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• W2103238084 abstract "WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with KD values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of “permanent” cartilage structures during development and in mature cartilages. WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with KD values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of “permanent” cartilage structures during development and in mature cartilages. The extracellular matrix (ECM) 3The abbreviations used are: ECM, extracellular matrix; VWA, von Willebrand factor A; GAG, glycosaminoglycan; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; En, embryonic day n; FGF, fibroblast growth factor.3The abbreviations used are: ECM, extracellular matrix; VWA, von Willebrand factor A; GAG, glycosaminoglycan; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; En, embryonic day n; FGF, fibroblast growth factor. is a complex and dynamic three-dimensional environment that plays fundamental roles in morphogenesis and development, tissue structure, repair, and metastasis (1Boudreau N. Bissell M.J. Curr. Opin. Cell. Biol. 1998; 10: 640-646Crossref PubMed Scopus (313) Google Scholar). The tissue-specific expression of collagen types and specialized ECM components results in the formation of architecturally precise interacting networks with unique functional and biological characteristics. A diverse range of ECM components have been described including more than 20 distinct collagen subtypes and a large number of proteoglycans and noncollagenous proteins. Many of these matrix proteins are modular in structure in that they are composed of protein domains, which is important in generating the multifunctionality that is characteristic of ECM proteins (2Hohenester E. Engel J. Matrix Biol. 2002; 21: 115-128Crossref PubMed Scopus (161) Google Scholar, 3Bork P. Downing A.K. Kieffer B. Campbell I.D. Q. Rev. Biophys. 1996; 29: 119-167Crossref PubMed Google Scholar). One of these domains, found in a growing number of ECM proteins involved in supramolecular structures, is the A domain first described in von Willebrand factor (VWA domain). VWA domains are found in a diverse range of ECM proteins including collagens (types VI, VII, XII, XIV, XX, XXI, XXVII, and XXVIII), matrilins, cochlin, polydom, AMACO (VWA-like domains related to those in matrilins and collagens), and the extracellular portions of nine transmembrane α-integrin chains (4Whittaker C.A. Hynes R.O. Mol. Biol. Cell. 2002; 13: 3369-3387Crossref PubMed Scopus (526) Google Scholar, 5Sengle G. Kobbe B. Morgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2003; 278: 50240-50249Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 6Veit G. Kobbe B. Keene D.R. Paulsson M. Koch M. Wagener R. J. Biol. Chem. 2006; 281: 3494-3504Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) We recently identified a new member of the von Willebrand factor A domain superfamily, WARP (von Willebrand factor A domain-related protein), that may have evolved from a collagen-like molecule (7Fitzgerald J. Ting S.T. Bateman J.F. FEBS Lett. 2002; 517: 61-66Crossref PubMed Scopus (33) Google Scholar, 8Fitzgerald J. Bateman J.F. FEBS Lett. 2003; 552: 91-94Crossref PubMed Scopus (7) Google Scholar). The WARP protein comprises a single N-terminal VWA domain containing a putative metal ion-dependent adhesion site motif, two fibronectin type III repeats, and a unique C-terminal segment. Our studies demonstrated WARP expression by chondrocytes, and in transfected cells WARP is a secreted glycoprotein that can form multimeric structures (7Fitzgerald J. Ting S.T. Bateman J.F. FEBS Lett. 2002; 517: 61-66Crossref PubMed Scopus (33) Google Scholar). We report here that in mouse cartilage WARP defines the presumptive articular cartilage prior to joint cavitation and subsequently during development is present in articular cartilage as well as in several fibrocartilage elements. Biochemical analysis