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• W2078974495 abstract "The cartilage oligomeric matrix protein (COMP) and matrilins are abundant non-collagenous proteins in the cartilage extracellular matrix. In the presence of calcium, COMP and matrilin-1 elute together in the gel filtration of cartilage extracts and can be co-immunoprecipitated. In a screen for ligands of matrilin-1, -3, and -4 using an ELISA-style binding assay, COMP was identified as a prominent binding partner for all three, indicating a conservation of the COMP interaction among matrilins. The interaction of COMP and matrilin-4 is saturable, and an apparent KD of 1 nm was determined. However, only the full-length COMP and the full-length matrilin-4 proteins showed a strong interaction, indicating that the oligomeric structures markedly increase the affinity. Mutations in COMP or matrilin-3 cause related forms of human chondrodysplasia, and the COMP mutation D469Δ, which is found in patients with pseudoachondroplasia, has been shown to cause a reduced calcium binding. Despite this, the mutation causes only a slight decrease in matrilin-4 binding. This indicates that impaired binding of COMP to matrilins does not cause the pseudoachondroplasia phenotype but rather that matrilins may be coretained in the rough endoplasmatic reticulum where COMP accumulates in the chondrocytes of patients. The cartilage oligomeric matrix protein (COMP) and matrilins are abundant non-collagenous proteins in the cartilage extracellular matrix. In the presence of calcium, COMP and matrilin-1 elute together in the gel filtration of cartilage extracts and can be co-immunoprecipitated. In a screen for ligands of matrilin-1, -3, and -4 using an ELISA-style binding assay, COMP was identified as a prominent binding partner for all three, indicating a conservation of the COMP interaction among matrilins. The interaction of COMP and matrilin-4 is saturable, and an apparent KD of 1 nm was determined. However, only the full-length COMP and the full-length matrilin-4 proteins showed a strong interaction, indicating that the oligomeric structures markedly increase the affinity. Mutations in COMP or matrilin-3 cause related forms of human chondrodysplasia, and the COMP mutation D469Δ, which is found in patients with pseudoachondroplasia, has been shown to cause a reduced calcium binding. Despite this, the mutation causes only a slight decrease in matrilin-4 binding. This indicates that impaired binding of COMP to matrilins does not cause the pseudoachondroplasia phenotype but rather that matrilins may be coretained in the rough endoplasmatic reticulum where COMP accumulates in the chondrocytes of patients. IntroductionAlthough the cartilage extracellular matrix contains only a limited number of structural proteins, many aspects of their assembly into a functional matrix remain to be described. Cartilage collagen fibrils and aggrecan, assembled into large aggregates, form the major networks that provide tensile strength and elasticity, whereas other matrix proteins are believed to mediate additional steps in the matrix assembly. Of these proteins, COMP 1The abbreviations used are: COMP, cartilage oligomeric matrix protein; TC, C-terminal COMP domain; ELISA, enzyme-linked immunosorbent assay; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; T3, thrombospondin type 3 repeat-containing COMP domain; VWA, von Willebrand factor A. (1Oldberg A. Antonsson P. Lindblom K. Heinegård D. J. Biol. Chem. 1992; 267: 22346-22350Abstract Full Text PDF PubMed Google Scholar) and the four members of the matrilin family (for review see Ref. 2Deák F. Wagener R. Kiss I. Paulsson M. Matrix Biol. 1999; 18: 55-64Crossref PubMed Scopus (137) Google Scholar) are among the most abundant and share the feature of having an oligomeric structure, allowing polyvalent interactions.COMP is a homopentameric member of the thrombospondin protein family (for review see Ref. 3Adams J.C. Annu. Rev. Cell Dev. Biol. 2001; 17: 25-51Crossref PubMed Scopus (319) Google Scholar). Its N-terminal α-helical coiled-coil oligomerization domain is followed by four epidermal growth factor-like domains, eight calcium-binding thrombospondin type 3 (T3) repeats, and a globular domain (TC) at the C terminus. COMP is predominantly found in the extracellular matrix of cartilage, tendons, and ligaments and has been shown to bind to collagens I, II, and IX (4Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 6Holden P. Meadows R.S. Chapman K.L. Grant M.E. Kadler K.E. Briggs M.D. J. Biol. Chem. 2001; 276: 6046-6055Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Mutations in COMP can lead to two related human diseases, pseudoachondroplasia and multiple epiphyseal dysplasia (7Briggs M.D. Chapman K.L. Hum. Mutat. 