Fibulin-1 Acts as a Cofactor for the Matrix Metalloprotease ADAMTS-1
2005; Elsevier BV; Volume: 280; Issue: 41 Linguagem: Inglês
10.1074/jbc.m506980200
ISSN1083-351X
AutoresNathan V. Lee, Juan Carlos Rodrı́guez-Manzaneque, Shelley N.-M. Thai, Waleed O. Twal, Alfonso Luque, Karen M. Lyons, W. Scott Argraves, M. Luisa Iruela‐Arispe,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoADAMTS-1 is a metalloprotease that has been implicated in the inhibition of angiogenesis and is a mediator of proteolytic cleavage of the hyaluronan binding proteoglycans, aggrecan and versican. In an attempt to further understand the biological function of ADAMTS-1, a yeast two-hybrid screen was performed using the carboxyl-terminal region of ADAMTS-1 as bait. As a result, the extracellular matrix protein fibulin-1 was identified as a potential interacting molecule. Through a series of analyses that included ligand affinity chromatography, co-immunoprecipitation, pulldown assays, and enzyme-linked immunosorbent assay, the ability of these two proteins to interact was substantiated. Additional studies showed that ADAMTS-1 and fibulin-1 colocalized in vivo. Furthermore, fibulin-1 was found to enhance the capacity of ADAMTS-1 to cleave aggrecan, a proteoglycan known to bind to fibulin-1. We confirmed that fibulin-1 was not a proteolytic substrate for ADAMTS-1. Together, these findings indicate that fibulin-1 is a new regulator of ADAMTS-1-mediated proteoglycan proteolysis and thus may play an important role in proteoglycan turnover in tissues where there is overlapping expression. ADAMTS-1 is a metalloprotease that has been implicated in the inhibition of angiogenesis and is a mediator of proteolytic cleavage of the hyaluronan binding proteoglycans, aggrecan and versican. In an attempt to further understand the biological function of ADAMTS-1, a yeast two-hybrid screen was performed using the carboxyl-terminal region of ADAMTS-1 as bait. As a result, the extracellular matrix protein fibulin-1 was identified as a potential interacting molecule. Through a series of analyses that included ligand affinity chromatography, co-immunoprecipitation, pulldown assays, and enzyme-linked immunosorbent assay, the ability of these two proteins to interact was substantiated. Additional studies showed that ADAMTS-1 and fibulin-1 colocalized in vivo. Furthermore, fibulin-1 was found to enhance the capacity of ADAMTS-1 to cleave aggrecan, a proteoglycan known to bind to fibulin-1. We confirmed that fibulin-1 was not a proteolytic substrate for ADAMTS-1. Together, these findings indicate that fibulin-1 is a new regulator of ADAMTS-1-mediated proteoglycan proteolysis and thus may play an important role in proteoglycan turnover in tissues where there is overlapping expression. Proteolytic events within the extracellular matrix (ECM) 3The abbreviations used are: ECM, extracellular matrix; ADAMTS, a disintegrin-like and metalloprotease with thrombospondin type 1 motifs; BSA, bovine serum albumin; PMSF, phenylmethanesulfonyl fluoride; TBS, Tris-buffered saline; mAb, monoclonal antibody. 3The abbreviations used are: ECM, extracellular matrix; ADAMTS, a disintegrin-like and metalloprotease with thrombospondin type 1 motifs; BSA, bovine serum albumin; PMSF, phenylmethanesulfonyl fluoride; TBS, Tris-buffered saline; mAb, monoclonal antibody. are essential for developmental morphogenesis, homeostasis of adult tissues, and pathological conditions such as wound healing and tumor angiogenesis (1Parks W.C. Wound Repair Regen. 