demonstrated that WARP exists as disulfide-bonded multimers in cartilage tissue. Furthermore, we show that WARP can strongly interact with perlecan, providing a mechanism by which WARP is integrated into the extracellular matrix of cartilage. cDNA Constructs and Cell Culture—The hexahistidine-tagged WARP constructs in the pCEP4 vector (7Fitzgerald J. Ting S.T. Bateman J.F. FEBS Lett. 2002; 517: 61-66Crossref PubMed Scopus (33) Google Scholar) containing single Cys-to-Ser mutations at amino acid 369 or 393 and the double mutant (C369S/C393S) were generated by strand overlap extension PCR (9Chan D. Weng Y.M. Hocking A.M. Golub S. McQuillan D.J. Bateman J.F. J. Biol. Chem. 1996; 271: 13566-13572Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The wild-type and mutant His-WARP cDNAs were transfected into HEK 293-EBNA cells using FuGENE 6 transfection reagent (Roche Applied Science) and grown as described (7Fitzgerald J. Ting S.T. Bateman J.F. FEBS Lett. 2002; 517: 61-66Crossref PubMed Scopus (33) Google Scholar). WARP Immunoprecipitations—Transfected cells were grown to confluence in 35-mm dishes and labeled for 18 h with 300 μCi of l-[35S]methionine (1388 Ci/mmol; PerkinElmer Life Sciences) in Dulbecco's modified Eagle's medium as described previously (7Fitzgerald J. Ting S.T. Bateman J.F. FEBS Lett. 2002; 517: 61-66Crossref PubMed Scopus (33) Google Scholar). Briefly, the cell and medium fractions were collected in the presence of protease inhibitors, and His-tagged WARP was immunoprecipitated from the cell and medium fractions with anti-His antibody (Roche Applied Science) (0.5 μg/ml) and protein A-Sepharose overnight. Following washing, the immunoprecipitated material was denatured and fractionated on a 7.5% (v/v) SDS-polyacrylamide gel and subjected to fluorography. Cartilage Sample Preparation—Femoro-tibial joint and rib cartilage was dissected from newborn mice and powdered in a liquid nitrogen-cooled Spex freezer mill and dissolved in neutral salt buffer (40 mm Tris-HCl, pH 7.5, 10 mm EDTA containing Complete protease inhibitor mixture; Roche Applied Science). Following sonication, the soluble material was collected (fraction 1), and the insoluble material was treated overnight at 37 °C with 0.02 units of chondroitinase ABC (Seikagaku) and 1 unit of hyaluronidase (Sigma) to cleave hyaluronan and chondroitin sulfate GAG chains. Following washing the soluble material was collected (fraction 2), and the insoluble pellet was dissolved in 6 m GuHCl, 40 mm Tris-HCl, pH 7.5, 10 mm EDTA containing protease inhibitors for 5 h at 4 °C and then centrifuged at room temperature for 20 min. The supernatant was saved as soluble fraction 3, the matrix components were precipitated with 95% ethanol, and the pellet was washed with 70% ethanol. The samples were then freeze-dried and resuspended in 200 μl of 8 m urea, 4% CHAPS, 40 mm Tris base, and 2 mm tributyl phosphine (Bio-Rad). For some experiments the reducing agent tributyl phosphine was omitted. Antibodies—Polyclonal antisera against the VWA domain and C-terminal domains of WARP were produced commercially in rabbits (Institute of Medical and Veterinary Science, Adelaide, Australia) using the GST-VWA domain (amino acids 21-212) and the maltose-binding protein-C-terminal domain (amino acids 389-415) fusion proteins expressed in bacteria as antigens. The antisera bound to the fusion proteins in a dose-dependent manner in a standard enzyme-linked immunosorbent assay (not shown). The anti-VWA domain WARP polyclonal antibody was further affinity-purified against the antigen immobilized on a nitrocellulose membrane as described elsewhere (10Michalczyk A.A. Allen J. Blomeley R.C. Ackland M.L. Biochem. J. 2002; 364: 105-113Crossref PubMed Scopus (64) Google Scholar) and used in immunohistochemical staining experiments at a final concentration of 1 μg/ml. Briefly, the GST-VWA domain fusion protein was transferred to nitrocellulose following SDS-PAGE. The band was visualized by staining