2002; 19: 465-478Crossref PubMed Scopus (223) Google Scholar).The matrilins constitute a recently discovered family of extracellular matrix proteins (2Deák F. Wagener R. Kiss I. Paulsson M. Matrix Biol. 1999; 18: 55-64Crossref PubMed Scopus (137) Google Scholar). Matrilin-2 and -4 have broad tissue distributions in both dense and loose connective tissue, whereas matrilin-1 and -3 are expressed almost exclusively in skeletal tissues. In the extracellular matrix of cultured cells, matrilins are deposited in fibrillar networks. All matrilins have a similar modular composition, with von Willebrand factor A (VWA)-like and epidermal growth factor-like domains followed by a C-terminal coiled-coil domain. It is thought that matrilins play a role in mediating interactions between major components of the extracellular matrix such as collagens and proteoglycans (8Hauser N. Paulsson M. Heinegård D. Mörgelin M. J. Biol. Chem. 1996; 271: 32247-32252Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and the three forms abundant in cartilage, namely matrilin-1, -3, and –4, are indeed associated with native collagen type VI microfibrils extracted from rat chondrosarcoma tissue (9Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegård D. Mörgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Here the matrilins are bound to the small leucinerich repeat proteoglycans biglycan and decorin, which, in turn, interact with the N-terminal globular domains of the collagen VI molecules. When located at the periphery of the microfibrillar complex, matrilins mediate interactions with aggrecan or collagen II. Matrilin-3 is strongly up-regulated in human osteoarthritic cartilage (10Pullig O. Weseloh G. Klatt A.R. Wagener R. Swoboda B. Osteoarthr. Cartil. 2002; 10: 253-263Abstract Full Text PDF PubMed Scopus (61) Google Scholar), and a missense mutation in the human matrilin-3 gene was recently found to coincide with hand osteoarthritis in a group of patients in the Icelandic population (11Stefansson S.E. Jonsson H. Ingvarsson T. Manolescu I. Jonsson H.H. Olafsdottir G. Palsdottir E. Stefansdottir G. Sveinbjornsdottir G. Frigge M.L. Kong A. Gulcher J.R. Stefansson K. Am. J. Hum. Genet. 2003; 72: 1448-1459Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). As in the case of COMP, mutations in the matrilin-3 gene have been shown to cause multiple epiphyseal dysplasia (12Chapman K.L. Mortier G.R. Chapman K. Loughlin J. Grant M.E. Briggs M.D. Nat. Genet. 2001; 28: 393-396Crossref PubMed Scopus (155) Google Scholar). In the present study we have identified an interaction between COMP and members of the matrilin family that is likely to be of importance both in extracellular matrix assembly and in the pathogenesis of chondrodysplasias.EXPERIMENTAL PROCEDURESGel Filtration Analysis—The extraction of matrix proteins from bovine cartilage was performed essentially as described earlier (13DiCesare P.E. Mörgelin M. Carlson C.S. Pasumarti S. Paulsson M. J. Orthop. Res. 1995; 13: 422-428Crossref PubMed Scopus (94) Google Scholar). In brief, fresh cartilage (∼100 g, wet weight) from adult bovine shoulder joints was homogenized using a Moulinex MiniPro homogenizer at full speed in 200 ml of prechilled extraction buffer (0.15 m NaCl, 10 mm Tris-HCl, pH 7.4, and 1 mm phenylmethylsulfonyl fluoride) without EDTA. Tissue residue was collected by centrifugation at 18,000 × g at 4 °C for 20 min. This wash cycle was repeated twice, each time with 10 min of homogenization. The tissue residue was then suspended in 200 ml of the same buffer containing, in addition, 10 mm EDTA and extracted for 2 h with stirring at 4 °C. The extraction with the EDTA-containing buffer was repeated twice, and the extracts were pooled.The crude extract was cleared by filtration and concentrated 100-fold by ultrafiltration. Gel filtration was performed using a pre-packed Superose 6 column (Amersham Biosciences) equilibrated with extraction buffer. For detection of the calcium-dependent co-elution of matrix proteins, the concentrated cartilage extract was dialyzed against extraction buffer without EDTA and, finally, against extraction buffer containing 2 mm CaCl2, which was also used during gel filtration. Fractions of 1 ml were collected and analyzed by SDS-PAGE using 10% polyacrylamide gels. Protein bands were visualized by silver stain. Peptide mapping and identification of protein bands by MALDI-TOF mass spectrometry was performed as described previously (14Sengle G. Kobbe B. Mörgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2003; 278: 50240-50249Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar).Immunoblotting—After SDS-PAGE, protein bands were transferred to a nitrocellulose membrane (Schleicher & Schuell) and incubated with a mouse monoclonal antibody against human COMP (12-C4) (15Vilim V. Voburka Z. Vytasek R. Senolt L. Tchetverikov I. Kraus V.B. Pavelka K. Clin. Chim. Acta. 2003; 