1999; 7: 423-432Crossref PubMed Scopus (329) Google Scholar, 2Heissig B. Hattori K. Friedrich M. Rafii S. Werb Z. Curr. Opin. Hematol. 2003; 10: 136-141Crossref PubMed Scopus (149) Google Scholar, 3Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3592) Google Scholar). ADAMTS-1 (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs) belongs to a family of metalloproteases involved in the processing of ECM proteins such as procollagen types I and II (4Fernandes 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, 5Li S.W. Arita M. Fertala A. Bao Y. Kopen G.C. Langsjo T.K. Hyttinen M.M. Helminen H.J. Prockop D.J. Biochem. J. 2001; 355: 271-278Crossref PubMed Scopus (100) Google Scholar), von Willebrand factor (6Levy 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, 7Zheng X. Chung D. Takayama T.K. 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Biochem. J. 2004; Google Scholar).A large percentage of mice that lack ADAMTS-1 die perinatally (45%), and those that survive display urinary track obstructions and kidney pathologies (13Shindo 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 (270) Google Scholar, 14Mittaz L. Russell D.L. Wilson T. Brasted M. Tkalcevic J. Salamonsen L.A. Hertzog P.J. Pritchard M.A. Biol. Reprod. 2004; 70: 1096-1105Crossref PubMed Scopus (144) Google Scholar). In addition, most ADAMTS-1 null females are infertile because of abnormalities in the ovaries and endometrium (13Shindo 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 (270) Google Scholar). Evaluation of secondary follicles in the ovaries of ADAMTS-1 null mice revealed poor cumulus expansion and defective ovulation. These anomalies were attributed to incomplete versican proteolysis (15Russell D.L. Doyle K.M. Ochsner S.A. Sandy J.D. Richards J.S. J. Biol. Chem. 2003; 278: 42330-42339Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Thus, a concrete understanding of the array of ADAMTS-1 biological functions will likely rely on insights from its substrates and regulation of catalytic function.The fibulins are a family of secreted glycoproteins that are components of elastic matrix fibers and basement membranes. Currently, there are six members of the fibulin family (16Argraves W.S. Greene L.M. Cooley M.A. Gallagher W.M. EMBO Rep. 2003; 4: 1127-1131Crossref PubMed Scopus (248) Google Scholar). The prototypic member, fibulin-1, contains the signature structural features of all fibulin family members, a series of epidermal growth factor-like repeats followed by a fibulin-type module at its carboxyl terminus (16Argraves W.S. Greene L.M. Cooley M.A. Gallagher W.M. EMBO Rep. 2003; 4: 1127-1131Crossref PubMed Scopus (248) Google Scholar).Functionally, fibulin-1 binds to many ECM proteins, including laminin, fibrinogen, fibronectin, nidogen-1, and endostatin, and to the proteoglycans, aggrecan and versican (17Timpl R. Brown J.C. Matrix Biol. 1994; 14: 275-281Crossref PubMed Scopus (408) Google Scholar, 18Godyna S. Diaz-Ricart M. Argraves W.S. Blood. 