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PubMed2026

Activation of pro-survival autophagy by a small molecule promoting p62 oligomerization.

Autophagy is a critical mechanism of cellular quality control, orchestrated by selective autophagy receptor (SAR) proteins. Pharmacologically enhancing the cargo-targeting capacity of SARs presents an attractive but underexplored strategy for the precise therapeutic activation of autophagy. Here, we characterize SQ-1, a small-molecule activator of autophagy that engages the prototypical SAR protein p62/sequestosome-1 (SQSTM1). We show that SQ-1 sensitizes p62 to oxidation and promotes its disulfide-mediated oligomerization in response to mitochondrial reactive oxygen species (ROS). This ROS-dependent activation of p62-mediated selective autophagy enhances the clearance of ROS-generating mitochondria and restores cell viability in models of Niemann-Pick type C1 disease, which is marked by impaired autophagic flux. In summary, the unique mode of action of SQ-1 enables self-regulated autophagy activation, offering a potential therapeutic strategy for lysosomal storage disorders and a broader spectrum of age-related diseases characterized by defective autophagy.

Niemann-Pick type C1 disease↗
PubMed2005

Serum concentration of cartilage oligomeric matrix protein (COMP) is sensitive to physiological cyclic loading in healthy adults.

OBJECTIVE: To test the hypothesis that physiological cyclic loading during a 30-min walking exercise causes an increase in serum cartilage oligomeric matrix protein (COMP) concentration in a healthy population. METHODS: Blood samples (5 ml) were drawn from 10 physically active adults immediately before and after, and 0.5h, 1.5h, 3.5h and 5.5h after a 30-min walking exercise on a level outdoor walking track at self-selected normal speed. On a separate day, blood samples were drawn from the same 10 subjects during 6h while they were resting in a chair. Serum COMP concentrations were determined using a commercial enzyme-linked immunosorbent assay (COMP ELISA). An activity monitor was used to record basic time-distance measurements of gait. Serum COMP concentrations within the exercise protocol and within the resting protocol were compared using separate repeated measures analyses of variance (alpha=0.05). RESULTS: In the exercise protocol, a first increase (9.7%; P=0.003) occurred immediately after the walking exercise. A second increase in serum COMP concentration (7.0%; P=0.024) occurred 5.5h after the walking exercise. In the resting protocol, the concentration at baseline was significantly higher than at all subsequent time points (8.2%; P<0.050). Serum COMP concentration decreased from the 3.5-h to the 5.5-h sample (-4.8%; P=0.012). CONCLUSIONS: Even a moderate walking activity can significantly influence serum COMP concentration. The immediate response points to a diffusion time of COMP fragments from cartilage to the blood of 30 min or less. The response at 5.5h indicates a metabolic delay for COMP in the range of 5h to 6h.

Adult↗
PubMed2005

In vivo human Cartilage oligomeric matrix protein (COMP) promoter activity.

Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein whose function is unknown. Mutations in COMP cause pseudoachondroplasia and multiple epiphyseal dysplasia, two skeletal dysplasias which are associated with intracellular retention of COMP in chondrocytes. In contrast, COMP null mice are normal suggesting gene redundancy or that the detrimental effect is associated with mutant COMP rather than the absence of functional COMP. To define the elements that regulate COMP transcription and tissue-specificity, we have evaluated the human COMP promoter driving fusion gene expression in vitro and in vivo. COMP promoter activity is higher in rat chondrosarcoma cells (RCS) than in a fibroblast cell line. In RCS cells, expression of a reporter gene containing 1.7 kb of the human COMP promoter was three-fold higher than all shorter COMP promoter constructs. In transgenic mice, 1.7 kb of the human COMP promoter is active early in development in the limbs, spine, and eye. As development progresses, promoter activity diminishes in the eye and migrates from the center to the ends of the long bones. On the other hand, while 375 bp of the human COMP promoter is sufficient for proper tissue-specific expression, levels are less than those found with the 1.7-COMP promoter. The expression pattern of both promoters recapitulates endogenous cartilage COMP expression in mice. Our findings indicate that the elements required for chondrocyte-specific expression lie within 375 bp of the translational start site, while DNA enhancer elements are located between 1.0 to 1.7 kb.

Animals↗
PubMed2006

Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts.

Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.

Animals↗
PubMed2003

Mutation (D472Y) in the type 3 repeat domain of cartilage oligomeric matrix protein affects its early vesicle trafficking in endoplasmic reticulum and induces apoptosis.