with 0.1% Ponceau S (w/v) in 5% acetic acid and cut out. The nitrocellulose strip was destained, blocked with 3% BSA in PBS, and incubated with the anti-VWA antiserum overnight at 4 °C. Following washing, bound antibody was eluted with 0.1 m glycine-HCl, pH 2.5, neutralized by the addition of 1 m Tris-HCl, pH 8.0. The polyclonal anti-C-terminal antiserum was used at a dilution of 1:1000 in immunohistochemistry and immunoblots and 1:200 in solid phase assays. Serum from the same rabbit, taken prior to immunization with the WARP fusion protein, was used as a negative control. For immunogold electron microscopy experiments, a polyclonal antibody raised in sheep against full-length recombinant WARP (described under “Recombinant Proteins “) was used. An antibody to bovine matrilin-1 was kindly provided by Prof. Mats Paulsson (University of Cologne, Cologne, Germany) and was used at a dilution of 1:3000 for immunohistochemistry or 1:500 for immunoblotting. Affinity-purified antibody against mouse perlecan domain V (11Brown J.C. Sasaki T. Gohring W. Yamada Y. Timpl R. Eur. J. Biochem. 1997; 250: 39-46Crossref PubMed Scopus (145) Google Scholar) was used at a final concentration of 2 μg/ml in immunohistochemical experiments. A monoclonal antibody against perlecan (MAB 1948; Chemicon), was used in the immunogold experiments. WARP Immunoblotting—Fractions 1, 2, and 3 (20 μg) were denatured by heating at 95 °C for 5 min, separated on a 10% polyacrylamide gel, and transferred to Immobilon™-P polyvinylidene difluoride membrane (Millipore). The membrane was blocked in 5% milk powder in PBS for 1 h and then incubated in antibody buffer (0.5% milk powder in PBS with 0.1% Tween 20) containing either WARP (1:1000 dilution) or matrilin-1 antisera for 1 h. Following three washes in PBS with 0.1% Tween 20, anti-rabbit IgG horseradish peroxidase secondary antibody (Dako) was added at a dilution of 1:10,000 in antibody buffer and incubated for 1 h. Following washing, the signal was developed with ECL Plus Western blotting detection system (Amersham Biosciences) and autoradiography using X-Omat film. WARP Immunohistochemistry—Embryonic mouse tissues were surgically removed and frozen in Tissue-Tek O.C.T. compound (Circle Scientific). Tissues from post-natal mice were fixed in Histochoice (Amresco) overnight at 4 °C before decalcification in PBS with 7% (w/v) EDTA. 10-μm sections were mounted and fixed in 95% ethanol, 5% acetic acid or in Histochoice (Amresco). To facilitate antibody penetration into the ECM, sections were treated with 0.2% hyaluronidase (bovine, type IV; Sigma) and treated with 0.3% H2O2 (v/v) in methanol to inactivate endogenous peroxidases. After blocking with 1% BSA (w/v) in PBS, the sections were immunolabeled with the rabbit antisera or preimmune serum and detected using the Vectastain Elite ABC kit (Vector Laboratories). Bound antibodies were visualized using Sigma-Fast DAB tablets, and sections were counterstained with hematoxylin. For immunogold electron microscopy, native suprastructural fragments were isolated from human articular cartilage and placed on grids. Samples were doubly immunostained with polyclonal sheep antisera against full-length WARP (or the VWA domain or C-terminal WARP, described above) and a monoclonal antibody against perlecan and colloidal gold-labeled secondary antibodies of different sizes and analyzed with transmission electron microscopy as described (12Kassner A. Hansen U. Miosge N. Reinhardt D.P. Aigner T. Bruckner-Tuderman L. Bruckner P. Grassel S. Matrix Biol. 2003; 22: 131-143Crossref PubMed Scopus (90) Google Scholar). In Situ Hybridization—15-μm cryosections were fixed with 4% paraformaldehyde in PBS for 15 min. Nonspecific probe binding was blocked by carbethoxylation with 0.1% active diethyl pyrocarbonate in PBS twice for 15 min before prehybridization in buffer containing 50% formamide, 5× SSC, and 40 μg/ml salmon sperm for 2 h at 58°C. Hybridization solution was prepared by adding a 1-kb digoxigenin-labeled riboprobe complementary to the 3′-untranslated region of WARP, or the sense orientation negative control, to prehybridization solution at 400 ng/ml and denatured at 80 °C. The sections were hybridized at 58 °C overnight and washed with 2× SSC and 1× SSCfor 1 h at 65 °C. Immunodetection was performed as described elsewhere (13Braissant O. Wahli W. Biochemica. 