1996; 88: 2569-2577Crossref PubMed Google Scholar, 19Tran H. VanDusen W.J. Argraves W.S. J. Biol. Chem. 1997; 272: 22600-22606Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Aspberg A. Adam S. Kostka G. Timpl R. Heinegard D. J. Biol. Chem. 1999; 274: 20444-20449Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 21Miosge N. Sasaki T. Timpl R. FASEB J. 1999; 13: 1743-1750Crossref PubMed Scopus (127) Google Scholar, 22Olin A.I. Morgelin M. Sasaki T. Timpl R. Heinegard D. Aspberg A. J. Biol. Chem. 2001; 276: 1253-1261Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Mice lacking fibulin-1 die perinatally and display vascular anomalies in the kidney in addition to extensive hemorrhage in several organs, likely related to abnormalities in endothelial cell interactions with subendothelial ECM (23Kostka G. Giltay R. Bloch W. Addicks K. Timpl R. Fassler R. Chu M.L. Mol. Cell Biol. 2001; 21: 7025-7034Crossref PubMed Scopus (138) Google Scholar).Using a series of biochemical approaches, we have found that fibulin-1 binds to ADAMTS-1 and enhances its catalytic activities toward aggrecan. These findings, together with those showing that fibulin-1 can bind to aggrecan, led us to speculate that fibulin-1 acts to enhance ADAMTS-1-mediated proteolysis by facilitating a ternary complex formation.EXPERIMENTAL PROCEDURESYeast Two-hybrid Screening—The Matchmaker Two-hybrid System 2 (Clontech Laboratories Inc. Palo Alto, CA) was used following the manufacturer's recommendations. A cDNA fragment encoding amino acids 827-951 of human ADAMTS-1 (GenBank™ accession number AF060152) was amplified by PCR using the primers 5′-CACCTACTTCGTACATATGAAGAAG-3′ and 5′-CTTAAACCAGTCGACTGCATTCTG-3′ and inserted into the vector pAS2-1 previously digested with NdeI and SalI restriction enzymes. The resulting construct encoded a GAL4BD-ADAMTS-1827-951 chimeric protein. A library of prey proteins from human placenta was obtained from Clontech Laboratories Inc. Both bait and prey vectors were simultaneously introduced into Saccharomyces cerevisiae CG-1945 cells and double transformants selected on synthetic minimal medium lacking tryptophan, leucine, and histidine. Colonies were restreaked and tested for β-galactosidase activity using a filter assay. DNA from positive colonies were amplified by PCR using Matchmaker 5′- and 3′-AD LD-Insert Screening Amplimers and the resulting amplicons sequenced.Pulldown Assay—Fibulin-1 or BSA (5 μg each) were individually covalently linked to magnetic M450 tosyl-activated dynabeads (DYNAL, Brown Deer, WI) by overnight incubation in 100 mm phosphate buffer, pH 7.4, at 37 °C. The beads were subsequently washed with 200 mm Tris, pH 8.5, with 0.1% BSA and incubated overnight at 4 °C with ADAMTS-1 (2 μg) in a volume of 400 μl. Dynabeads were magnetically collected and washed with phosphate-buffered saline to remove unbound proteins. Bound proteins were released using sample buffer containing β-mercaptoethanol and separated on 10% SDS-polyacrylamide gels, transferred to Optitran nitrocellulose membrane (Schleicher and Schuell, Keene, NH), and immunoblotted with guinea pig anti-ADAMTS-1 polyclonal antibody (GPB).Co-immunoprecipitation Analysis—Fragments of human full-term placenta were homogenized in cold lysis buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 5 mm iodoacetamide, 0.5 mm CaCl2, 0.5 mm MgCl2, 0.02% NaN3, 1 mm PMSF, complete protease inhibitor mixture tablets following the manufacturer's direction (Roche Applied Science), 1% Triton X-100 using a Waring blender. The lysate was incubated with protein G-agarose beads (Roche Applied Science) overnight at 4 °C to absorb resin-binding proteins. Aliquots of the lysates (1 ml) were then incubated with antibodies to fibulin-1 (mAb 3A11), ADAMTS-1 (GPB), or mouse IgG (Sigma) for 3 h at 4°C. Antibody complexes were precipitated for 1 h at 4°C using either protein G-agarose (Roche Applied Science) for mouse IgGs or protein A-agarose (Roche Applied Science) for GPB. The agarose-bound complexes were pelleted by centrifugation at 1,000 × g and the pellets washed with 10 mm Tris, pH 8.0, 150 mm NaCl, and 0.5% Nonidet P-40. Bound proteins were released using sample buffer containing β-mercaptoethanol and separated on 10% SDS-polyacrylamide gels, transferred to Optitran nitrocellulose membrane (Schleicher and Schuell), and analyzed by immunoblotting.Conditioned medium from fibulin-1C-expressing HT1080 cells cultured in the absence of serum was collected and precleared with protein G-agarose beads (Roche Applied Science) for 16 h at 4 °C. Fibulin-1 monoclonal antibody (mAb 3A11) was added to 1-ml aliquots and incubated for 4 h at 4°C. Immunocomplexes were pelleted with protein G-agarose beads and the pellets washed extensively using 10 mm Tris, pH 8.0, 150 mm NaCl, and 0.5% Nonidet P-40 buffer. These fibulin-1-immobilized immunocomplexes were incubated with ADAMTS-1 (87-kDa form) for 2 h at 4 °C and unbound protein removed by washing with 10 mm Tris, pH 8.0, 150 mm NaCl, and 0.5% Nonidet P-40 buffer. After several washes, bound proteins were released using sample buffer containing β-mercaptoethanol, separated on 10% SDS-polyacrylamide gels, and transferred to Optitran nitrocellulose membrane (Schleicher and Schuell). The presence of ADAMTS-1 and fibulin-1 was evaluated by immunoblotting with GPB and mAb 3A11, respectively.Enzyme-linked Immunosorbent Assay—Purified ADAMTS-1 (2.5 μg/ml) or fibulin-1 (10 μg/ml) in binding buffer (150 mm NaCl, 0.5 mm CaCl2, 0.5 mm MgCl2, 25 mm Tris, pH 8.2) was coated on Costar high binding 96-well plates for 16 h at 4 °C. Coated wells were incubated with 2% BSA in TBS with 0.5 mm CaCl2, 0.5 mm MgCl2 for 90 min at room temperature. Wells coated with ADAMTS-1 were incubated with fibulin-1 (10 μg/ml) in TBS with 0.5% BSA, 0.5 mm CaCl2, and 0.5 mm MgCl2 and vice versa for 90 min at room temperature. Plates were washed with TBS with 0.5% BSA, 0.5 mm CaCl2, 0.5 mm MgCl2. Bound proteins were detected with either mouse monoclonal antibodies against ADAMTS-1 (mAb 3E4C6B4) or fibulin-1 (mAb 3A11), and anti-mouse-IgG-alkaline phosphatase conjugate. Colorimetric reactions were performed using p-nitrophenyl phosphate substrate (Sigma) and read at λ = 405 nm using a SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA).Solid Binding Assays—Microtiter plate wells were coated with human fibronectin, ADAMTS-1, or BSA at 3 μg/ml in TBS, pH 8.0, for 2 h at 37 °C and then rinsed three times with TBS, pH 7.4. Unbound sites were blocked for 1 h at room temperature with 5% nonfat milk in TBS, 1 mm CaCl2, and 1 mm MgCl2, pH 7.4. Wells were then rinsed three times with TBS, pH 7.4, and incubated with varying concentrations of fibulin-1 (ranging from 100 to 0.137 μg/ml) for 3-4 h at 37 °C in TBS containing 3% nonfat milk, 0.1% Tween 20, 1 mm CaCl2, and 1 mm MgCl2. After rinsing with TBS, 0.1% Tween 20 and 1 mm CaCl2 and 1 mm MgCl2, pH 7.4 (wash buffer), wells were incubated with anti-fibulin-1 monoclonal antibody (mAb 3A11) at 1 μg/ml in TBS, pH 7.4, containing 3% nonfat milk and 0.1% Tween 20, 1 mm CaCl2, and 1 mm MgCl2. Wells were then rinsed with wash buffer three times and incubated for 1 h at room temperature with anti-mouse horseradish peroxidase-conjugated IgG (1:5000 dilution, Amersham Biosciences) diluted in TBS, pH 7.4, containing 3% nonfat milk and 0.1% Tween 20. The wells were rinsed three times with wash buffer, and detection was performed using TMB substrate (KPL, Gaithersburg, MD) with color development measured spectroscopically at λ = 650 nm. Curve fitting of data was performed