328: 59-69Crossref PubMed Scopus (38) Google Scholar) or a rabbit polyclonal antibody against human matrilin-1. Primary antibodies were detected using anti-mouse or anti-rabbit IgG horseradish peroxidase conjugates and the ECL chemoluminescence system (Amersham Biosciences).Immunoprecipitation—A bovine cartilage extract was dialyzed extensively against distilled water containing 10 μm EDTA to capture endogenous calcium. CaCl2 was then added at concentrations as indicated in Fig. 1C, and samples were incubated for 4 h at 4 °C with protein A-Sepharose (Amersham Biosciences) pre-treated with matrilin-1 antibody. After precipitation the protein A-Sepharose was washed three times with PBS, and co-precipitated COMP was detected by immunoblotting using a monoclonal antibody to COMP (12-C4).Expression and Purification of Recombinant Proteins—The recombinant expression in human embryonic kidney 293/Epstein-Barr virus nuclear antigen cells and conventional chromatographic purification as well as the use of affinity chromatography in the purification of tagged proteins have been described previously (5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 16Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 17Klatt A.R. Nitsche D.P. Kobbe B. Mörgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2000; 275: 3999-4006Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Spitznagel L. Nitsche D.P. Paulsson M. Maurer P. Zaucke F. Biochem. J. 2004; 377: 479-487Crossref PubMed Google Scholar, 19Thur J. The Effects of Chondrodysplasia-associated Mutations on the Structure and Function of the Cartilage Oligometric Matrix Protein. University of Cologne, Cologne, Germany2000Google Scholar). Denaturing solvents were avoided in all steps. Tags were not removed prior to binding assays. The cDNAs encoding full-length matrilin-3 and monomeric matrilin-4 lacking the coiled-coil domain were amplified by PCR using primers that inserted an SpeI restriction site at the 5′-end and a NotI site at the 3′-end, respectively (matn3 forward, 5′-GCCCACTAGTCCGTTTGGCCCGCGCGAGC-3′, and matn3 reverse, 5′-CAATGCGGCCGCACGATGTACTTGTCCATATTC-3′; matn4m forward, 5′-GCCACTAGTCCAGCTCCAGTCTGCAGGTCC-3′, and matn4m reverse, 5′-CAATGCGGCCGCCTCTGGGCAAATGCTGCCTTTG-3′). After digestion with SpeI and NotI, the amplified cDNA fragments were inserted into the expression vector pCEP-Pu-StrepII-tag (C-terminal) in-frame with the sequence of the signal peptide of BM40 (20Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (201) Google Scholar). The cDNA encoding matrilin-4 VWA1 and VWA2 was amplified by PCR using primers that inserted an NheI restriction site at the 5′-end and a NotI site 3′ to the stop codon (matn4A1 forward, 5′-GCCACTAGTCTGCTATAATGGGCCCCTGGATT-3′, and matn4A1 reverse, 5′-CAATGACTGCGGCCGCTTACCCGCACAGGCGGCCCTG-3′; matn4A2 forward, 5′-GCCCACTAGTCTGCCGGGAGGGCCACGTG-3′, and matn4A2 reverse, 5′-CAATGACTGCGGCCGCTTAGCAAATGCTGCCTTTGAGATTC-3′). After digestion with NheI and NotI, the amplified cDNA fragments were inserted into the expression vector pCEP-Pu-BM40-His6 (N-terminal) in-frame with the sequence of the signal peptide of BM40 (20Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (201) Google Scholar). The recombinant plasmids carrying cDNAs encoding full-length matrilin-3 and matrilin-4 VWA1 and VWA2 were transfected into human embryonic kidney 293/Epstein-Barr virus nuclear antigen cells (Invitrogen) using FuGENE6 (Roche). Transfected cells were selected with 1 μg/ml puromycin and grown to confluency. Secretion of recombinant proteins into the cell culture medium was confirmed by SDS-PAGE performed on medium samples from transfected cells and by immunoblotting using specific antisera directed against matrilin-3 and matrilin-4, respectively. The serum-free cell culture supernatant containing the C-terminally Strep-tag II-tagged matrilin-3 was applied to a Strep-tactin column (1.5 ml; IBA) and eluted with 2.5 mm desthiobiotin and 10 mm Tris-HCl, pH 8. Purification of the recombinant His6-tagged matrilin-4 proteins from serum-free cell culture supernatants was carried out using TALON metal affinity columns (Clontech) following the supplier's protocol. C-terminally Strep-tag II-tagged matrilin-4 full-length wild type protein was expressed and purified as described earlier (16Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).The recombinant full-length wild type COMP, the truncated COMP carrying the T3 and TC domains, the COMP TC domain alone, and the COMP full-length protein carrying the D469Δ mutation were expressed carrying a N-terminal His6-Myc-factorX tag as described (18Spitznagel L. Nitsche D.P. Paulsson M. Maurer P. Zaucke F. Biochem. J. 2004; 377: 479-487Crossref PubMed Google Scholar). The COMP monomer was expressed without a tag and subsequently purified on a Q-Sepharose FastFlow ion exchange column (Amersham Biosciences) and a Sepharose CL-6B gel filtration column (Amersham Bioscience) as described (19Thur J. The Effects of Chondrodysplasia-associated Mutations on the Structure and Function of the Cartilage Oligometric Matrix Protein. University of Cologne, Cologne, Germany2000Google Scholar). The COMP and matrilin-4 coiled-coil domains were recombinantly expressed in Escherichia coli and purified as described earlier (21Efimov V.P. Engel J. Malashkevich V.N. Proteins. 