Cartilage Oligomeric Matrix Protein Interacts with Type IX Collagen, and Disruptions to These Interactions Identify a Pathogenetic Mechanism in a Bone Dysplasia Family
2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês
10.1074/jbc.m009507200
ISSN1083-351X
AutoresPaul Holden, Roger S. Meadows, Kathryn Chapman, Michael E. Grant, Karl E. Kadler, Michael D. Briggs,
Tópico(s)Cell Adhesion Molecules Research
ResumoCartilage oligomeric matrix protein (COMP) and type IX collagen are key structural components of the cartilage extracellular matrix and have important roles in tissue development and homeostasis. Mutations in the genes encoding these glycoproteins result in two related human bone dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, which together comprise a “bone dysplasia family.” It has been proposed that these diseases have a similar pathophysiology, which is highlighted by the fact that mutations in either the COMP or the type IX collagen genes produce multiple epiphyseal dysplasia, suggesting that their gene products interact. To investigate the interactions between COMP and type IX collagen, we have used rotary shadowing electron microscopy and real time biomolecular (BIAcore) analysis. Analysis of COMP-type IX collagen complexes demonstrated that COMP interacts with type IX collagen through the noncollagenous domains of type IX collagen and the C-terminal domain of COMP. Furthermore, peptide mapping identified a putative collagen-binding site that is associated with known human mutations. These data provide evidence that disruptions to COMP-type IX collagen interactions define a pathogenetic mechanism in a bone dysplasia family. Cartilage oligomeric matrix protein (COMP) and type IX collagen are key structural components of the cartilage extracellular matrix and have important roles in tissue development and homeostasis. Mutations in the genes encoding these glycoproteins result in two related human bone dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, which together comprise a “bone dysplasia family.” It has been proposed that these diseases have a similar pathophysiology, which is highlighted by the fact that mutations in either the COMP or the type IX collagen genes produce multiple epiphyseal dysplasia, suggesting that their gene products interact. To investigate the interactions between COMP and type IX collagen, we have used rotary shadowing electron microscopy and real time biomolecular (BIAcore) analysis. Analysis of COMP-type IX collagen complexes demonstrated that COMP interacts with type IX collagen through the noncollagenous domains of type IX collagen and the C-terminal domain of COMP. Furthermore, peptide mapping identified a putative collagen-binding site that is associated with known human mutations. These data provide evidence that disruptions to COMP-type IX collagen interactions define a pathogenetic mechanism in a bone dysplasia family. pseudoachondroplasia cartilage oligomeric matrix protein multiple epiphyseal dysplasia extracellular matrix rough endoplasmic reticulum high molecular weight low molecular weight collagenous noncollagenous response units C-terminal domain of cartilage oligomeric matrix protein polyacrylamide gel electrophoresis bovine serum albumin Tris-buffered saline The skeletal dysplasias are a diverse group of genetic diseases affecting primarily the development of the osseous skeleton, and range in severity from relatively mild to severe and lethal forms (1Rimoin D.L. Lachman R.S. Rimoin D.L. Connor J.M. Pyeritz R.E. Emery and Rimoin's Principles and Practice of Medical Genetics. 3rd Ed. 2. Churchill Livingstone, Edinburgh1997: 2779-2816Google Scholar). There are over 200 unique well characterized phenotypes (2International Working Group on Constitutional Diseases of Bone Am. J. Med. Genet. 1998; 79: 82-376Crossref PubMed Google Scholar), and many of these conditions have been grouped into “bone dysplasia families” on the basis of similar clinical and radiographic presentation with the supposition that they will share a common disease pathophysiology (3Spranger J. Pathol. Immunopathol. Res. 1988; 7: 76-80Crossref PubMed Scopus (55) Google Scholar). While there has been great progress in identifying many of the genes involved in these diseases (4Mundlos S. Olsen B.R. FASEB J. 1997; 11: 227-233Crossref PubMed Scopus (125) Google Scholar, 5Dreyer S.D. Zhou G. Lee B. Clin. Genet. 