using Sigmaplot 2001 software (SPSS Inc., Chicago, IL).ADAMTS-1 Affinity Chromatography—ADAMTS-1 (2 mg) was coupled to 2 ml of CNBr-activated CL-4B-Sepharose (Amersham Biosciences) following the manufacturer's instructions. Placental tissue (30 g) was extracted in 100 ml of lysis buffer (20 mm Tris-HCl, 150 mm NaCl, 0.5 mm MgCl, 0.5 mm CaCl2, 1% Triton X-100, 0.02% NaN3, 1 mm PMSF, 0.2 units/ml aprotinin, and 5 mm iodoacetamide, pH 8.0) using a Waring blender. The lysate was centrifuged at 3,000 × g for 30 min at 4 °C and then ultracentrifuged at 100,000 × g for 1 h at 4 °C. The lysate was precleared by passing through a 2-ml precolumn of glycine-Sepharose CL-4B (pre-equilibrated in lysis buffer) and loaded onto a 2-ml column of ADAMTS-1-Sepharose CL-4B (pre-equilibrated in lysis buffer) at a flow rate of 0.5 ml/min (Biologic Work station; Bio-Rad). The column was washed sequentially with 15 ml of lysis buffer and 15 ml of washing buffer (20 mm ethanolamine, 0.2% Triton X-100, 150 mm NaCl, 0.5 mm MgCl, 0.5 mm CaCl2, 1 mm PMSF, pH 9). Bound proteins were eluted with a gradient starting at 20 mm ethanolamine, 0.2% n-octylglucoside, 150 mm NaCl, 0.5 mm MgCl2, 0.5 mm CaCl2, 1 mm PMSF, pH 9, and finishing at 20 mm ethanolamine, 0.2% n-octylglucoside, 1 m NaCl, 0.5 mm MgCl2, 0.5 mm CaCl2, 1 mm PMSF, pH 12. Fractions of 1 ml were collected and neutralized with 0.1 volume of 1 mm Tris, pH 6.7. Eluted proteins were separated by SDS-PAGE and subjected to immunoblotting with anti-fibulin-1 (mAb 3A11).Analysis of ADAMTS-1, Fibulin-1, and Aggrecan Expression in Embryonic Kidneys—Kidneys harvested from E15.5 embryos were fixed in 2% paraformaldehyde overnight, embedded in paraffin, and sectioned at 5 μm. Sections were deparaffinized and incubated with blocking buffer (BB, 5% goat serum and 2.5% BSA in PBS) for 2 h at room temperature. Sections were incubated with guinea pig anti-ADAMTS-1 (GP12) at 1:200 dilution in 0.5× BB, rabbit anti-fibulin-1 (Rb1323) (24Argraves W.S. Dickerson K. Burgess W.H. Ruoslahti E. Cell. 1989; 58: 623-629Abstract Full Text PDF PubMed Scopus (104) Google Scholar) at 10 μg/ml in 0.5× BB, rabbit anti-aggrecan G1 domain or TASELE neo-epitope at 1:200 dilution in 0.5× BB for 2 h at room temperature. Sections were then incubated with anti-guinea pig IgG conjugated to biotin (Vector Labs, Burlingame, CA) at 1:250 dilution in 0.5× BB for 1.5 h, followed by incubation with streptavidin conjugated to CY3 (Sigma) at 1:100 dilution in 0.5× BB and anti-rabbit IgG conjugated to fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, PA) at 1:150 dilution in 0.5× BBfor 1 h at room temperature. Fluorescence immunohistochemistry was analyzed using a Bio-Rad MRC 1024ES laser confocal microscope.Anti-ADAMTS-1 GP12 antibody was raised in guinea pig by BIOSOURCE (Camarillo, CA) using purified ADAMTS-1 protein as immunizing antigen. Antibody specificity was tested against conditioned medium and cell lysates from T47D cells transfected with ADAMTS-1 or empty vector construct.Digestion of Fibulin-1 with ADAMTS-1—Fibulin-1 (1 μg) was incubated with ADAMTS-1 at 1:1, 1:2, and 1:3 molar ratio in 50 mm Tris, pH 7.4, 10 mm CaCl2, 80 mm NaCl overnight at 37 °C in a total volume of 60 μl. Digests were separated in 10% SDS-PAGE electrophoresis under reducing conditions using β-mercaptoethanol and followed by immunoblotting with antibodies to the amino terminus (mAb 3A11) or the carboxyl terminus (mAb 5D12) of fibulin-1.Fibulin-1 was biotinylated with Sulfo-NHS-LC-Biotin (Pierce