A novel, somewhat basic noncollagenous protein was purified from guanidine hydrochloride extracts of human articular cartilage using cesium chloride density gradient centrifugation, followed by ion-exchange chromatography at pH 5, and gel filtration on two serially coupled columns of Superose 6 and Superdex 200.The protein of 91.5 kDa contains a single polypeptide chain substituted with N-linked oligosaccharides. It appeared unique to cartilage as studied by enzyme-linked immunosorbent assay and immunoblots of various tissue extracts. Its concentration in articular cartilages showed some variability with age being lower in young individuals. It represents a chondrocyte product, since it is synthesized by articular chondrocytes in explant cultures. Interestingly, the distribution of the protein in the articular cartilage provides important information on the nature of chondrocytes at different compartments in the tissue. Thus, chondrocytes in the middle/ deeper layers of the tissue in particular, appeared to have produced the protein and deposited it in the interterritorial matrix. The protein was neither seen in the superficial nor in the deepest regions of the articular cartilage. Based on its immunolocalization we have named this protein CILP (cartilage intermediate layer protein).Articular cartilage is a heterogeneous tissue where the cells are arranged in layers of matrix that have a different composition and function depending on their location from the articular surface to the subchondral bone (1, 2). The extracellular matrix is also arranged into compartments around the cells: pericellular (closest to the cell), territorial (extending around individual or groups of chondrocytes), and the interterritorial matrix (furthest away from the cells). The matrix is produced by the chondrocytes and contains, as major constituents, fibrilforming collagens and large aggregating proteoglycans that are assembled into highly organized structures (1, 3-5).Collagen confers tensile properties to the tissue, whereas proteoglycans have a key role in the normal resilience and load dissipation of the cartilage. There is also a minor population of non-collagenous proteins for which no functional role has yet been identified. They may have roles in maintaining the tissue homeostasis by the regulation of matrix assembly, cell recognition, and cell attachment. They may also have a part to play in balancing the processes of cartilage repair and degradation, as well as in disease processes where degradation outbalances repair and loss of tissue ensues.To identify matrix constituents involved in these processes, it is imperative to identify, isolate, and characterize these matrix proteins. The present paper describes the isolation and partial characterization of a 91.5-kDa single chain protein from human articular cartilage. This protein was selected, since it appeared to change in osteoarthritis. The protein was found to be restricted in its tissue distribution to cartilage and furthermore, to specific zones within the tissue. We su...

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Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn 281 with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser 54 . The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.Cartilage matrix consists of fibrillar networks, primarily of collagen II and highly negatively charged molecules of aggrecan. There are also a number of noncollagenous glycoproteins that apparently contribute to the regulation of tissue assembly and properties. Among them is the family of the leucine-rich repeat (LRR) 1 proteins, which contains several members found in the extracellular matrix. There are currently 11 known members of this family. These molecules share a common structure with a central stretch of LRRs. This LRR domain is flanked by disulfide bridged loops, with 4 cysteine residues preceding the LRR domain and 2 on its C-terminal side. Apart from chondroadherin, these proteins also contain divergent amino-terminal extensions with features unique for the different proteins. Based on amino acid sequence and gene organization the family can be divided into four distinct groups.Decorin (1) and biglycan (2) constitute the first group (class I). These proteins have 10 LRRs and carry one and two chondroitin or dermatan sulfate chains, respectively. The glycosaminoglycan chains are linked to serine residues in the amino terminus. The molecules in this group are secreted with a propeptide.The second group (class II) consists of fibromodulin (3), lumican (4), keratocan (5), PRELP (6), and osteoadherin (7). Like the class I proteins they consist of 10 LRRs. With the exception of PRELP, they all carry polylactosamine or keratan sulfate chains linked to the LRR region and sulfated tyrosine residues in the amino-terminal extension. In contrast, the amino terminus of PRELP has a cluster of positively charged amino acid residues that mediates binding to heparan sulfate (8). Unlike all other family members, osteoadh...

The cDNA cloning and expression in vitro and in eukaryotic cells of a novel protein isolated from human articular cartilage, cartilage intermediate layer protein (CILP) is described. A single 4.2-kilobase mRNA detected in human articular cartilage encodes a polypeptide of 1184 amino acids with a calculated molecular mass of 132.5 kDa. The protein has a putative signal peptide of 21 amino acids, and is a proform of two polypeptides. The amino-terminal half corresponds to CILP (molecular mass of 78.5 kDa, not including posttranslational modifications) and the carboxyl-terminal half corresponds to a protein homologous to a porcine nucleotide pyrophosphohydrolase, NTPPHase (molecular mass of 51.8 kDa, not including post-translational modifications). CILP has 30 cysteines and six putative N-glycosylation sites. The human homolog of porcine NTPPHase described here contains 10 cysteine residues and two putative N-glycosylation sites. In the precursor protein the NTPPHase region is immediately preceded by a tetrapeptide conforming to a furin proteinase cleavage consensus sequence. Expression of the fulllength cDNA in a cell-free translation system and in COS-7 or EBNA cells indicates that the precursor protein is synthesized as a single polypeptide chain that is processed, possibly by a furin-like protease, into two polypeptides upon or preceding secretion.Articular cartilage is a tissue that varies in composition with site, e.g. from the surface to the deep zone and also from the pericellular environment close to the cells to the more distant interterritorial matrix (1, 2). The major constituents of the matrix are collagens (reviewed in Ref.3) and large aggregating proteoglycans that contain large numbers of chondroitin sulfate chains (reviewed in Ref. 4).A third set of molecules are the non-collagenous glycoproteins including several members of the family of leucine-rich repeat proteins and the thrombospondins. The leucine-rich repeat proteins include decorin, biglycan, fibromodulin, and lumican, all with the capacity to bind to collagen (for Refs. see Ref. 1). These molecules are found along surfaces of collagen fibrils in the tissue. Other members of this family that do not bind to collagen include chondroadherin (5) and PRELP (6).The thrombospondin family includes the pentameric COMP, which is one of the more abundant cartilage matrix proteins (7,8). Furthermore, thrombospondin-1 has been identified in articular cartilage (9).There are also a number of other proteins that may not be part of the described families. One such protein is the cartilage intermediate layer protein (CILP), 1 previously identified and purified from human articular cartilage (25). In most cases the structure and function of these proteins are not known. However, detailed knowledge of their structure, function, and variability in normal conditions as well as in disease is of fundamental importance to define mechanisms and events occurring in joint disease.Interestingly, in joint disease, such as osteoarthrosis there are major alterations in t...

Background: To understand molecular processes underlying cartilage destruction, we analyzed COMP fragments released into synovial fluid in joint diseases. Results: Twelve novel COMP neoepitopes have been identified. Conclusion:The release of COMP neoepitopes provides means for monitoring disease progression. Significance: Based on the specificity, selectivity, and sensitivity of each neoepitope, a new generation of biomarkers for cartilage destruction can be developed.