Contributions of the ionization states of acidic residues to the stability of the coiled coil domain of matrilin-1. (1/320)

The pKa values of eight glutamic acid residues in the homotrimeric coiled coil domain of chicken matrilin-1 have been determined from 2D H(CA)CO NMR spectra recorded as a function of the solution pH. The pKa values span a range between 4.0 and 4.7, close to or above those for glutamic acid residues in unstructured polypeptides. These results suggest only small favorable contributions to the stability of the coiled coil from the ionization of its acidic residues.  (+info)

Enhancement of cell adhesion and spreading by a cartilage-specific noncollagenous protein, cartilage matrix protein (CMP/Matrilin-1), via integrin alpha1beta1. (2/320)

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.  (+info)

Matrilin-2, a large, oligomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks. (3/320)

Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.  (+info)

Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts. (4/320)

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.  (+info)

Assembly of a novel cartilage matrix protein filamentous network: molecular basis of differential requirement of von Willebrand factor A domains. (5/320)

Cartilage matrix protein (CMP) is the prototype of the newly discovered matrilin family, all of which contain von Willebrand factor A domains. Although the function of matrilins remain unclear, we have shown that, in primary chondrocyte cultures, CMP (matrilin-1) forms a filamentous network, which is made up of two types of filaments, a collagen-dependent one and a collagen-independent one. In this study, we demonstrate that the collagen-independent CMP filaments are enriched in pericellular compartments, extending directly from chondrocyte membranes. Their morphology can be distinguished from that of collagen filaments by immunogold electron microscopy, and mimicked by that of self-assembled purified CMP. The assembly of CMP filaments can occur from transfection of a wild-type CMP transgene alone in skin fibroblasts, which do not produce endogenous CMP. Conversely, assembly of endogenous CMP filaments by chondrocytes can be inhibited specifically by dominant negative CMP transgenes. The two A domains within CMP serve essential but different functions during network formation. Deletion of the A2 domain converts the trimeric CMP into a mixture of monomers, dimers, and trimers, whereas deletion of the A1 domain does not affect the trimeric configuration. This suggests that the A2 domain modulates multimerization of CMP. Absence of either A domain from CMP abolishes its ability to form collagen-independent filaments. In particular, Asp22 in A1 and Asp255 in A2 are essential; double point mutation of these residues disrupts CMP network formation. These residues are part of the metal ion-dependent adhesion sites, thus a metal ion-dependent adhesion site-mediated adhesion mechanism may be applicable to matrilin assembly. Taken together, our data suggest that CMP is a bridging molecule that connects matrix components in cartilage to form an integrated matrix network.  (+info)

The noncollagenous domain 1 of type X collagen. A novel motif for trimer and higher order multimer formation without a triple helix. (6/320)

In this study, we test the hypothesis that the carboxyl noncollagenous (NC1) domain of collagen X is sufficient to direct multimer formation without a triple helix. Two peptides containing the NC1 domain of avian collagen X have been synthesized using a bacterial expression system and their properties characterized. One peptide consists only of the NC1 domain, and the other is a chimeric molecule with a noncollagenous A domain of matrilin-1 fused to the N terminus of NC1. The NC1 peptide alone forms a 45-kDa trimer under native conditions, suggesting that NC1 contains all the information for trimerization without any triple helical residues. This trimeric association is highly thermostable without intermolecular disulfide bonds. This indicates that the NC1 domain contributes to the remarkable structural stability of collagen X. Chemical cross-linking of the NC1 trimer results in a series of varying sized multimers, the smallest of which is a trimer. Therefore the NC1 trimer is sufficient to form higher order multimers. The chimeric A-NC1 peptide forms a homotrimer by itself, and a series of heterotrimers with the NC1 peptide via the NC1 domain. Thus the NC1(X) domain directs multimer formation, even in a noncollagenous molecule.  (+info)

A new animal model for relapsing polychondritis, induced by cartilage matrix protein (matrilin-1). (7/320)

Relapsing polychondritis (RP) differs from rheumatoid arthritis (RA) in that primarily cartilage outside diarthrodial joints is affected. The disease usually involves trachea, nose, and outer ears. To investigate whether the tissue distribution of RP may be explained by a specific immune response, we immunized rats with cartilage matrix protein (matrilin-1), a protein predominantly expressed in tracheal cartilage. After 2-3 weeks, some rats developed a severe inspiratory stridor. They had swollen noses and/or epistaxis, but showed neither joint nor outer ear affection. The inflammatory lesions involved chronic active erosions of cartilage. Female rats were more susceptible than males. The disease susceptibility was controlled by both MHC genes (f, l, d, and a haplotypes are high responders, and u, n, and c are resistant) and non-MHC genes (the LEW strain is susceptible; the DA strain is resistant). However, all strains mounted a pronounced IgG response to cartilage matrix protein. The initiation and effector phase of the laryngotracheal involvement causing the clinical symptoms were shown to depend on alphabeta T cells. Taken together, these results represent a novel model for RP: matrilin-1-induced RP. Our findings also suggest that different cartilage proteins are involved in pathogenic models of RP and RA.  (+info)

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage? (8/320)

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.  (+info)