Biotechnology) following the manufacturer's protocol. Biotinylated fibulin-1 was then dialyzed in PBS to remove unbound biotin. Soluble biotinylated fibulin-1 was incubated with 2× molar ratio of ADAMTS-1, catalytically inactive ADAMTS-1 (zinc mutant), or buffer control in 50 mm Tris, pH 7.4, 10 mm CaCl2, 80 mm NaCl overnight at 37 °C in a total volume of 60 μl. Biotinylated fibulin-1 was also incubated in fibronectin-coated 96-well plates. Fibronectin-immobilized fibulin-1 was subsequently incubated with 2 molar ratio of ADAMTS-1 or zinc mutant in 50 mm Tris, pH 7.4, 10 mm CaCl2, 80 mm NaCl overnight at 37 °C in a total volume of 60 μl. Digests were separated on SDS containing 10% polyacrylamide gels under reducing conditions with β-mercaptoethanol. Biotinylated proteins were detected with avidin-conjugated horseradish peroxidase (Vector Labs).Proteoglycan Digestion—Rat aggrecan was a generous gift from Dr. John Sandy (Shriners Hospital for Children, Tampa, FL). Aggrecan is further purified to obtain the intact form and removed processed forms; however, 250-kDa fragments are frequent contaminants, and this degree of contamination varies from 10 to 50%. Thus, controls for each purification lot are always included. Aggrecan (7.5 μg) either alone or in the presence of ADAMTS-1 (0.5-1 μg) was incubated in 60 μl of 50 mm Tris, pH 7.4, 10 mm CaCl2, 80 mm NaCl for 2 h at 37°C containing fibulin-1, fibronectin, vitronectin, fibrinogen, or BSA (1 μg each). The reaction mixtures were deglycosylated in 50 mm sodium acetate, 50 mm Tris, 10 mm EDTA, pH 7.6 with 1.8-15 milliunits of chondroitinase ABC (Sigma) at 37 °C for 1 h prior to separation on 10% SDS-PAGE electrophoresis in reducing conditions with β-mercaptoethanol and immunoblotting. Rat aggrecan was also digested with ADAMTS-1 in the presence of varying amounts of fibulin-1 (0.35, 0.7, or 1.5 μg) or laminin-1 (0.7, 1.5, or 3.0 μg) for 2 h at 37°C (60-μl reaction volume) followed by chondroitinase digestion as mentioned above. Digests were resolved on 10% SDS-PAGE or 4-12% gradient polyacrylamide gels (Invitrogen). The extent of aggrecan cleavage was determined by immunoblotting using rabbit anti-G1 aggrecan antibody (a generous gift from Dr. John Sandy). ADAMTS-1 was detected in immunoblotting with GPB, laminin-1 was detected with anti-laminin (Sigma), and fibulin-1 was detected with mAb 3A11 antibodies.RNA Isolation and Northern Analysis—Total RNA was isolated from several organs at E16.5, E17.5, and E18.5. Purification of mRNA was performed as previously described (25Thai S.N. Iruela-Arispe M.L. Mech. Dev. 2002; 115: 181-185Crossref PubMed Scopus (61) Google Scholar). Fractionation of mRNA (2 μg/lane) was performed on agarose/formaldehyde gels, and subsequent transfer was done onto nylon membranes. Probes included a 951-bp cDNA fragment for ADAMTS-1 and a 360-bp fragment encoding the 3′-end of fibulin-1.Generation of ADAMTS-1 Null Mice—Bacterial artificial chromosome (BAC) clone number 413009 carrying genomic fragments of adamts1 from mouse strain 129/SvJ was purchased from Genome Systems, Inc. (St. Louis, MI). An 8.4-kb EcoRI fragment extending from the 5′-untranslated region (UTR) to exon 2 and a 6.6-kb HindIII fragment spanning from intron 7 to the 3′-UTR were obtained after digestion of BAC clone number 413009 and were subsequently subcloned into pGEM11ZF(+) and pGEM7ZF(+) (Promega, Madison, WI), respectively. The targeting construct was generated by subcloning a 3.2-kb HindIII-XbaI fragment spanning the 5′-UTR to part of