1996; 24: 259-262Crossref PubMed Scopus (34) Google Scholar, 22Frank S. Schulthess T. Landwehr R. Lustig A. Mini T. Jeno P. Engel J. Kammerer R.A. J. Biol. Chem. 2002; 277: 19071-19079Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).Purification of Matrilin-1—Matrilin-1 was purified from fetal bovine rib cartilage under native conditions using 0.25 m NaCl, 5 mm Tris-HCl, pH 7.4, 2 mm phenylmethylsulfonyl fluoride, 2 mm N-ethylmaleimide, and 10 mm EDTA as extraction buffer. After the removal of proteoglycans on a DEAE-Sepharose FastFlow column (Amersham Biosciences), the flow-through was concentrated on an SP-Sepharose FastFlow column (Amersham Bioscience), and the matrilin-1-containing eluate fractions were submitted to gel filtration on a Sepharose CL-6B column (Amersham Bioscience) as described (8Hauser N. Paulsson M. Heinegård D. Mörgelin M. J. Biol. Chem. 1996; 271: 32247-32252Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).ELISA-style Ligand Binding Assay—All solutions used contained 1 mm CaCl2. Proteins in the panel used for ligand screening were kind gifts of M. Aumailley (laminin-1 and fibronectin), U. Hansen (collagen XI), M. Koch (collagens XII, XIV, and XXII), M. Mörgelin (chondroadherin and the proline arginine-rich end leucine-rich repeat protein), N. Smyth (decorin), and F. Zaucke (COMP) or were purchased from Sigma (collagens I and II). Purified proteins were diluted in Tris-buffered saline, pH 7.4, and coated at 10 μg/ml (500 ng/well) overnight at 4 °C onto 96-well plates (Nunc Maxisorb). After washing with Tris-buffered saline and 05% Tween 20, plates were blocked for 2 h at room temperature with Tris-buffered saline and 5% milk powder. Fluid phase ligands were diluted to concentrations between 0.01 and 50 nm for titration experiments or to 20 nm for single point determinations and incubated for 1 h at room temperature. After extensive washing with Tris-buffered saline and 0.05% Tween 20, bound ligands were detected with specific polyclonal rabbit antisera directed against matrilin-1 (23Hauser N. Paulsson M. J. Biol. Chem. 1994; 269: 25747-25753Abstract Full Text PDF PubMed Google Scholar), matrilin-3 (17Klatt A.R. Nitsche D.P. Kobbe B. Mörgelin M. Paulsson M. Wagener R. J. Biol. Chem. 2000; 275: 3999-4006Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), matrilin-4 (16Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and COMP (13DiCesare P.E. Mörgelin M. Carlson C.S. Pasumarti S. Paulsson M. J. Orthop. Res. 1995; 13: 422-428Crossref PubMed Scopus (94) Google Scholar), respectively, followed by swine anti-rabbit immunoglobulin horseradish peroxidase conjugates (Dako Cytomation) and tetramethylbenzidine as substrate. Absorption was measured at 450 nm after stopping the reaction with 10% sulfuric acid.RESULTSCOMP and Matrilin-1 Co-elute in Gel Filtration Performed in the Presence of CaCl2—To identify the components of the cartilage extracellular matrix that interact in a calcium-dependent manner, the gel filtration elution profile of a bovine cartilage extract obtained in the presence of 2 mm CaCl2 was compared with that of an extract chromatographed in the absence of divalent cations. A comparison of fractions by reducing SDS-PAGE revealed that a band with an apparent molecular mass of 60 kDa was markedly shifted toward the void volume in the presence of calcium where it co-eluted with a band of 100-kDa apparent molecular mass (Fig. 1A). By in-gel trypsin digestion followed by MALDI-TOF mass spectrometry fingerprint analysis (not shown) and immunoblot analysis (Fig. 1B) the 60-kDa protein was identified as matrilin-1, and the 100-kDa protein was identified as COMP by immunoblot (Fig. 1B). The COMP peak was also shifted slightly toward the void volume in the presence of calcium, indicating an increase in mass of the COMP-containing complex. The formation of a calcium-dependent complex between COMP and matrilin-1 was confirmed by the co-precipitation of COMP when matrilin-1 was immunoprecipitated from a cartilage extract (Fig. 1C). The interaction between COMP and matrilin-1 was already saturated by the addition of calcium at a concentration of 1 μm.Screen for Matrilin Ligands Using an ELISA-style Binding Assay—A panel of different purified proteins and proteoglycans from the cartilage extracellular matrix was tested for interactions with matrilin-1, -3, and -4, the three matrilins most abundant in cartilage, in an ELISA-style ligand binding assay. COMP was identified as the protein giving the highest signal (Fig. 2). The fact that all three matrilins showed significant binding to COMP indicated a conservation of the COMP interaction among matrilins.Fig. 2Screen for matrilin ligands using an ELISA-style ligand binding assay. A panel of different proteins and proteoglycans from cartilage extracellular matrix was tested for interactions with matrilin-1, -3, and -4. The proteins in the panel (10 μg/ml) were coated to the plastic and incubated with full-length matrilin-1 (A), matrilin-3 (B), and matrilin-4 (C) (concentration equalling 50 nm; matrilin-1, 8.7 μg/ml; matrilin-3, 10.7 μg/ml; and matrilin-4, 11.0 μg/ml) in the presence of 1 mm CaCl2 for 1 h. Binding was detected in an ELISA-style manner using specific antisera directed against the matrilins. Coll, collagen; ChAd, chondroadherin; PRELP, proline arginine-rich end leucine-rich repeat protein. Abs., absorbance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of the Interaction between Matrilin-4 and COMP—For detailed interaction studies we decided to focus on matrilin-4, as its relatively good solubility and, therefore, comparatively high purification yields allowed more extensive experimentation. Titration of the interaction of matrilin-4 with COMP in the ELISA-style binding assay showed saturable binding and indicated an apparent KD of 1 nm (Fig. 3).Fig. 3Concentration-dependence of the COMP-matrilin-4 interaction determined in an ELISA-style ligand binding assay. Different concentrations of full-length matrilin-4 were incubated in the presence of 1 mm CaCl2 with full-length wild type COMP coated onto the plastic from a 10 μg/ml solution. Binding was detected in an ELISA-style manner using a specific antiserum directed against matrilin-4. Abs., absorbance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Shorter constructs of COMP and matrilin-4 were tested in ELISA-style ligand binding assays with each of the proteins being studied both in the fluid and on the solid phase. When full-length COMP was coated and different matrilin-4 proteins were used as ligands in the fluid phase (Fig. 4, A and B), the matrilin-4 full-length protein showed the strongest binding, followed by a moderate binding of a matrilin-4 monomer lacking the coiled-coil oligomerization domain. However, the single VWA domains as well as the isolated coiled-coil domain did not bind to the immobilized COMP. In an inverted assay we coated full-length matrilin-4 and the shorter forms using both full-length wild type COMP and a mutant form (D469Δ) as ligands in the fluid phase (Fig. 4C). The COMP D469Δ mutation found in chondrodysplasia patients results in a decreased binding capacity for calcium (5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 24Maddox B.K. Mokashi A. Keene D.R. Bächinger H.P. J. Biol. Chem. 2000; 275: 11412-11417Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). When matrilin-4 was coated, the D469Δ mutant bound to the same extent as did wild type COMP (Figs. 4C and 5C). The binding to matrilin-4 was moderately decreased by the mutation when the mutant COMP was immobilized (Fig. 5B). Taken together, the results indicate only a mild influence of the mutation on matrilin-4 binding, whereas the addition of EDTA completely abolished the interaction (not shown). Significant binding to full-length matrilin-4 was seen, whereas only weak binding to both VWA domains was detected, and no binding to the matrilin-4 coiled-coil domain was found (Fig. 4, C and D).Fig. 4Binding of full-length and truncated matrilin-4 to COMP and D469Δ COMP in an ELISA-style ligand binding assay. A, full-length, wild type COMP was coated onto plastic and incubated in the blocked wells with full-length, wild type matrilin-4 (Matn-4 fl wt), the matrilin-4 coiled-coil domain (Matn-4 CC), and the matrilin-4 VWA1 (Matn-4 A1) as well as the matrilin-4 VWA2 (Matn-4 A2) domain. B, full-length wild type COMP was coated and incubated in the blocked wells with full-length, wild type matrilin-4 (Matn-4 fl wt) and the matrilin-4 monomer (Matn-4 mono). C, full-length matrilin-4 (Matn-4 fl wt), the matrilin-4 VWA1 domain (Matn-4 A1), and the matrilin-4 VWA2 (Matn-4 A2) domain were coated and incubated with full-length wild type COMP (solid bars) and D469Δ COMP (open bars) in the blocked wells. D, full-length, wild type matrilin-4 (Matn-4 fl wt), the matrilin-4 monomer (Matn-4 mono), and the matrilin-4 coiled-coil domain (Matn-4 CC) were coated to plastic and incubated with full-length wild type COMP. In all assays the proteins were coated onto plastic at 10 μg/ml, and the ligands in the liquid phase were used at 20 nm in the presence of 1 mm CaCl2. Binding was detected in an ELISA-style manner using antisera to