1998; 1: 10-16Google Scholar). Recombinant Proteins—Recombinant His-WARP was purified from the media of transfected 293-EBNA cells using nickel ion affinity chromatography as described for decorin (14Goldoni S. Owens R.T. McQuillan D.J. Shriver Z. Sasisekharan R. Birk D.E. Campbell S. Iozzo R.V. J. Biol. Chem. 2004; 279: 6606-6612Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Fractions enriched for WARP dimer were used for subsequent interaction analyses (see Fig. 5). Recombinant mouse perlecan domain IA containing heparan sulfate (15Costell M. Mann K. Yamada Y. Timpl R. Eur. J. Biochem. 1997; 243: 115-121Crossref PubMed Scopus (68) Google Scholar); mutant domain I lacking heparan sulfate (16Sasaki T. Costell M. Mann K. Timpl R. FEBS Lett. 1998; 435: 169-172Crossref PubMed Scopus (35) Google Scholar); domains II (17Costell M. Sasaki T. Mann K. Yamada Y. Timpl R. FEBS Lett. 1996; 396: 127-131Crossref PubMed Scopus (31) Google Scholar); subdomains III-1, III-2, and III-3 (18Schulze B. Mann K. Battistutta R. Wiedemann H. Timpl R. Eur. J. Biochem. 1995; 231: 551-556Crossref PubMed Scopus (44) Google Scholar); subdomains IV-1 and IV-2 (19Hopf M. Gohring W. Kohfeldt E. Yamada Y. Timpl R. Eur. J. Biochem. 1999; 259: 917-925Crossref PubMed Scopus (152) Google Scholar); and domain V (11Brown J.C. Sasaki T. Gohring W. Yamada Y. Timpl R. Eur. J. Biochem. 1997; 250: 39-46Crossref PubMed Scopus (145) Google Scholar) were expressed in 293-EBNA cells and purified as described. Protein concentrations were determined by amino acid analysis. Full-length perlecan was purified from mouse Engelbreth-Holm-Swarm tumor (20Paulsson M. Yurchenco P.D. Ruben G.C. Engel J. Timpl R. J. Mol. Biol. 1987; 197: 297-313Crossref PubMed Scopus (109) Google Scholar). Solid Phase Binding Assay—Multiwell plates (96 wells, Immulon 4; Dynatech) were coated with recombinant proteins (1 or 5 μg/ml, 50 μl/well) or heparin (1 μg/ml) (Sigma) isolated from porcine intestinal mucosa in enzyme-linked immunosorbent assay coating buffer (50 mm sodium carbonate, pH 9.2) overnight at room temperature. Following four wash buffer (0.15 m NaCl, 0.05% Tween 20) rinses, nonspecific binding sites were blocked for 1 h with Tris-buffered saline (0.15 m NaCl, 50 mm Tris, pH 7.4) containing 1% BSA. Incubations with recombinant WARP (0-200 nm) were carried out in Tris-buffered saline for 1 h. In some experiments 5 mm CaCl2 or 10 mm EDTA was included in WARP incubations and the subsequent washes. Incubation with the primary polyclonal antibodies (diluted 1:200) against the soluble ligands was followed by incubation with anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (diluted 1:1000; Dako). Color was developed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in citrate buffer (0.1% (w/v) ABTS, 0.03% H2O2, 61 mm citrate, 77 mm Na2HPO4.2H2O pH 4.0), and color yields were determined at 405 nm using a microtitre plate reader (model 450; Bio-Rad). Nonspecific binding to 1% BSA was subtracted from each absorbance reading. Surface Plasmon Resonance Analysis—The BIAcore 3000 system (BIAcore AB) was used to characterize interactions between WARP and perlecan and WARP self-association. Purified recombinant proteins were immobilized to a CM5 sensor chip at 25 °C at a flow rate of 5 μl/min. The sensor chip was activated with a 35-μl mixture of 50 mm N-hydroxysuccinimide and 200 mm N-ethyl-N′-(dimethylaminopropyl) carbodiimide. WARP (30 μg/ml) in 5 mm sodium malate, pH 6.0, or perlecan subdomain III-2 (30 μg/ml) in sodium citrate, pH 3.35, were coupled. The remaining active groups were blocked with 1 