intron 1, a 3.6-kb BamHI-BamHI fragment encompassing exon 9 and the 3′-UTR and a 2.0-kb flox-PGK-Neo (XbaI) into pGEM11ZF(+). The flox-PGK-Neo construct was a generous gift from Dr. K. Lyons. The resulting targeting construct was linearized with XhoI and HindIII to remove the pGEM11ZF(+) plasmid prior to being introduced into 129/SvJ-derived embryonic stem cells by electroporation at the UCLA Molecular Genetics and Technology Center. Embryonic stem cells were then selected in growth medium containing G418 (300 μg/ml), and homologous recombinants were identified by Southern blot analysis. Chimeric males were generated from two independent targeted embryonic stem cell clones injected into C57BL/6 blastocysts (UC Irvine Transgenic Mouse Facility, Irvine, CA). Male chimeras were crossbred with BALB/c females, and germ line transmission was obtained from both independent embryonic stem clones.PCR Genotyping of ADAMTS-1 Mice—Genomic DNA was isolated from tails of embryos using a standard phenol:chloroform extraction protocol. Wild-type locus was amplified using primers that anneal to the 5′-end (5′-TGCCACACCTCACTGCTTAC-3′) and the 3′-end of exon 9 (5′-TTAATGGACACCCTGCTTCC-3′). Targeted locus was amplified using primers to the 3′-end of exon 9 as mentioned and the 5′-PGK-neo cassette sequence (5′-TATCGCCGCTCCCGATTC-3′). PCR was performed using HOTMASTER Taq polymerase (Eppendorf AG, Hamburg, Germany) with annealing temperature of 62 °C for 35 cycles.RESULTSIdentification of Fibulin-1D by Yeast Two-hybrid Screening as an ADAMTS-1-binding Protein—A yeast two-hybrid screen of a human placenta cDNA library was conducted to identify proteins capable of interacting with ADAMTS-1. A fragment of the carboxyl-terminal portion of human ADAMTS-1 containing the two last thrombospondin type I repeats (amino acid residues 827-951) was used as a bait to screen for potential binding proteins (Fig. 1). Among several positive clones identified, one clone, designated F3.3.2, contained a 1371-bp insert that corresponded to nucleotides 1439-2810 of a human fibulin-1D cDNA (GenBank™ accession number AF126110). The sequence encoded by clone F3.3.2 corresponds to the carboxyl-end of fibulin-1D (amino acid residues 476-703), including the two last epidermal growth factor-like elements and the carboxyl-terminal fibulin-type module (Fig. 1).Evaluation of the ADAMTS-1-Fibulin-1 Binding Interaction—The interaction between ADAMTS-1 and fibulin-1 was further validated by several biochemical approaches. Tosyl-activated magnetic beads coupled to fibulin-1 were able to bind to purified ADAMTS-1 in solution, whereas BSA-coated magnetic beads did not (Fig. 2A). Fibulin-1C immunoprecipitated from conditioned medium with mAb 3A11 was able to pull down ADAMTS-1 (Fig. 2B, lane 2). Anti-fibulin-1 mAb 3A11 only recovered trace amounts of ADAMTS-1 when in the absence of fibulin-1C (Fig. 2B, lane 5). In addition, the interaction was tested by enzyme-linked immunosorbent assay using purified proteins. As shown in Fig. 2C, fibulin-1 bound to immobilized ADAMTS-1. The reciprocal experiment was also performed, and ADAMTS-1 was found to bind to immobilized fibulin-1 (Fig. 2C). The binding affinity between these two proteins was also determined. Fibronectin was used as a positive control because it has been shown to interact with fibulin-1 (19Tran H. VanDusen W.J. Argraves W.S. J. Biol. Chem. 1997; 272: 22600-22606Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 26Balbona K. Tran H. Godyna S. Ingham K.C. Strickland D.K. Argraves W.S. J. Biol. Chem. 1992; 267: 20120-20125Abstract Full Text PDF PubMed Google Scholar). 