matrilin-4 (A and B) or COMP (C and D). Abs., absorbance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Binding of full-length, truncated, and D469Δ COMP to matrilin-4 in an ELISA-style ligand binding assay. A and B, full-length, wild type COMP (COMP fl wt), monomeric COMP (COMP mono), truncated COMP consisting of T3-repeats and the TC domain (COMP T3+TC), the COMP TC domain (COMP TC), the COMP coiled-coil domain (COMP CC), and the full-length COMP mutant D469Δ (COMP D469D) were coated onto plastic at 10 μg/ml and incubated with matrilin-4 in the blocked wells at 20 nm. C, matrilin-4 was coated onto plastic at 10 μg/ml and incubated with COMP proteins (abbreviations as in panels A and B) in the blocked wells at 20 nm. All assays were carried out in the presence of 1 mm CaCl2. Binding was detected in an ELISA-style manner using antisera to matrilin-4 (A and B) or COMP (C). Abs., absorbance.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Truncated COMP proteins were also tested for binding to matrilin-4 (Fig. 5). The ELISA plate was coated with monomeric COMP lacking the N-terminal coiled-coil oligomerization domain, a protein comprising the T3 repeats together with the TC domain and the isolated TC and the coiled-coil domain in addition to full-length pentameric forms of wild type and mutant (D469Δ) COMP (Fig. 5, A and B). After incubation with full-length matrilin-4 in the fluid phase, strong signals were detected for both full-length wild type and mutant COMP. A weak signal was seen with the coiledcoil domain alone, whereas the monomeric COMP and the shorter constructs showed no significant binding. The addition of EDTA abolished binding (not shown). When the plate was coated with full-length matrilin-4 in an inverted assay (Fig. 5C), full-length COMP as well as the mutant COMP bound strongly, whereas monomeric COMP, the T3+TC domain pair, and the TC domain alone showed only weak interactions.DISCUSSIONIn the present study we have identified and characterized a calcium-dependent interaction between COMP and matrilins, proteins thought to mediate assembly of the cartilage extracellular matrix. The identification of a complex between COMP and matrilin-1 in CaCl2-containing cartilage extracts indicates a physiological role for a COMP-matrilin interaction. Both COMP and matrilins have been shown to associate with cartilage collagen fibrils (4Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 25Winterbottom N. Tondravi M.M. Harrington T.L. Klier F.G. Vertel B.M. Goetinck P.F. Dev. Dyn. 1992; 193: 266-276Crossref PubMed Scopus (91) Google Scholar). COMP binds to collagens I, II, and IX (4Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 6Holden P. Meadows R.S. Chapman K.L. Grant M.E. Kadler K.E. Briggs M.D. J. Biol. Chem. 2001; 276: 6046-6055Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and matrilin-1 has been localized on the surface of collagen II-containing fibrils (25Winterbottom N. Tondravi M.M. Harrington T.L. Klier F.G. Vertel B.M. Goetinck P.F. Dev. Dyn. 1992; 193: 266-276Crossref PubMed Scopus (91) Google Scholar). Furthermore, matrilins are components of the collagen VI-containing microfibrils found in cartilage (9Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegård D. Mörgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Here the leucine-rich-repeat proteoglycans decorin and biglycan bind to the N-terminal non-collagenous domains of collagen VI molecules and then, in turn, also to matrilins. The matrilins at the periphery of the microfibrillar structure show association with both collagen II molecules and aggrecan and presumably act as adaptors connecting the microfibrils to the other macromolecular networks. The interactions between COMP and matrilins are, at least in part, likely to take place at the surface of the collagen fibrils and may be of importance in creating a “perifibrillar” matrix of a defined composition. They also may be of relevance for the regulation of collagen fibrillogenesis and for interactions between collagen fibrils, issues to be the subjects of further studies.An indication of a pathophysiological relevance for the COMP-matrilin interaction comes from the fact that mutations in either COMP, collagen IX, or matrilin-3 have been shown to cause the related pseudoachondroplasia or multiple epiphyseal dysplasia forms of chondrodysplasia (7Briggs M.D. Chapman K.L. Hum. Mutat. 2002; 19: 465-478Crossref PubMed Scopus (223) Google Scholar). In pseudoachondroplasia patients carrying mutations in COMP as well as in cell culture models of this disease, COMP is retained in the rough endoplasmatic reticulum of the chondrocytes, and collagen IX co-accumulates, presumably due to the COMP-collagen IX interaction (24Maddox B.K. Mokashi A. Keene D.R. Bächinger H.P. J. Biol. Chem. 2000; 275: 11412-11417Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26Dinser R. Zaucke F. Kreppel F. Hultenby K. Kochanek S. Paulsson M. Maurer P. J. Clin. Investig. 