1998; 54: 464-473Crossref PubMed Scopus (24) Google Scholar), we still have a very limited understanding of the precise cell matrix pathology of individual phenotypes and the relationship between pathogenetic mechanisms within specific bone dysplasia families.Pseudoachondroplasia (PSACH)1and multiple epiphyseal dysplasia (MED) comprise a bone dysplasia family; they are clinically similar diseases characterized by varying degrees of short-limbed dwarfism, joint laxity, and early onset degenerative joint disease (1Rimoin D.L. Lachman R.S. Rimoin D.L. Connor J.M. Pyeritz R.E. Emery and Rimoin's Principles and Practice of Medical Genetics. 3rd Ed. 2. Churchill Livingstone, Edinburgh1997: 2779-2816Google Scholar). Mild and severe forms of PSACH can be recognized (6Maroteaux P. Stanescu R. Stanescu V. Fontaine G. Eur. J. Pediatr. 1980; 133: 227-231Crossref PubMed Scopus (18) Google Scholar, 7Rimoin D.L. Rasmussen I.M. Briggs M.D. Roughly P.J. Gruber H.E. Warman M.L. Olsen B.R. Hsia Y.E. Yuen J. Reinker K. Garber A.P. Grover J. Lachman R.S. Cohn D.H. Hum. Genet. 1994; 93: 42-236Crossref PubMed Scopus (37) Google Scholar), and MED presents with considerable clinical variability where traditionally the mild Ribbing and severe Fairbank forms have been used to define the phenotypic spectrum (8Beighton P. McKusick V.A. McKusick's Heritable Disorders of Connective Tissue. 5th Ed. Mosby, St. Louis1993: 557-689Google Scholar).PSACH results almost exclusively from mutations in the gene encoding cartilage oligomeric matrix protein (COMP) (9Briggs M.D. Hoffman S.M.G. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Cekleniak J.A. Knowlton R.G. Cohn D.H. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (423) Google Scholar, 10Briggs M.D. Mortier G.R. Cole W.G. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 11Hecht J.T. Nelson L.D. Crowder E. Wang Y. Elder F.F. Harrison W.R. Francomano C.A. Prange C.K. Lennon G.G. Deere M. Lawler J. Nat. Genet. 1995; 10: 325-329Crossref PubMed Scopus (312) Google Scholar). COMP is a pentameric glycoprotein found in the extracellular matrix (ECM) of cartilage (12Hedbom E. Antonsson P. Hjerpe A. Aeschlimann D. Paulsson M. Rosa-Pimentel E. Sommarin Y. Wendel M. Oldberg A. Heinegard D. J. Biol. Chem. 1992; 267: 6132-6136Abstract Full Text PDF PubMed Google Scholar), tendon (13DiCesare P. Hauser N. Lehman D. Pasumarti S. Paulsson M. FEBS Lett. 1994; 354: 237-240Crossref PubMed Scopus (216) Google Scholar), and ligament, where it is thought to play a major role in tissue development and homeostasis through interactions with cells (14DiCesare P.E. Morgelin M. Mann K. Paulsson M. Eur. J. Biochem. 1994; 223: 927-937Crossref PubMed Scopus (123) Google Scholar) and other ECM components such as type I and type II collagen (15Rosenberg K. Olsson H. Morgelin M. Heinegard D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). It is a member of the thrombospondin gene family (16Oldberg A. Antonsson P. Lindblom K. Heinegard D. J. Biol. Chem. 1992; 267: 22346-22350Abstract Full Text PDF PubMed Google Scholar,17Newton G. Weremowicz S. Morton C.C. Copeland N.G. Gilbert D.J. Jenkins N.A. Lawler J. Genomics. 1994; 24: 435-439Crossref PubMed Scopus (136) Google Scholar) and is a modular protein comprising an amino-terminal domain, calcium binding domains (type II and type III repeats), and a large carboxyl domain situated at the distal termini of the pentamer. The majority of the mutations identified in the COMP gene are located within exons encoding the calcium binding type III repeats and are postulated to produce qualitative defects to the protein and a reduction in Ca2+ binding (18Maddox B.K. Mokashi A. Keene D.R. Bachinger H.P. J. Biol. Chem. 2000; 275: 11412-11417Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This results in the retention of abnormal COMP pentamers within the rough endoplasmic reticulum (RER) by an undetermined “protein quality control mechanism” (19Maddox B.K. Keene D.R. Sakai L.Y. Charbonneau N.L. Morris N.P. Ridgway C.C. Boswell B.A. Sussman M.D. Horton W.A. Bachinger H.P. Hecht J.T. J. Biol. Chem. 1997; 272: 30993-30997Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 20Delot E. Brodie S.G. King L.M. Wilcox W.R. Cohn D.H. J. Biol. Chem. 1998; 273: 26692-26697Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Hecht J.T. Montufar-Solis D. Decker G. Lawler J. Daniels K. Duke P.J. Matrix Biol. 1998; 17: 625-633Crossref PubMed Scopus (80) Google Scholar, 22Hecht J.T. Deere M. Putnam E. Cole W. Vertel B. Chen H. Lawler J. Matrix Biol. 1998; 17: 269-278Crossref PubMed Scopus (94) Google Scholar). Interestingly, type IX collagen has been found to colocalize with abnormal COMP in RER vesicles, but the specificity of these intracellular interactions is unknown. Recently, we were the first to identify mutations in one of the exons encoding the carboxyl terminus, thus confirming an important role for this domain in the structure and/or function of COMP (10Briggs M.D. Mortier G.R. Cole W.G. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Electron microscopy of labrum ligament from a PSACH patient with a COMP mutation shows severe disruption to collagen fibril orientation, variable fibril diameters, and numerous fused fibrils, confirming an important role for COMP in collagen fibrillogenesis. 2P. Holden and M. D. Briggs, manuscript in preparation. 2P. Holden and M. D. Briggs, manuscript in preparation.Some forms of MED are allelic with PSACH and also result from qualitative defects in COMP (9Briggs M.D. Hoffman S.M.G. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Cekleniak J.A. Knowlton R.G. Cohn D.H. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (423) Google Scholar, 10Briggs M.D. Mortier G.R. Cole W.G. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar); however, as a reflection of its clinical variability, MED is genetically heterogeneous and can result from mutations in the genes encoding type IX collagen (COL9A2 and COL9A3) (23Bonnemann C.G. Cox G.F. Shapiro F. Wu J.J. Feener C.A. Thompson T.G. Anthony D.C. Eyre D.R. Darras B.T. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1212-1217Crossref PubMed Scopus (89) Google Scholar, 24Holden P. Canty E.G. Mortier G.R. Zabel B. Spranger J. Carr A. Grant M.E. Loughlin J.A. Briggs M.D. Am. J. Hum. Genet. 1999; 65: 31-38Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Muragaki Y. Mariman E.C. van Beersum S.E. Perala M. van Mourik J.B. Warman M.L. Olsen B.R. Hamel B.C. Nat. Genet. 1996; 12: 103-105Crossref PubMed Scopus (173) Google Scholar, 26Paassilta P. Lohiniva J. Annunen S. Bonaventure J. Le Merrer M. Pai L. Ala-Kokko L. Am. J. Hum. Genet. 1999; 64: 1036-1044Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Type IX collagen is closely associated with type II collagen fibrils, where it binds in an anti-parallel orientation to type II collagen molecules (27Olsen B.R. Int. J. Biochem. Cell Biol. 1997; 29: 555-558Crossref PubMed Scopus (83) Google Scholar). Type IX collagen is a member of the FACIT (fibril-associated collagen withinterrupted triple helices) group of collagens and is a heterotrimer α1(IX)α2(IX)α3(IX) of polypeptides derived from three distinct genes (COL9A1,COL9A2, and COL9A3). Type IX collagen comprises three collagenous (COL) domains separated by four noncollagenous (NC) domains and has long been thought to act as a molecular bridge between collagen fibrils and other cartilage matrix components (28Smith Jr., G.N. Brandt K.D. J. Rheumatol. 1992; 19: 14-17PubMed Google Scholar). The COL3 and NC4 domains project out from the fibril surface, providing ideal sites for these interactions. All of the mutations identified in theCOL9A2 (24Holden P. Canty E.G. Mortier G.R. Zabel B. Spranger J. Carr A. Grant M.E. Loughlin J.A. Briggs M.D. Am. J. Hum. Genet. 1999; 65: 31-38Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Muragaki Y. Mariman E.C. van Beersum S.E. Perala M. van Mourik J.B. Warman M.L. Olsen B.R. Hamel B.C. Nat. Genet. 1996; 12: 103-105Crossref PubMed Scopus (173) Google Scholar) and COL9A3 (23Bonnemann C.G. Cox G.F. Shapiro F. Wu J.J. Feener C.A. Thompson T.G. Anthony D.C. Eyre D.R. Darras B.T. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1212-1217Crossref PubMed Scopus (89) Google Scholar, 24Holden P. Canty E.G. Mortier G.R. Zabel B. Spranger J. Carr A. Grant M.E. Loughlin J.A. Briggs M.D. Am. J. Hum. Genet. 1999; 65: 31-38Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Muragaki Y. Mariman E.C. van Beersum S.E. Perala M. van Mourik J.B. Warman M.L. Olsen B.R. Hamel B.C. Nat. Genet. 1996; 12: 103-105Crossref PubMed Scopus (173) Google Scholar, 26Paassilta P. Lohiniva J. Annunen S. Bonaventure J. Le Merrer M. Pai L. Ala-Kokko L. Am. J. Hum. Genet. 1999; 64: 1036-1044Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) genes are in the splice donor or acceptor sites of exon 3. These result in the skipping of exon 3, leading to an in-frame deletion of 12 amino acid residues from equivalent regions of the COL3 domain of the α2(IX) and α3(IX) chains. The restricted localization of these mutations suggests a possible role for this region of the COL3 domain of type IX collagen in the proposed interactions with other components of the cartilage ECM.The observations that mutations in COMP (9Briggs M.D. Hoffman S.M.G. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Cekleniak J.A. Knowlton R.G. Cohn D.H. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (423) Google Scholar, 10Briggs M.D. Mortier G.R. Cole W.G. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and type IX collagen genes (23Bonnemann C.G. Cox G.F. Shapiro F. Wu J.J. Feener C.A. Thompson T.G. Anthony D.C. Eyre D.R. Darras B.T. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1212-1217Crossref PubMed Scopus (89) Google Scholar, 24Holden P. Canty E.G. Mortier G.R. Zabel B. Spranger J. Carr A. Grant M.E. Loughlin J.A. Briggs M.D. Am. J. Hum. Genet. 1999; 65: 31-38Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Muragaki Y. Mariman E.C. van Beersum S.E. Perala M. van Mourik J.B. Warman M.L. Olsen B.R. Hamel B.C. Nat. Genet. 1996; 12: 103-105Crossref PubMed Scopus (173) Google Scholar, 26Paassilta P. Lohiniva J. Annunen S. Bonaventure J. Le Merrer M. Pai L. Ala-Kokko L. Am. J. Hum. Genet. 1999; 64: 1036-1044Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) result in phenotypes within the MED disease spectrum, that COMP interacts with triple helical type I and type II collagen (15Rosenberg K. Olsson H. Morgelin M. Heinegard D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and that abnormal collagen fibril morphology is associated with COMP gene mutations provided a rationale for investigating potential interactions between COMP and type IX collagen.In this paper, we show that COMP can interact with type IX collagen. These interactions are mediated through the C-terminal domain of COMP and the noncollagenous domains of type IX collagen. A putative collagen-binding domain in COMP is located between residues 579 and 595, and mutations in this region are likely to disrupt these interactions. Overall, these data provide evidence that disruptions to molecular interactions between two key components of the cartilage extracellular matrix might produce distinct clinical phenotypes that share a common disease pathophysiology and belong to the same bone dysplasia family.DISCUSSIONCOMP and type IX collagen are important structural components of the cartilage ECM with fundamental roles in collagen fibrillogenesis, tissue development, and homeostasis. We have used rotary shadowing electron microscopy and BIAcore analysis to show that COMP can interact with native type IX collagen. These interactions are mediated through the C-terminal domain of COMP and the noncollagenous domains of type IX collagen (NC1–4). Using BIAcore, we demonstrated qualitatively that COMP can interact with native type IX collagen and to a certain extent the pepsin-derived HMW fragment but not the LMW fragment. These data collectively suggest that each of the noncollagenous domains of type IX collagen are involved in interactions with COMP. The use of recombinant Ct-COMP in BIAcore studies confirmed the rotary shadowing EM findings that the C-terminal domain of COMP mediates interaction with type IX collagen. Furthermore, the use of peptide inhibition assays aided in the identification of a putative collagen-binding site between residues 579 and 595 of COMP. This region of COMP has previously been shown to contain mutations resulting in skeletal dysplasia, providing a direct link between these fundamental interactions and human disease.Overall, these findings support recent data indicating that the C-terminal domain of COMP can bind to collagen I/II and procollagen I/II molecules in the presence of divalent cations (15Rosenberg K. Olsson H. Morgelin M. Heinegard D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Using a solid-phase binding assay, Rosenberg and colleagues determined that interactions between COMP and collagen I/II displayed a preference for Zn2+, with binding saturated at 0.5 mm. They subsequently characterized these interactions further using BIAcore and rotary shadowing transmission electron microscopy with 1 mmZn2+ (15Rosenberg K. Olsson H. Morgelin M. Heinegard D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). We performed similar experiments to study interactions between COMP and type IX collagen, which appear also to be mediated by the C-terminal domain of COMP in the presence of 1 mm Zn2+. Whereas Rosenberg and co-workers demonstrated that COMP bound to the collagenous regions of types I and II collagen at four sites