96-well plates were coated with human fibronectin, ADAMTS-1, or BSA (negative control) at 3 μg/ml and subsequently allowed to bind fibulin-1 at varying concentrations. Based on a fit of the data to a form of the binding isotherm (26Balbona K. Tran H. Godyna S. Ingham K.C. Strickland D.K. Argraves W.S. J. Biol. Chem. 1992; 267: 20120-20125Abstract Full Text PDF PubMed Google Scholar), fibulin-1 bound to ADAMTS-1 with an apparent dissociation constant (Kd) of 1.0 μm as compared with Kd of 0.43 μm for fibulin-1 binding to fibronectin (Fig. 2D). As previously shown for fibulin-1-fibronectin interactions (19Tran H. VanDusen W.J. Argraves W.S. J. Biol. Chem. 1997; 272: 22600-22606Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), saturable binding was not achieved between fibulin-1 and immobilized ADAMTS-1. The basis for this may relate to the fact that fibulin-1 can self associate (19Tran H. VanDusen W.J. Argraves W.S. J. Biol. Chem. 1997; 272: 22600-22606Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar); thus, with increased binding of solution phase fibulin-1 to the immobilized ADAMTS-1, additional fibulin-1 binding sites are created.FIGURE 2Demonstration of ADAMTS-1-fibulin-1 binding. A, immunoblot of ADAMTS-1 bound to Dynal magnetic beads incubated with fibulin-1 (fib-1) or BSA. B, immunoblot of ADAMTS-1 (87-kDa) using GPB pulled down with fibulin-1C immunoprecipitated from conditioned medium of cells expressing fibulin-1C. Immunoblot of fibulin-1C (fib-1C) was done with mAb 3A11. C, enzyme-linked immunosorbent assays in which the first series shows the binding to soluble 87-kDa ADAMTS-1 to immobilized fibulin-1 or BSA, and the second series shows the binding of soluble fibulin-1 to immobilized 87-kDa ADAMTS-1 or BSA. IMM, immobilized; Sol, soluble. D, enzyme-linked immunosorbent assay showing binding of a range of fibulin-1 concentrations to immobilized ADAMTS-1, fibronectin (FN), or BSA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The interaction between fibulin-1 and ADAMTS-1 was also tested using ADAMTS-1-Sepharose affinity chromatography of placental extracts. As shown in Fig. 3A, low ionic strength buffer released weakly interacting fibulin-1 (fractions 3-13). Subsequent exposure to higher pH and high ionic strength buffer released tightly bound fibulin-1 (fraction 22). No fibulin-1 binding was observed when the plain Sepharose column used to preclear the extract was exposed to the same elution protocol.FIGURE 3Fibulin-1 interaction detected by ADAMTS-1 affinity chromatography and co-immunoprecipitation analysis. A, anti-fibulin-1 immunoblot of fractions eluted from ADAMTS-1-Sepharose column after application of placental extract. B, immunoblots showing that ADAMTS-1 co-immunoprecipitates with fibulin-1 and conversely that fibulin-1 co-immunoprecipitates with ADAMTS-1. EXP, exposure.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, we performed co-immunoprecipitation analyses using placental extracts and fibulin-1 monoclonal antibodies. These experiments revealed that ADAMTS-1 could be co-immunoprecipitated with fibulin-1 (Fig. 3B, lane 3). Conversely, fibulin-1 was found to co-immunoprecipitate with ADAMTS-1 antibodies (Fig. 3B, lane 1, GPB). ADAMTS-1 or fibulin-1 were not present in immunoprecipitations using control mouse IgG (lane 2). Together these findings support the conclusion that fi
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