-1, we aligned the prodomains of ADAMTS family members because conserved residues could potentially be required or implicated in these processes. Fig. 5 shows the alignment of the 19 human ADAMTS prodomains using hierarchical clustering. Only that portion of the prodomains with the highest sequence homology is shown, and identical amino acids found in >12 ADAMTS prodomains are termed "consensus" residues. This alignment identifies 15 conserved residues and motifs in the prodomain of ADAMTS besides the furin recognition sequences found at the C-terminal end of the prodomains. Site-directed mutagenesis was performed to investigate the effect of replacing these conserved amino acids in the prodomain of ADAMTS-1. Fig. 6A illustrates the different Ala mutants of ADAMTS-1 used in the study. Pulse-chase analysis was performed in human embryonic kidney-293 cells transfected with nine different ADAMTS-1 prodomain mutants (Fig. 6B). First, we found that the zymogen of every mutant expressed was detected in cell extracts. The T111A, V112A, and P116A mutants essentially behaved similarly to the wild-type ADAMTS-1 with regards to their processing pattern. Replacing residues 137-144 by Ala (ADAMTS-1/8A) completely abolished the processing of the 110-kDa zymogen form into the 87-kDa active moiety, but this zymogen form failed to be detected in the media. For the C106A and C125A mutants, processing was greatly reduced and, similar to ADAMTS-1/8A mutants, no detectable forms were observed in the media. Conversely, significantly lower amounts of processed forms of Y108A, G110A, and C181A were found in the media. Intriguingly, although zymogen forms of these mutants were detected intracellularly, they were not observed as secreted products as was the furin recognition sequence mutant R235A (Fig. 3B), indicating that zymogen activation is not a prerequisite for secretion. Moreover, it can be seen that an overall reduction of immunoreactive material can be observed for these mutants, suggesting that a percentage of these proteins may undergo inefficient folding leading to degradation. Nevertheless, those immunoreactive m
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