A novel COMP mutation in a Chinese patient with pseudoachondroplasia

A 2.75-year-old Chinese boy presented with typical clinical features of pseudoachondroplasia, including disproportionate short-limb short stature, brachydactyly, genu varus and waddling gait. Radiologically, tubular bones were short with widened metaphyses, irregular and small epiphyses; anterior tonguing or beaking of vertebral bodies were characteristic. DNA sequencing analysis of the COMP gene revealed a heterozygous mutation (c.1511G>A, p.Cys504Tyr) in the patient but his parents were unaffected without this genetic change. The missense mutation (c.1511G>A) was not found in 100 healthy controls and has not been reported previously. Our findings expand the spectrum of known mutations in COMP leading to pseudoachondroplasia.     

Apoptosis staining in cultured pseudoachondroplasia chondrocytes

Pseudoachondroplasia (PSACH) is a skeletal dysplasia caused by a mutation in cartilage oligomeric matrix protein (COMP), a glycoprotein of normal cartilage matrix. PSACH chondrocytes have a distinctive phenotype with enlarged rER cisternae containing COMP, aggrecan, type IX collagen, and chaperone proteins. Ultrastructural studies suggested that this accumulation compromises cell function, hastening cell death, and consequently reducing the number of cells in the growth plate contributing to linear bone growth. Using the alginate bead system, we cultured control and PSACH chondrocytes for twenty weeks and one year to determine the effect of the mutation on size and number of cartilage nodules; and the presence of apoptotic cell death (TUNEL assay). At 20 weeks, beads containing PSACH or control chondrocytes did not differ in size and number of cartilage nodules or number of TUNEL-positive cells. After one year, nodule number, size and percent cartilage per bead were significantly less in PSACH nodules, and the number of cells staining positive for apoptosis was significantly greater than in controls (71.8% vs. 44.6%). The increase in apoptosis in PSACH nodules correlates with a decrease in growth of cartilage, supporting our hypothesis that death of damaged cells contributes to the growth plate defects in PSACH.     

COMP mutations: domain-dependent relationship between abnormal chondrocyte trafficking and clinical PSACH and MED phenotypes

Mutations in cartilage oligomeric matrix protein (COMP) produce clinical phenotypes ranging from the severe end of the spectrum, pseudoachondroplasia (PSACH), which is a dwarfing condition, to a mild condition, multiple epiphyseal dysplasia (MED). Patient chondrocytes have a unique morphology characterized by distended rER cisternae containing lamellar deposits of COMP and other extracellular matrix proteins. It has been difficult to determine why different mutations give rise to variable clinical phenotypes. Using our in vitro cell system, we previously demonstrated that the most common PSACH mutation, D469del, severely impedes trafficking of COMP and type IX collagen in chondrocytic cells, consistent with observations from patient cells. Here, we hypothesize that PSACH and MED mutations variably affect the cellular trafficking behavior of COMP and that the extent of defective trafficking correlates with clinical phenotype. Twelve different recombinant COMP mutations were expressed in rat chondrosarcoma cells and the percent cells with ER-retained COMP was assessed. For mutations in type 3 (T3) repeats, trafficking defects correlated with clinical phenotype; PSACH mutations had more cells retaining mutant COMP, while MED mutations had fewer. In contrast, the cellular trafficking pattern observed for mutations in the C-terminal globular domain (CTD) was not predictive of clinical phenotype. The results demonstrate that different COMP mutations in the T3 repeat domain have variable effects on intracellular transport, which correlate with clinical severity, while CTD mutations do not show such a correlation. These findings suggest that other unidentified factors contribute to the effect of the CTD mutations. J. Cell. Biochem. 103: 778-787, 2008. (c) 2007 Wiley-Liss, Inc.     

Novel and recurrent COMP (cartilage oligomeric matrix protein) mutations in pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are common skeletal dysplasias with impaired enchondral ossification and premature degenerative joint disease. The two disorders were in the past considered to be distinct clinical entities; however, recent studies have proven that both diseases can result from mutations of the gene encoding cartilage oligomeric matrix protein (COMP). To characterize further COMP mutations and investigate phenotype-genotype relationships, we screened this gene in 15 patients with PSACH or MED by directly sequencing polymerase chain reaction products from genomic DNA. We identified ten mutations involving conserved residues among the eight calmodulin-like repeats of the gene product: seven were novel missense mutations in exons 9, 10, 11, 13 or 14, and the other three resulted from deletion of one of the five GAC repeats in exon 13. We have found that the GAC repeats in the 7th calmodulin-like repeat in exon 13 represent a hot-spot for mutation, and that mutations in the 7th calmodulin-like repeat produce severe PSACH phenotypes while mutations elsewhere in the gene exhibit mild PSACH or MED phenotypes. These genotype-phenotype correlations may facilitate molecular diagnosis and classification of PSACH and MED, and provide insight into the relationship between structure and function of the COMP gene product. Received: 1 June 1998 / Accepted: 22 October 1998     