m ethanolamine HCl, pH 8.5. A control surface containing no immobilized ligand was prepared by activation and blocking in the absence of ligand. The immobilization resulted in 3500-4200 resonance units for WARP and 3600 resonance units for perlecan subdomain III-2. Binding studies were performed in HEPES-buffered saline buffer (10 mm HEPES, pH 7.4, 0.15 m NaCl, 5 mm CaCl2, 0.005% P-20) at a flow rate of 5 μl/min, and the binding sites of the immobilized ligands were regenerated using a 12-s pulse of either 30 or 50 mm NaOH at 50 μl/min. WARP was immobilized (3900 response units) to the C1 biosensor surface in 10 mm sodium acetate, pH 5.0. The perlecan domain 1 binding studies were performed as described above except that WARP surface was regenerated using a 6s pulse of 10 mm NaOH and the experiment was carried out at a flow rate of 30 μl/min. For the kinetic analyses, the flow rates were performed at 30 μl/min. In one experiment WARP was coupled directly to the sensor chip (990 response units). Alternatively WARP (30 μg/ml, 5-min injection) was bound to immobilized anti-His antibody (9000 response units). After subtraction of blank curves, the association (ka) and dissociation (kd) rate constants were determined simultaneously using global curve fits to the equation for 1:1 Langmuir binding in the BIAevaluation 3.2 software (BIAcore AB). Distinct Localization of WARP in Joint Cartilage during Development— The spatial distribution of WARP in the developing mouse knee joint was assessed during development at E15.5 and E18.5 and later in the 2- and 6-week-old knee joint. Immunohistochemistry was performed using the polyclonal antibody raised to the WARP C-terminal domain, with the corresponding preimmune serum used as a negative control. The results were verified by immunostaining with the WARP anti-VWA domain antibody (data not shown). At E15.5, the joint cavity between the cartilage rudiments of the femur and tibia had not formed. The femur and tibia anlagen were separated by the joint interzone, consisting of two chondrogenous layers separated by an intermediate zone of mesenchymal cells, which will differentiate to form the articular surfaces and the joint cavity. At E15.5 WARP was present in the chondrogenous layers of the joint interzone of the femur and tibia (Fig. 1A) but was absent from the underlying epiphyseal cartilage and the noncartilaginous intermediate layer of the future joint space. In comparison, antibodies against another VWA domain superfamily member used as a marker for developing cartilaginous structures, matrilin-1, strongly labeled the cartilage anlage of the developing femur and tibia but not the joint interzone (Fig. 1B). In E18.5 mice, WARP protein was restricted to ∼6-10 cell layers extending from the articular surface (Fig. 1C). Matrilin-1 was absent from this developing articular zone but was abundant throughout the epiphyses of the femur and tibia (Fig. 1D). No staining was detected with the preimmune serum (not shown). To determine the site of WARP mRNA expression during early joint development, in situ hybridization was performed on frontal sections from an E14.5 mouse knee joint. Consistent with the distribution of WARP protein, WARP mRNA was expressed by cells of the two chondrogenous layers of the joint interzone separating the femur and patella but was absent from the mesenchymal intermediate layer of the interzone and from the epiphyseal cartilage (Fig. 1E). No signal was seen with the sense orientation negative control (Fig. 1F). In 2-week-old mice, after development of the secondary ossification center, WARP localized to the pericellular matrix primarily around superficial zone chondrocytes of the femoral and tibial articular cartilage (Fig. 1, H and J) and could also be detected in the superficial layers of the meniscus (Fig. 1H) and the patella (not shown). In 6-week-old articular cartilage, the distribution of WARP was clearly restricted to the pericellular matrix, with no detectable