2002; 110: 505-513Crossref PubMed Scopus (61) Google Scholar). It is not clear at present how the mutations in matrilin-3 result in multiple epiphyseal dysplasia. Possibly, mutations in any of these three interacting proteins, namely COMP, collagen IX, or matrilin-3, result in the retention of a similar set of molecules independent of which protein actually harbors the mutation. This may explain why changes in any of these three proteins lead to a similar clinical phenotype. Indeed, in a recent review of the etiology of chondrodysplasias, Briggs and Chapman mentioned preliminary data on a high affinity binding of matrilin-3 to COMP in this context, but, in contrast to our results, the interaction was described as being cation-independent (7Briggs M.D. Chapman K.L. Hum. Mutat. 2002; 19: 465-478Crossref PubMed Scopus (223) Google Scholar). In all our experiments the binding was completely abolished by the addition of EDTA. The calcium binding to COMP is well studied, and it has been determined that the T3 repeats can bind 14–17 Ca2+ ions (5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 24Maddox B.K. Mokashi A. Keene D.R. Bächinger H.P. J. Biol. Chem. 2000; 275: 11412-11417Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The chondrodysplasia-causing D469Δ mutation leads to a partial loss of calcium binding sites and also to a slightly reduced affinity for collagens I, II, and IX (5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In the presence of calcium the mutant protein does not show the compact shape of wild type COMP but instead a more extended conformation (27Chen H. Deere M. Hecht J.T. Lawler J. J. Biol. Chem. 2000; 275: 26538-26544Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Nevertheless, it was shown that the secondary structure of COMP is not dramatically affected and that the collagen affinity is only slightly diminished (5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), indicating that the structure of the individual domains is at least partially retained and that the remaining Ca2+ binding sites are sufficient for ligand binding. This could explain why the D469Δ mutation only mildly influences the COMP-matrilin interaction in our experiments. The matrilin-4 binding was moderately reduced only in assays where the mutant COMP was immobilized, whereas binding of wild type and mutant COMP to immobilized matrilin-4 was comparable. Thus, it is likely that mutant COMP retained in the rough endoplasmic reticulum would have the capacity to interact with matrilins and reduce their secretion.Full-length COMP binds to full-length matrilin-4 with high affinity (Fig. 3). When either full-length COMP or monomeric COMP lacking the oligomerization domain was immobilized on ELISA plates, only the full-length, pentameric protein bound matrilin-4. In contrast, immobilized full-length matrilin-4 is able to bind the COMP monomer, although this interaction appears to be much weaker than that between the oligomers. Immobilized full-length COMP binds monomeric matrilin-4, but the amount bound is significantly decreased as compared with the oligomeric form. The preference for the oligomeric protein forms indicates a multivalent binding mechanism. This is of interest because both COMP and matrilins have been shown to be prone to degradation by proteolysis (16Klatt A.R. Nitsche D.P. Kobbe B. Macht M. Paulsson M. Wagener R. J. Biol. Chem. 2001; 276: 17267-17275Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 28Neidhart M. Hauser N. Paulsson M. DiCesare P.E. Michel B.A. Häuselmann H.J. Br. J. Rheumatol. 1997; 36: 1151-1160Crossref PubMed Scopus (211) Google Scholar). It could be that limited proteolysis, which in both cases occurs at sites close to the coiled-coil oligomerization domains and releases almost complete but monomeric subunits, also regulates COMP-matrilin interactions in vivo. The cooperative nature of the interaction did, however, hamper our attempts to determine the protein domains in COMP and matrilin-4 that are directly involved in binding, as the truncated proteins employed gave only weak or no binding. It is likely that residual affinity remains also in truncated, monomeric proteins and that future experiments employing more sensitive binding assays or artificially oligomerized subunits will be more informative. IntroductionAlthough the cartilage extracellular matrix contains only a limited number of structural proteins, many aspects of their assembly into a functional matrix remain to be described. Cartilage collagen fibrils and aggrecan, assembled into large aggregates, form the major networks that provide tensile strength and elasticity, whereas other matrix proteins are believed to mediate additional