软骨寡聚基质蛋白荧光免疫层析定量检测方法建立

本研究旨在建立一种可用于类风湿性关节炎(rheumatoid arthritis, RA)辅助诊断的人软骨寡聚基质蛋白(cartilage oligomeric matrix protein, COMP)荧光层析检测方法。采用双抗体夹心法原理制备免疫层析试纸条,并进行性能评价及方法学对比。通过对临床样本的检测得到受试者工作特征(receiver operating characteristic, ROC)曲线,计算试纸条灵敏度、特异性、阳性和阴性预测值。线性范围为0.39–50.00ng/mL;批内批间变异系数均小于15%;试纸条37℃加速破坏20d,荧光信号强度变化范围在15%以内;与类风湿因子(rheumatoid factor,RF)、抗环瓜氨酸肽(anti-cyclic citrullinated peptide,anti-CCP)抗体无交叉反应;与ELISA试剂盒平行检测48份临床血清样本,相关性良好。采用本研究制备的试纸条检测样本,COMP区分RA患者和健康人的cut-off值为22.55 ng/mL (灵敏度为0.821,特异度为0.842,阳性预测值为0.741,阴性预...     

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.     

COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins.

Cloning and sequence analysis of cartilage oligomeric matrix protein (COMP) cDNA, representing a cartilage pentameric protein, revealed a protein of 755 amino acid residues with a calculated molecular mass of 82,700 Da. Expression of the cDNA in COS cells showed that COMP is a homopolymer composed of five identical disulfide-linked subunits. COMP is homologous to the carboxyl-terminal half of thrombospondin, and the homologies include 89% and 54% of the residues in COMP and thrombospondin, respectively. The similarities are most pronounced in the carboxyl-terminal domains and in the calcium binding type 3 repeat domains in which about 60% of the amino acid residues are identical. In the type 2/epidermal growth factor repeat domains the two proteins contain 41% identical residues. The sequence of the amino-terminal 84-amino acid residues is unique for COMP. Comparison of the amino acid sequences in the type 2 and type 3 repeat domains of COMP and the thrombospondins shows that COMP is the product of a unique gene and not the result of an alternatively spliced thrombospondin gene.     

Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage.

An Mr = 524,000 oligomeric protein was isolated from bovine cartilage and designated COMP (Cartilage Oligomeric Matrix Protein). The protein is composed of disulfide-bonded subunits with an apparent Mr of 100,000 each. It is markedly anionic, probably due to its high contents of aspartic acid and glutamic acid, as well as to its substitution with negatively charged carbohydrates. COMP was found in all cartilages analyzed, but could not be detected in other tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. Within a given cartilage, COMP shows a preferential localization to the territorial matrix surrounding the chondrocytes.     

A depth-dependent model of the pericellular microenvironment of chondrocytes in articular cartilage

Experimental studies suggest that the magnitude of chondrocyte deformation is much smaller than expected based on the material properties of extracellular matrix (ECM) and cells, and that this result could be explained by a structural unit, the chondron, that is thought to protect chondrocytes from large deformations in situ. We extended an existing numerical model of chondrocyte, ECM and pericellular matrix (PCM) to include depth-dependent structural information. Our results suggest that superficial zone chondrocytes, which lack a pericellular capsule (PC), are relatively stiff, and therefore are protected from excessive deformations, whereas middle and deep zone chondrocytes are softer but are protected by the PC that limits cell deformations in these regions. We conclude that cell deformations sensitively depend on the immediate structural environment of the PCM in a depth-dependent manner, and that the functional stiffness of chondrocytes in situ is much larger than experiments on isolated cells would suggest.     