interterritorial matrix staining (Fig. 1, K and M). Pericellular WARP immunolabeling was also seen in the meniscus, the patella, and the intermediate as well as superficial zones of the femoral and tibial articular cartilage. No immunostaining was detected in sections probed with the preimmune serum (Fig. 1, I and L). WARP Also Localizes to the Permanent Cartilages of Spine and Sternum—In the spine, immunostaining for WARP was restricted to the articular surfaces of the ribs and vertebrae at the costo-vertebral joint and barely detectable in the rib costal cartilage and vertebral body (Fig. 2A). WARP also exhibited a pericellular staining pattern throughout the annulus fibrosus of the intervertebral disc (Fig. 2B). In contrast, matrilin-1 antibodies strongly labeled the cartilage anlage of the developing vertebral bodies and ribs (Fig. 2, E and F) and was barely detectable in articular cartilage of the costo-vertebral joint and in the annulus fibrosus, in a distribution complementary to that of WARP. In the embryonic sternum, WARP exhibits a pericellular distribution in the fibrocartilaginous synarthrodial joint between each sternebrae (Fig. 2, C and D) and is absent from the anlage of the developing ribs and sternebrae. Strong matrilin-1 immunoreactivity was detected throughout the embryonic anlage of the ribs and sternebrae but was absent from the synarthrodial joint (Fig. 2, G and H). This distribution is complementary to that of WARP, a consistent observation from analysis of the developing joint and spine. WARP Forms Disulfide-bonded Multimers in Cartilage—To assess whether WARP forms multimeric structures in vivo, mouse cartilage preparations were serially extracted under progressively more denaturing conditions and the solubilized material in each extract subjected to immunoblot analysis using WARP antisera. Serial extraction of femorotibial joint cartilage, first with neutral salt buffer and then extraction of the insoluble material with hyaluronidase and chondroitinase ABC to degrade hyaluronan and chondroitin sulfate GAG chains and release interacting proteins, did not extract WARP (Fig. 3A, lanes 1 and 2). However, extraction of the remaining insoluble material with the strong denaturant 6 m guanidine HCl solubilized WARP protein, which was detected as a band on SDS-polyacrylamide gels migrating at ∼50 kDa when resolved under reducing conditions (lane 3), consistent with the predicted molecular mass of the WARP monomer (7Fitzgerald J. Ting S.T. Bateman J.F. FEBS Lett. 2002; 517: 61-66Crossref PubMed Scopus (33) Google Scholar). The ECM “adaptor” molecule, matrilin-1, was also present in the guanidine-soluble extract (lane 4), in agreement with published data (21Hauser N. Paulsson M. Heinegard D. Morgelin M. J. Biol. Chem. 1996; 271: 32247-32252Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The finding that WARP is solubilized by 6 m guanidine HCl suggests that, as with matrilin-1, WARP is a strongly interacting component of the cartilage ECM. When resolved without reduction, WARP migrated as disulfide-bonded oligomeric forms greater than 200 kDa (Fig. 3B, lane 1). Immunoblot analysis on conditioned media from cells transfected with WARP cDNA confirms that WARP assumes several multimeric forms, including a dimer at 100 kDa, that are reducible to a 50-kDa monomer (Fig. 3B, lanes 2-4). These multimers were also formed by purified recombinant WARP, confirming that these structures are likely to be oligomers of WARP rather than WARP bound to other molecules (lanes 5 and 6). The ability of WARP to self-associate was assessed in BIAcore biosensor experiments where WARP coupled to the sensor chip surface was challenged with WARP in solution (Fig. 3C). Soluble WARP (120-720 nm) bound to immobilized WARP in a dose-dependent manner. Global or local curve fitting using BIA evaluation software (v3.2) failed to fit these binding curves using the Langmuir 1:1 model, suggesting that the WARP-WARP interactions reflect