Cartilage oligomeric matrix protein (COMP) is a large pentameric extracellular glycoprotein found in cartilage, tendon, and synovium, and plays structural roles in cartilage as the fifth member of the thrombospondin family. Familial mutations in type 3 repeats of COMP are known to cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Although such mutations induce enlarged rough endoplasmic reticulum (rER) as a morphological change, the metabolic trafficking of mutated COMP remains unclear. In transfected COS7 cells, wild-type COMP was rapidly secreted into culture medium, while the great majority of COMP with the type 3 repeats mutation (D472Y) remained in the cells and a small portion of mutated COMP was secreted. This finding was followed up with a confocal study with an antibody specific to COMP, which demonstrated mutated COMP tightly associated with abnormally enlarged rER. Phosphorylated eIF2alpha, an ER stress protein, was expressed as a pathological reaction in virtually all COS7 cells expressing mutated but not wild-type COMP. Moreover, COS7 cells expressing mutated COMP exhibited significantly more apoptotic reaction than those expressing wild-type COMP. Pathological accumulation of COMP in rER and apoptosis in COS7 cells that were induced by the mutation (D472Y) in COMP imply that COMP mutations play a role in the pathogenesis of PSACH.

Achondroplasia↗
PubMed2003

Monoclonal antibodies to human cartilage oligomeric matrix protein: epitope mapping and characterization of sandwich ELISA.

BACKGROUND: Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids. METHODS: COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody. RESULTS: Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera. CONCLUSIONS: We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.

Antibodies, Monoclonal↗
PubMed2000

Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP).

Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.

Aggrecans↗
PubMed1997

Expression of cartilage oligomeric matrix protein by human synovium.

Human synovium was analyzed for the possible expression of cartilage oligomeric matrix protein (COMP). Immunostaining with polyclonal antiserum to COMP demonstrated positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. Western blot analysis using either polyclonal or monoclonal antibodies to human COMP confirmed the presence of COMP by immunoreactive bands with the same molecular mass (approximately 110 kDa) as purified articular cartilage COMP. PCR using oligonucleotides that span human COMP exons 7-13 revealed identical amplification products from cDNA prepared from either human chondrocytes or synovium. Northern blot analysis using a biotinylated-probe to human COMP, spanning exons 12-13, also reveal an identical hybridization product to either human chondrocyte or synovium total RNA. Human synovium should be considered as a potential tissue source of COMP in any investigation of biological markers of cartilage metabolism.

Base Sequence↗
PubMed2000

Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors.

TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain of cartilage oligomeric matrix protein (COMP). The affinity of Fas and CD40 (but not TNFR-1 and TRAIL-R2) to their ligands is increased by fusion to COMP, when compared to the respective Fc chimeras. In functional assays, Fas:COMP was at least 20-fold more active than Fas:Fc at inhibiting the action of sFasL, and CD40:COMP could block CD40L-mediated proliferation of B cells, whereas CD40:Fc could not. In conclusion, members of the TNF receptor family can display high specificity and excellent avidity for their ligands if they are adequately multimerized.

Animals↗
PubMed2001

Tissue distribution and measurement of cartilage oligomeric matrix protein in patients with magnetic resonance imaging-detected bone bruises after acute anterior cruciate ligament tears.

Histologic and immunostaining analyses were performed on articular cartilage/subchondral bone biopsy specimens overlying MRI-detected bone bruises in 12 patients with anterior cruciate ligament (ACL) tears. Staining with toluidine blue for proteoglycan revealed loss of staining from the superficial portion of the articular cartilage. Immunostaining for cartilage oligomeric matrix protein (COMP) showed an increased staining in the superficial matrix of the articular cartilage. Using polyclonal antisera against COMP, the authors performed a competitive enzyme-linked immunosorbent assay (ELISA) on the synovial fluid from the injured and uninjured knees. There was an approximately 10-fold higher synovial fluid COMP levels in injured knees. The COMP levels were greater in those patients who had synovial fluid samples harvested closer to the date of initial injury. Western blot analysis of the synovial fluid showed an increased presence of COMP degradation fragments from injured knees. These results are indicative of a significant injury to the articular cartilage, and may represent preclinical posttraumatic osteoarthritic lesions.

Adolescent↗
PubMed2001

Analysis of the promoter region of human cartilage oligomeric matrix protein (COMP).

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein expressed in cartilage, ligament, and tendon. The importance of COMP in the matrix of these cells is underscored by the discovery that mutations in COMP cause the skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Here, we present the first report on the analysis of the human COMP promoter region in cartilage, ligament, and tendon cells. A 1.7-kb region of the COMP promoter has been cloned and sequenced and no TATA or CAAT boxes were found. Primer extension identified multiple transcription start sites. All four transcription start sites were utilized in chondrocytes with only three of them utilized in tendon and ligament cells. Differential regulation was observed for different parts of this 1.7-kb region with the 370-bp proximal region conveying the strongest promoter activity. The highest activity was observed in tendon and ligament. Finally, we provide evidence that the DNA binding protein SP1 plays a role in the regulation of COMP expression. These results indicate that COMP expression within these cells is regulated in a unique manner that differs from the expression of other extracellular matrix genes.

Base Sequence↗
PubMed2002

Matrix-matrix interaction of cartilage oligomeric matrix protein and fibronectin.