steps in the matrix assembly. Of these proteins, COMP 1The abbreviations used are: COMP, cartilage oligomeric matrix protein; TC, C-terminal COMP domain; ELISA, enzyme-linked immunosorbent assay; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; T3, thrombospondin type 3 repeat-containing COMP domain; VWA, von Willebrand factor A. (1Oldberg A. Antonsson P. Lindblom K. Heinegård D. J. Biol. Chem. 1992; 267: 22346-22350Abstract Full Text PDF PubMed Google Scholar) and the four members of the matrilin family (for review see Ref. 2Deák F. Wagener R. Kiss I. Paulsson M. Matrix Biol. 1999; 18: 55-64Crossref PubMed Scopus (137) Google Scholar) are among the most abundant and share the feature of having an oligomeric structure, allowing polyvalent interactions.COMP is a homopentameric member of the thrombospondin protein family (for review see Ref. 3Adams J.C. Annu. Rev. Cell Dev. Biol. 2001; 17: 25-51Crossref PubMed Scopus (319) Google Scholar). Its N-terminal α-helical coiled-coil oligomerization domain is followed by four epidermal growth factor-like domains, eight calcium-binding thrombospondin type 3 (T3) repeats, and a globular domain (TC) at the C terminus. COMP is predominantly found in the extracellular matrix of cartilage, tendons, and ligaments and has been shown to bind to collagens I, II, and IX (4Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Thur J. Rosenberg K. Nitsche D.P. Pihlajamaa T. Ala-Kokko L. Heinegård D. Paulsson M. Maurer P. J. Biol. Chem. 2001; 276: 6083-6092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 6Holden P. Meadows R.S. Chapman K.L. Grant M.E. Kadler K.E. Briggs M.D. J. Biol. Chem. 2001; 276: 6046-6055Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Mutations in COMP can lead to two related human diseases, pseudoachondroplasia and multiple epiphyseal dysplasia (7Briggs M.D. Chapman K.L. Hum. Mutat. 2002; 19: 465-478Crossref PubMed Scopus (223) Google Scholar).The matrilins constitute a recently discovered family of extracellular matrix proteins (2Deák F. Wagener R. Kiss I. Paulsson M. Matrix Biol. 1999; 18: 55-64Crossref PubMed Scopus (137) Google Scholar). Matrilin-2 and -4 have broad tissue distributions in both dense and loose connective tissue, whereas matrilin-1 and -3 are expressed almost exclusively in skeletal tissues. In the extracellular matrix of cultured cells, matrilins are deposited in fibrillar networks. All matrilins have a similar modular composition, with von Willebrand factor A (VWA)-like and epidermal growth factor-like domains followed by a C-terminal coiled-coil domain. It is thought that matrilins play a role in mediating interactions between major components of the extracellular matrix such as collagens and proteoglycans (8Hauser N. Paulsson M. Heinegård D. Mörgelin M. J. Biol. Chem. 1996; 271: 32247-32252Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and the three forms abundant in cartilage, namely matrilin-1, -3, and –4, are indeed associated with native collagen type VI microfibrils extracted from rat chondrosarcoma tissue (9Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegård D. Mörgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Here the matrilins are bound to the small leucinerich repeat proteoglycans biglycan and decorin, which, in turn, interact with the N-terminal globular domains of the collagen VI molecules. When located at the periphery of the microfibrillar complex, matrilins mediate interactions with aggrecan or collagen II. Matrilin-3 is strongly up-regulated in human osteoarthritic cartilage (10Pullig O. Weseloh G. Klatt A.R. Wagener R. Swoboda B. Osteoarthr. Cartil. 2002; 10: 253-263Abstract Full Text PDF PubMed Scopus (61) Google Scholar), and a missense mutation in the human matrilin-3 gene was recently found to coincide with hand osteoarthritis in a group of patients in the Icelandic population (11Stefansson S.E. Jonsson H. Ingvarsson T. Manolescu I. Jonsson H.H. Olafsdottir G. Palsdottir E. Stefansdottir G. Sveinbjornsdottir G. Frigge M.L. Kong A. Gulcher J.R. Stefansson K. Am. J. Hum. Genet. 2003; 72: 1448-1459Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). As in the case of COMP, mutations in the matrilin-3 gene have been shown to cause multiple epiphyseal dysplasia (12Chapman K.L. Mortier G.R. Chapman K. Loughlin J. Grant M.E. Briggs M.D. Nat. Genet. 2001; 28: 393-396Crossref PubMed Scopus (155) Google Scholar). In the present study we have identified an interaction between COMP and members of the matrilin family that is likely to be of importance both in extracellular matrix assembly and in the pathogenesis of chondrodysplasias." @default.
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• W2078974495 title "Interactions between the Cartilage Oligomeric Matrix Protein and Matrilins" @default.
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