complex self-associations, consistent with WARP multimerization. To test whether one or both Cys residues are involved in multimer stabilization, the two C-terminal cysteines were mutated to serine residues separately and in tandem (Fig. 3D), and their effects on multimerization were assessed in transfected cells (Fig. 3E). Because the majority of WARP is present in the medium fraction rather than the cell fraction of all four transfected cell lines, the introduced mutations did not result in intracellular accumulation of the mutant protein. In cells expressing the wild-type chain, WARP migrated primarily as dimeric and higher order forms with several discrete bands greater than 250 kDa (lane 2) that were identified as WARP in immunoblotting experiments (Fig. 3B). The C1 and C2 single cysteine mutants both migrated as a monomer and a dimer with no higher molecular mass structures evident (lanes 3-6). As expected, disulfide-bonded dimers or multimers were not detected in the C1C2 double mutant, which migrated only as a monomer (lanes 7 and 8). These data suggest that both Cys residues can participate in disulfide bond formation and that both Cys residues are required to stabilize the higher molecular mass WARP oligomers. WARP Co-localizes with Perlecan—Our finding that WARP forms multimeric structures in the cartilage extracellular environment raises the possibility that, like other multimeric components such as matrilins, WARP interacts with different cartilage ECM molecules. Because it has been reported that perlecan, a large multidomain proteoglycan that is essential for skeletal development, is a component of the cartilage pericellular environment (22SundarRaj N. Fite D. Ledbetter S. Chakravarti S. Hassell J.R. Journal of Cell Science. 1995; 108: 2663-2672Crossref PubMed Google Scholar, 23Melrose J. Smith S. Ghosh P. Whitelock J. J. Histochem. Cytochem. 2003; 51: 1331-1341Crossref PubMed Scopus (39) Google Scholar, 24Iozzo R.V. Cohen I.R. Grassel S. Murdoch A.D. Biochem. J. 1994; 302: 625-639Crossref PubMed Scopus (338) Google Scholar, 25Timpl R. Experientia. 1993; 49: 417-428Crossref PubMed Scopus (122) Google Scholar, 26Costell M. Gustafsson E. Aszodi A. Morgelin M. Bloch W. Hunziker E. Addicks K. Timpl R. Fassler R. J. Cell Biol. 1999; 147: 1109-1122Crossref PubMed Scopus (537) Google Scholar), we assessed whether WARP and perlecan co-localize in articular cartilage. We performed immunohistochemistry on mouse articular cartilage from 6-week-old mice and immunogold electron microscopy on human articular cartilage extract using antisera against WARP and monoclonal antibodies against perlecan (Fig. 4). In 6-week-old mice perlecan was present in articular cartilage and specifically localized to the chondrocyte pericellular region (Fig. 4, A and B) in a pattern that was strikingly similar to that of WARP (Figs. 1M and 4C). Immunogold electron microscopy was performed on extracts of human articular cartilage using an antibody against recombinant full-length WARP (18-nm particles) and a monoclonal anti-perlecan antibody (12-nm particles) (Fig. 4D). Both molecules were frequently found in close association within the amorphous extrafibrillar material, providing further evidence that WARP and perlecan co-localize in articular cartilage. The immunogold EM experiments were repeated using the VWA domain and C-terminal WARP antisera, and the same pattern of co-localization with perlecan was demonstrated (data not shown). WARP Interacts with Perlecan—We have established that WARP is a multimeric ECM component that localizes to the same connective" @default.
• W2103238084 created "2016-06-24" @default.
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• W2103238084 date "2006-03-01" @default.
• W2103238084 modified "2023-10-17" @default.
• W2103238084 title "WARP Is a Novel Multimeric Component of the Chondrocyte Pericellular Matrix That Interacts with Perlecan" @default.
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