Recent work indicates that cartilage oligomeric matrix protein (COMP) plays an important role in extracellular matrix assembly and matrix-matrix protein interactions. In order to identify the proteins in extracellular matrix that interact with COMP, we used an ELISA-based solid-phase binding assay, which revealed a specific, high-affinity interaction between COMP and fibronectin. This interaction is concentration-dependent and saturable, and appears to occur under physiologically relevant conditions. Electron microscopy after negative staining and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nm from the N-terminal domain of fibronectin, which can be inhibited by the presence of a polyclonal antibody specific for the C-terminal heptadecapeptide of COMP. This interaction is further demonstrated in vivo by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture, and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry.

Animals↗
PubMed2003

Cleavage of cartilage oligomeric matrix protein (thrombospondin-5) by matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs.

Cartilage oligomeric matrix protein (COMP) is a pentameric glycoprotein present in cartilage, tendon and ligament. Fragments of the molecule are present in the diseased cartilage, synovial fluid and serum of patients with knee injuries, osteoarthritis and rheumatoid arthritis. Although COMP is a substrate for several matrix metalloproteinases (MMPs), the enzymes responsible for COMP degradation in vivo have yet to be identified. In this study we utilised well-established bovine cartilage culture models to examine IL-1alpha-stimulated COMP proteolysis in the presence and absence of MMP inhibitors. COMP was released from bovine nasal cartilage, in response to IL-1alpha, at an intermediate time between proteoglycans and type II collagen, when soluble MMP levels in the culture medium were undetectable. The major fragment of COMP released following IL-1alpha-stimulation migrated with an apparent molecular mass of approximately 110 kDa (Fragment-110) and co-migrated with both the major fragment present in human arthritic synovial fluid samples and the product of COMP cleavage by purified MMP-9. However, the broad-spectrum MMP and ADAM inhibitor BB94 only partially inhibited the formation of Fragment-110 and failed to inhibit COMP release significantly. Therefore the results of these studies indicate a role for proteinases other than MMPs in the degradation of COMP in bovine cartilage. It was further demonstrated that purified COMP was cleaved by ADAMTS-4, but not ADAMTS-1 or -5, to yield a fragment which co-migrated with Fragment-110. Therefore this is the first demonstration of COMP as a substrate for ADAMTS-4, although it remains to be determined whether this enzyme plays a role in COMP degradation in vivo.

ADAM Proteins↗
PubMed1998

Characterization of cartilage oligomeric matrix protein (COMP) in human normal and pseudoachondroplasia musculoskeletal tissues.

Cartilage oligomeric matrix protein (COMP), the fifth member of the -thrombospondin gene family, is an extracellular matrix calcium-binding protein. The importance of COMP is underscored by the finding that mutations in COMP cause the human dwarfing condition, pseudoachondroplasia (PSACH). Here, we report the results of human tissue distribution and cell secretion studies of human COMP. COMP is expressed and secreted by cultured monolayer chondrocyte, tendon and ligament cells, and COMP secretion is not restricted to a differentiated chondrocyte phenotype. Whereas COMP is retained in the endoplasmic reticulum that accumulates within PSACH chondrocytes in vivo, COMP is not retained intracellularly in the dedifferentiated PSACH chondrocytes in cultures. These results lend further support to the hypothesis that retention of COMP is related to the terminal PSACH chondrocyte phenotype, processing of proteins related to extracellular matrix formation, and maintenance in cartilage.

Achondroplasia↗
PubMed1998

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.

Achondroplasia↗
PubMed1995

Mutations in exon 17B of cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is a well characterized dwarfing condition mapping to chromosome 19p12-13.1. Cartilage oligomeric matrix protein (COMP), a cartilage specific protein, maps to the same location within a contig that spans the PSACH locus. Using single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing we have identified COMP mutations in eight familial and isolated PSACH cases. All mutations involve either a single base-pair change or a three base-pair deletion in exon 17B. Six mutations delete or change a well conserved aspartic acid residue within the calcium-binding type 3 repeats. These results demonstrate that mutations in the COMP gene cause pseudochondroplasia.

Achondroplasia↗
PubMed1995

Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage oligomeric matrix protein gene.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are dominantly inherited chondrodysplasias characterized by short stature and early-onset osteoarthrosis. The disease genes in families with PSACH and MED have been localized to an 800 kilobase interval on the short arm of chromosome 19. Recently the gene for cartilage oligomeric matrix protein (COMP) was localized to chromosome 19p13.1. In three patients with these diseases, we identified COMP mutations in a region of the gene that encodes a Ca++ binding motif. Our data demonstrate that PSACH and some forms of MED are allelic and suggest an essential role for Ca++ binding in COMP structure and function.

Achondroplasia↗
PubMed2004

Characterization of a pseudoachondroplasia-associated mutation (His587-->Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP).

We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.

Arginine↗