Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

Proteomic analysis of tendon extracellular matrix reveals disease stage-specific fragmentation and differential cleavage of COMP (cartilage oligomeric matrix protein).

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1ß and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1ß enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.

The C-terminal peptide of chondroadherin modulates cellular activity by selectively binding to heparan sulfate chains.

Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH(359), now shown to bind to heparin with a K(D) of 13 µm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their ß(1) integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.

Chondroadherin fragmentation mediated by the protease HTRA1 distinguishes human intervertebral disc degeneration from normal aging.

Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.

Quantitative proteomic analysis of eight cartilaginous tissues reveals characteristic differences as well as similarities between subgroups.

Human synovial joints display a characteristic anatomic distribution of arthritis, e.g. rheumatoid arthritis primarily affects the metacarpophalangeal and proximal finger joints, but rarely the distal finger joints, whereas osteoarthritis occurs in the distal and proximal finger joints. Pelvospondylitis has a selective localization to the spine and sacroiliac joints. Is this tropism due to differences between the cartilages at the molecular level? To substantiate this concept the present study provides a background detailed compositional analysis by relative quantification of extracellular matrix proteins in articular cartilages, meniscus, intervertebral disc, rib, and tracheal cartilages on samples from 5-6 different individuals using an optimized approach for proteomics. Tissue extraction followed by trypsin digestion and two-dimensional LC separations coupled to tandem mass spectrometry, relative quantification with isobaric labeling, iTRAQ(TM), was used to compare the relative abundance of about 150 proteins. There were clear differences in protein patterns between different kinds of cartilages. Matrilin-1 and epiphycan were specific for rib and trachea, whereas asporin was particularly abundant in the meniscus. Interestingly, lubricin was prominent in the intervertebral disc, especially in the nucleus pulposus. Fibromodulin and lumican showed distributions that were mirror images of one other. Analyses of the insoluble residues from guanidine extraction revealed that a fraction of several proteins remained unextracted, e.g. asporin, CILP, and COMP, indicating cross-linking. Distinct differences in protein patterns may relate to different tissue mechanical properties, and to the intriguing tropism in different patterns of joint pathology.

PRELP protein inhibits the formation of the complement membrane attack complex.

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.

Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans.

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.

Immunolocalization of collagens (I and III) and cartilage oligomeric matrix protein in the normal and injured equine superficial digital flexor tendon.

This is a descriptive study of tendon pathology with different structural appearances of repair tissue correlated to immunolocalization of cartilage oligomeric matrix protein (COMP) and type I and III collagens and expression of COMP mRNA. The material consists of nine tendons from seven horses (5-25 years old; mean age of 10 years) with clinical tendinopathy and three normal tendons from horses (3, 3, and 13 years old) euthanized for non-orthopedic reasons. The injured tendons displayed different repair-tissue appearances with organized and disorganized fibroblastic regions as well as areas of necrosis. The normal tendons presented distinct immunoreactivity for COMP and expression of COMP mRNA and type I collagen in the normal aligned fiber structures, but no immunolabeling of type III collagen. However, immunoreactivity for type III collagen was present in the endotenon surrounding the fiber bundles, where no expression of COMP could be seen. Immunostaining for type I and III collagens was present in all of the pathologic regions indicating repair tissue. Interestingly, the granulation tissues showed immunostaining for COMP and expression of COMP mRNA, indicating a role for COMP in repair and remodeling of the tendon after fiber degeneration and rupture. The present results suggest that not only type III collagen but also COMP is involved in the repair and remodeling processes of the tendon.

Early increase in serum-COMP is associated with joint damage progression over the first five years in patients with rheumatoid arthritis.

BACKGROUND: Currently available biomarkers for the early tissue process leading to joint damage in rheumatoid arthritis are insufficient and lack prognostic accuracy, possibly a result of variable activity of the disease over time. This study represents a novel approach to detect an altered activity of the disease process detected as increasing serum-COMP levels over a short time and whether this would correlate with joint damage progression over the first 5 years of disease. METHODS: In all, 349 patients from the Swedish BARFOT early RA study were examined. Serum-COMP was analysed by ELISA at diagnosis and after 3 months. Based on changes in serum-COMP levels, three subgroups of patients were defined: those with unchanged levels (change ≤ 20%) (N=142), decreasing levels (> 20%) (N=173) and increasing levels (> 20%) (N=34). Radiographs of hands and feet were obtained at inclusion, after 1, 2 and 5 years and scored according to Sharp van der Heijde (SHS). Radiographic progression was defined as increase in SHS by ≥5.8. RESULTS: The group of patients with increasing COMP levels showed higher median change in total SHS and erosion scores at 1, 2 and 5 year follow-up compared with the groups with stable or decreasing COMP levels. Furthermore, the odds ratio of radiographic progression was 2.8 (95% CI 1.26-6.38) for patients with increasing COMP levels vs. patients with unchanged levels.The group of patients with increasing COMP levels had higher ESR at inclusion but there were no baseline differences between the groups for age, gender, disease duration, disease activity (DAS28), function (HAQ), CRP, nor presence of rheumatoid factor or anti-CCP. Importantly, neither did changes over the 3-month period in DAS28, HAQ, ESR nor CRP differ between the groups and these variables did not correlate to joint damage progression. CONCLUSION: Increasing serum-COMP levels between diagnosis and the subsequent 3 months in patients with early RA represents a novel indicator of an activated destructive process in the joint and is a promising tool to identify patients with significant joint damage progression during a 5-year period.

Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment.

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)ß(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)ß(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)ß(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.

NC4 Domain of cartilage-specific collagen IX inhibits complement directly due to attenuation of membrane attack formation and indirectly through binding and enhancing activity of complement inhibitors C4B-binding protein and factor H.

Collagen IX containing the N-terminal noncollagenous domain 4 (NC4) is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and noncollagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein, matrilins, fibromodulin, and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3, and C9 and to directly inhibit C9 polymerization and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together, our results suggest that collagen IX and its interactions with matrix components are important parts of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis.

Extracellular matrix alterations and acute inflammation; developing in parallel during early induction of pulmonary fibrosis.

Pulmonary fibrosis is a hallmark of several systemic diseases such as systemic sclerosis. Initiation and early development is not well characterized, as initiation usually is unnoticed in patients, yet fibrosis has been considered a late event, occurring after an inflammatory phase. By utilizing an animal model, the starting point can be defined and the initiation process and early development thoroughly investigated. To investigate these processes from a systemic perspective, we choose a systemic administration route, instead of the more commonly used local administration. The aim of this work was to study the initiation of pulmonary fibrosis in an animal model and to investigate early alterations in connective tissue, cell turnover and acute immune response in lung parenchyma. Animals were injected subcutaneously with bleomycin, three times a week (w) for 1-4w (controls received saline). Total collagen was histologically assessed by Picro Sirius Red and Masson's Trichrome, collagen production by antibodies directed against N-terminal of procollagens I and III, proliferation by labeling with proliferating cell nuclear antigen, apoptosis by TUNEL and innate immunity by detecting neutrophils and macrophages. Total collagen was significantly increased at 1, 2 and 4w compared with controls. Procollagen I, was increased at 1w and remained increased, whereas procollagen III-staining was increased at 2w, compared with controls. Myofibroblasts were increased at all times as were proliferation, whereas apoptosis was increased from 2w. Neutrophils peaked at 1w (2779±820 cells/mm²) and gradually decreased, whereas macrophages peaked at 2w (135±29 cells/mm²). Subcutaneously administered bleomycin induces rapid alterations in connective tissue and cell turnover, suggesting a plasticity of the connective tissue. A transient neutrophilia is detected and increased number of macrophages likely represents a clearance process of said neutrophils. The study suggests fibrosis initiation and acute inflammation to occur in parallel in this model.

Serglycin inhibits the classical and lectin pathways of complement via its glycosaminoglycan chains: implications for multiple myeloma.

Serglycin (SG) is a proteoglycan expressed by hematopoietic cells and is constitutively secreted by multiple myeloma (MM) cells. SG participates in the regulation of various inflammatory events. We found that SG secreted by human MM cell lines inhibits both the classical and lectin pathways of complement, without influencing alternative pathway activity. The inhibitory effect of SG is due to direct interactions with C1q and mannose-binding lectin (MBL). C1q-binding is mediated through the glycosaminoglycan moieties of SG, whereas MBL binds additionally to SG protein core. Interactions between SG and C1q as well as MBL are diminished in the presence of chondroitin sulfate type E. In addition, we localized the SG-binding site to the collagen-like stalk of C1q. Interactions between SG and C1q as well as MBL are ionic in character and only the interaction with MBL was found to be partially dependent on the presence of calcium. We found the serum levels of SG to be elevated in patients with MM compared to healthy controls. Moreover, we found that SG expressed from myeloma plasma cells protects these cells from complement activation induced by treatment with anti-thymocyte immunoglobulins. This might protect myeloma cells during immunotherapy and promote survival of malignant cells.

The streptococcal collagen-binding protein CNE specifically interferes with alphaVbeta3-mediated cellular interactions with triple helical collagen.

Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α(V)ß(3)-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α(V)ß(3)-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α(V)ß(3)-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding ß(1) integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α(V)ß(3)-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α(V)ß(3)-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.