located at 0 (C-terminal), 126, 206, and 300 nm (N-terminal) (15Rosenberg K. Olsson H. Morgelin M. Heinegard D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), we have shown that COMP appears to bind exclusively to the noncollagenous domains of type IX collagen. We used BIAcore analysis to confirm that type II collagen interacted with COMP in our system and then used peptide inhibition assays to show that this binding could also be specifically disrupted with peptide 579–595 (but not 713–725). These data suggest that type I, II, and IX collagen interactions are mediated through the same (or closely located) region of the C-terminal domain of COMP.The majority of mutations in the COMP gene are within exons encoding the calcium binding (type III repeat) domain (9Briggs M.D. Hoffman S.M.G. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Cekleniak J.A. Knowlton R.G. Cohn D.H. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (423) Google Scholar, 10Briggs M.D. Mortier G.R. Cole W.G. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 11Hecht J.T. Nelson L.D. Crowder E. Wang Y. Elder F.F. Harrison W.R. Francomano C.A. Prange C.K. Lennon G.G. Deere M. Lawler J. Nat. Genet. 1995; 10: 325-329Crossref PubMed Scopus (312) Google Scholar) and are predicted to result in qualitative defects to COMP (18Maddox B.K. Mokashi A. Keene D.R. Bachinger H.P. J. Biol. Chem. 2000; 275: 11412-11417Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) leading to the retention of misfolded protein in the RER, a matrix deficient in COMP, and ultimately cell death (21Hecht J.T. Montufar-Solis D. Decker G. Lawler J. Daniels K. Duke P.J. Matrix Biol. 1998; 17: 625-633Crossref PubMed Scopus (80) Google Scholar). Interestingly, analysis of chondrocytes from PSACH cartilage shows the accumulation of type IX collagen along with COMP in the RER, suggesting that interactions, possibly specific, occur between these molecules prior to secretion (19Maddox B.K. Keene D.R. Sakai L.Y. Charbonneau N.L. Morris N.P. Ridgway C.C. Boswell B.A. Sussman M.D. Horton W.A. Bachinger H.P. Hecht J.T. J. Biol. Chem. 1997; 272: 30993-30997Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). During pentamerization, by random association, 97% of all pentamers will contain at least one abnormal monomer. The relative effect of different numbers of abnormal monomers on COMP pentamer secretion has yet to be determined, but immunohistochemical analysis of cartilage has shown that there is a significant reduction in the level of extracellular COMP that would be available for interactions with collagen (19Maddox B.K. Keene D.R. Sakai L.Y. Charbonneau N.L. Morris N.P. Ridgway C.C. Boswell B.A. Sussman M.D. Horton W.A. Bachinger H.P. Hecht J.T. J. Biol. Chem. 1997; 272: 30993-30997Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Interestingly, electron microscopy of labrum ligament from a PSACH patient with a mutation in the type III domain of COMP (G465S) shows a generalized disruption to tissue organization and abnormal collagen fibril morphology. Longitudinal sections show severe disruption to the orientation of collagen fibrils and differences in individual fibril thickness, whereas transverse sections show variable fibril diameter, irregular fibril surface, and numerous fused fibrils.2Overall, these data confirm a role for COMP in collagen fibrillogenesis and matrix assembly.We hypothesize that disruptions to COMP-type IX collagen interactions are a secondary component of the pathophysiology of the “PSACH-MED bone dysplasia family.” Disruption to these interactions can occur by one of two mechanisms; either mutations occur within the binding sites of these molecules or there is a reduction in the amount of one (or both) of these molecules in the ECM of cartilage. We have shown that a collagen binding site is located between residues 579 and 595, a region of COMP previously shown to contain mutations that cause either PSACH or MED. Four mutations have been identified within five residues, E583K, T585R, T585M, H587R, (10Briggs M.D. Mortier G.R. Cole W.G. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 36Deere M. Sanford T. Francomano C.A. Daniels K. Hecht J.T. Am. J. Med. Genet. 1999; 85: 486-490Crossref PubMed Scopus (52) Google Scholar, 37Deere M. Sanford T. Ferguson H.L. Daniels K. Hecht J.T. Am. J. Med. Genet. 