Serum cartilage oligomeric matrix protein (COMP) decreases in rheumatoid arthritis patients treated with infliximab or etanercept

Changes in serum cartilage oligomeric matrix protein (COMP) were studied during a 6-month period from initiation of treatment of rheumatoid arthritis patients with either infliximab or etanercept, to elucidate whether the favourable results of tissue protection reported in clinical trials are corroborated by changing levels of circulating COMP. Rheumatoid arthritis patients commencing treatment with infliximab (N = 32) or etanercept (N = 17) were monitored in accordance with a structured protocol. Only patients who were not receiving glucocorticoids or who were on a stable dose of oral prednisolone (<10 mg daily) were included. Serum COMP was measured by a sandwich immunoassay based on two monoclonal antibodies against human COMP in samples obtained at treatment initiation and at 3 and 6 months. Serum COMP decreased at 3 months in both infliximab- and etanercept-treated patients (P < 0.001 and <0.005, respectively) and remained low at 6 months. There was no significant correlation between changes in or concentrations of serum COMP and serum C-reactive protein at any time point. A decrease in serum COMP was seen both in ACR20 responders (patients meeting the American College of Rheumatology criteria for 20% improvement) and in nonresponders. The pattern of changes of serum COMP, a marker for cartilage turnover, in these patient groups supports the interpretation that infliximab and etanercept have a joint protective effect. Serum COMP has potential as a useful marker for evaluating tissue effects of novel treatment modalities in rheumatoid arthritis.     

Articular cartilage and changes in Arthritis: Noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage

Cartilage contains numerous noncollagenous proteins in its extracellular matrix, including proteoglycans. At least 40 such molecules have been identified, differing greatly in structure, distribution, and function. Some are present in only selected cartilages or cartilage zones, some vary in their presence with a person's development and age, and others are more universal in their expression. Some may not even be made by the chondrocytes, but may arise by absorption from the synovial fluid. In many cases, the molecules' function is unclear, but the importance of others is illustrated by their involvement in genetic disorders. This review provides a selective survey of these molecules and discusses their structure, function, and involvement in inherited and arthritic disorders.     

Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in relation to collagen fibrils in the equine tendon.

The structure and organisation of the extracellular matrix, and in particular the axial alignment of type I collagen fibrils, are essential for the tensile strength of tendons. The resident tenocytes synthesize and maintain the composition of the extracellular matrix, which changes with age and maturation. Other components of the extracellular matrix include less abundant collagen types II, III, V, VI, XII, proteoglycans and glycoproteins. Cartilage oligomeric matrix protein (COMP) is an abundant non-collagenous pentameric glycoprotein in the tendon, which can bind to collagen types I and II. The function of COMP in the tendon is not clear, but it may act as a catalyst in fibrillogenesis. Its concentration changes with age, maturation and load. The present study delineates the ultrastructural distribution of COMP and its correlation to collagen fibril thickness in different compartments in two flexor tendons from horses of different ages (foetus, 8 months, 3 years, 12 years). The immunolabeling for COMP was higher in the superficial digital flexor tendon compared with the deep digital flexor tendon and it increased with the age of the animal, with the highest concentration in the 3-year-olds. Fibril diameter differed between age groups and a more homogenous fibril population was found in the fetal tendons. A positive correlation between high COMP immunolabeling and the percentage of small fibrils (<60 nm) were present in the SDFT. COMP immunolabeling was enriched at the gap region of the collagen fibril. In situ hybridization revealed the strongest expression in tendons from the 3-year-old horses whereas there was no expression in foetal tendon.     

Disruption of extracellular matrix structure may cause pseudoachondroplasia phenotypes in the absence of impaired cartilage oligomeric matrix protein secretion

Pseudoachondroplasia and multiple epiphyseal dysplasia are two dominantly inherited chondrodysplasias associated with mutations in cartilage oligomeric matrix protein (COMP). The rarely available patient biopsies show lamellar inclusions in the endoplasmic reticulum. We studied the pathogenesis of these chondrodysplasias by expressing several disease-causing COMP mutations in bovine primary chondrocytes and found that COMP-associated chondrodysplasias are not exclusively storage diseases. Although COMP carrying the mutations D469Delta and D475N was retained within the endoplasmic reticulum, secretion of COMP H587R was only slightly retarded. All pseudoachondroplasia mutations impair cellular viability and cause a disruption of the extracellular matrix formed in alginate culture irrespective of the degree of cellular retention. The mutation D361Y associated with the clinically milder disease multiple epiphyseal dysplasia gave mild retention and limited matrix alterations, but the transfected cells showed normal viability. The effect of mutated COMP on matrix formation and cell-matrix interaction may be a major element in the pathogenesis of COMP-associated chondrodysplasias.     

Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium

Cell culture with serum-containing medium has potential problems associated with contamination of infectious agents. This study demonstrates for the first time the feasibility of regenerating cartilage tissues in vivo by implantation of chondrocytes cultured in vitro in a chemically-defined, serum-free medium. Chondrocytes cultured in the serum-free medium grew similarly to those in a serum-containing medium. Implantation of chondrocytes cultured in the serum-free medium and seeded on to polymer scaffolds resulted in the regeneration of cartilage tissues with histological aspects similar to those of cartilage tissues regenerated from chondrocytes cultured in serum-containing medium.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.