Regulation of complement by cartilage oligomeric matrix protein allows for a novel molecular diagnostic principle in rheumatoid arthritis.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA) and osteoarthritis (OA). This study was undertaken to investigate the ability of COMP to regulate complement, a capacity that has previously been shown for some other cartilage proteins. METHODS: Regulation of complement by COMP was studied using functional in vitro assays. Inter-actions between complement proteins and COMP were investigated by direct binding assay and electron microscopy. Circulating COMP and COMP-C3b complexes in serum and synovial fluid from RA and OA patients and healthy controls were measured with a novel enzyme-linked immunosorbent assay. RESULTS: We found in vivo evidence of complement activation by released COMP in the general circulation of patients with RA, but not patients with OA. COMP induced activation and deposition of C3b and C9 specifically via the alternative pathway of complement, which was attributable to direct interaction between COMP and properdin. Furthermore, COMP inhibited the classical and the lectin complement pathways due to direct interaction with the stalk region of C1q and mannose-binding lectin, respectively. CONCLUSION: COMP is the first extracellular matrix protein for which an active role in inflammation has been demonstrated in vivo. It can activate one complement pathway at the same time as it has the potential to inhibit another. The net outcome of these interactions is most likely determined by the type of released COMP fragments, which may be disease specific.

Complement inhibitor C4b-binding protein interacts directly with small glycoproteins of the extracellular matrix.

Components derived from cartilage have been suggested to maintain the inflammation in joints in arthritis. Small leucine-rich repeat proteins (SLRPs) are structural components of cartilage important in organizing the meshwork of extracellular matrix components. It has recently been shown that the SLRP fibromodulin interacts with complement initiator C1q, leading to complement activation. The complement response is limited since fibromodulin also interacts with the complement inhibitor factor H. We have now found that osteoadherin, chondroadherin, fibromodulin, and proline arginine-rich end leucine-rich repeat protein bind to the complement inhibitor C4b-binding protein (C4BP). Using direct binding assays with C4BP fragments and C4BP mutants lacking individual domains in combination with electron microscopy, we have demonstrated that mainly the central core of C4BP mediated binding to SLRPs. Binding of SLRPs to C4BP did not affect its ability to inhibit complement. Osteoadherin, fibromodulin, and chondroadherin, which bind C1q and activate complement, were found to cause significantly higher C9 deposition in C4BP-depleted serum compared with Igs, indicating that the level of complement activation initiated by SLRPs is regulated by simultaneous binding to C4BP. A similar dual binding of C1q and complement inhibitors was observed previously for other endogenous ligands (amyloid, prions, C-reactive protein, and apoptotic cells) but not for exogenous activators (bacteria-bound Igs). These interactions can be significant during inflammatory joint diseases, such as rheumatoid arthritis, where cartilage is degraded, and cartilage components are released into synovial fluid, where they can interact with factors of the complement system.

Increased cartilage oligomeric matrix protein concentrations in equine digital flexor tendon sheath synovial fluid predicts intrathecal tendon damage.

OBJECTIVES: To evaluate digital flexor tendon sheath (DFTS) synovial fluid cartilage oligomeric matrix protein (COMP) concentrations as a molecular marker for intrathecal pathology. STUDY DESIGN: Case control study. ANIMALS: Horses (n=46) with DFTS tenosynovitis; 23 fresh cadaver horses. METHODS: DFTS synovial fluid samples were collected from clinical cases with noninfected DFTS tenosynovitis and from control DFTS. Clinical and surgical findings were recorded, and dissection of control limbs was performed to confirm the DFTS to be grossly normal. Synovial fluid COMP was quantified using a homologous competitive inhibition ELISA. RESULTS: Abnormalities were identified tenoscopically: intrathecal tendon/ligament tearing was identified in 37 cases and 9 had other lesions. In control horses, synovial fluid COMP was higher in younger horses. Clinical cases with intrathecal tendon/ligament tearing had higher synovial fluid COMP than either clinical cases with other lesions, or controls. In horses ≥5 years old, the sensitivity and specificity of the assay was high for diagnosing intrathecal tendon/ligament tearing. CONCLUSIONS: COMP concentrations in DFTS synovial fluid were significantly greater than those in normal horses with noninfected tenosynovitis caused by intrathecal tendon/ligament tearing, but not by other lesions.

The tyrosine sulfate-rich domains of the LRR proteins fibromodulin and osteoadherin bind motifs of basic clusters in a variety of heparin-binding proteins, including bioactive factors.

The small leucine-rich repeat proteins, fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-sulfated tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N and C termini, respectively, and PRELP is known to bind to heparin via this domain. Another heparin-binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C terminus. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full-length osteoadherin. The tyrosine sulfate domains also bound heparin-binding proteins such as basic fibroblast growth factor-2, thrombospondin I, MMP13, the NC4 domain of collagen IX, and interleukin-10. Fibronectin with large heparin-binding domains did not bind, neither did CILP containing a heparin-binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the sulfated tyrosine residues shown by different binding properties of 10-kDa fragments subfractionated by ion-exchange chromatography. These interactions may sequester growth factors, cytokines, and matrix metalloproteinases in the extracellular matrix as well as contribute to its organization.

Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization.

The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).