1998; 80: 510-513Crossref PubMed Scopus (59) Google Scholar), suggesting that the motif EGTFH plays an important role in COMP-collagen interactions. We suggest that mutations in exons encoding the C-terminal domain of COMP are likely to have a less deleterious effect on the structure and folding of abnormal COMP, therefore not preventing its secretion into the extracellular matrix. In this case, interactions with type IX collagen are likely to be disrupted by mutations in the collagen-binding site of COMP.The cell matrix pathology of MED, resulting from eitherCOL9A2 or COL9A3 mutations, is unresolved. Previously, analysis of cartilage ultrastructure has suggested that there was no retention of abnormal type IX collagen within the RER of chondrocytes from some affected patients (38van Mourik J.B. Buma P. Wilcox W.R. Ultrastruct. Pathol. 1998; 22: 249-251Crossref PubMed Scopus (7) Google Scholar). However, recent data have shown that inclusion bodies can be present with a lamellar structure similar to that seen in chondrocytes from patients with COMP gene mutations (23Bonnemann C.G. Cox G.F. Shapiro F. Wu J.J. Feener C.A. Thompson T.G. Anthony D.C. Eyre D.R. Darras B.T. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1212-1217Crossref PubMed Scopus (89) Google Scholar). Finally, we have reported that a specific mutation in COL9A2 results in the degradation ofCOL9A2 mRNA from the mutant allele, and undoubtedly this would result in an overall reduction in type IX collagen within the ECM (24Holden P. Canty E.G. Mortier G.R. Zabel B. Spranger J. Carr A. Grant M.E. Loughlin J.A. Briggs M.D. Am. J. Hum. Genet. 1999; 65: 31-38Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Collectively, these data suggest that although several different mechanisms contribute to the pathophysiology of MED, all of them are likely to result in an ECM deficient in type IX collagen, thus disrupting important interactions between COMP and type IX collagen.In conclusion, we have shown that COMP interacts with type IX collagen and have identified the domains in each of these proteins that mediate these interactions. High resolution structural studies will be needed to map the binding sites with precision. Disruption to these interactions is likely to define a pathogenetic mechanism in a human bone dysplasia family, and this finding has major implications in understanding the cell matrix pathology of human skeletal dysplasias. The skeletal dysplasias are a diverse group of genetic diseases affecting primarily the development of the osseous skeleton, and range in severity from relatively mild to severe and lethal forms (1Rimoin D.L. Lachman R.S. Rimoin D.L. Connor J.M. Pyeritz R.E. Emery and Rimoin's Principles and Practice of Medical Genetics. 3rd Ed. 2. Churchill Livingstone, Edinburgh1997: 2779-2816Google Scholar). There are over 200 unique well characterized phenotypes (2International Working Group on Constitutional Diseases of Bone Am. J. Med. Genet. 1998; 79: 82-376Crossref PubMed Google Scholar), and many of these conditions have been grouped into “bone dysplasia families” on the basis of similar clinical and radiographic presentation with the supposition that they will share a common disease pathophysiology (3Spranger J. Pathol. Immunopathol. Res. 1988; 7: 76-80Crossref PubMed Scopus (55) Google Scholar). While there has been great progress in identifying many of the genes involved in these diseases (4Mundlos S. Olsen B.R. FASEB J. 1997; 11: 227-233Crossref PubMed Scopus (125) Google Scholar, 5Dreyer S.D. Zhou G. Lee B. Clin. Genet. 1998; 54: 464-473Crossref PubMed Scopus (24) Google Scholar), we still have a very limited understanding of the precise cell matrix pathology of individual phenotypes and the relationship between pathogenetic mechanisms within specific bone dysplasia families. Pseudoachondroplasia (PSACH)1and multiple epiphyseal dysplasia (MED) comprise a bone dysplasia family; they are clinically similar diseases characterized by varying degrees of short-limbed dwarfism, joint laxity, and early onset degenerative joint disease (1Rimoin D.L. Lachman R.S. Rimoin D.L. Connor J.M. Pyeritz R.E. Emery and Rimoin's Principles and Practice of Medical Genetics. 3rd Ed. 2. Churchill Livingstone, Edinburgh1997: 2779-2816Google Scholar). Mild and severe forms of PSACH can be recognized (6Maroteaux P. Stanescu R. Stanescu V. Fontaine G. Eur. J. Pediatr. 1980; 133: 227-231Crossref PubMed Scopus (18) Google Scholar, 7Rimoin D.L. Rasmussen I.M. Briggs M.D. Roughly P.J. Gruber H.E. Warman M.L. Olsen B.R. Hsia Y.E. Yuen J. Reinker K. Garber A.P. Grover J. Lachman R.S. Cohn D.H. Hum. 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