Among the noncollagenous proteins of cartilage, aggrecan has undoubtedly received the greatest attention, because of its high abundance in cartilage, its close association with the ability of the tissue to resist compression, and its modification in many cartilage disorders. Aggrecan belongs to the family of aggregating proteoglycans that form large, multimolecular complexes with hyaluronan . The family also includes versican, neurocan, and brevican, though of these only versican has been shown to be expressed in cartilage, and at much lower levels than aggrecan. All the members of the family have an amino-terminal globular domain, which is responsible for interaction with hyaluronan, and a carboxy-terminal globular domain, which has lectin-like homology. These features have resulted in the family being termed hyalectans or lecticans.
Aggrecan has an additional globular domain (G2) that is separated from the amino-terminal globular domain (G1) by a short, interglobular domain . The G2 domain is separated from the carboxy-terminal globular domain (G3) by a keratan sulfate attachment domain and two chondroitin sulfate (CS) attachment domains (CS1 and CS2). Over 100 CS and keratan sulfate chains may be present in the three glycosamino-glycan attachment domains, though it is not clear at present whether all potential attachment sites are always occupied or whether variation may occur among individuals. The high CS and keratan sulfate content of aggrecan and its ability to interact with hyaluronan are essential features for normal articular cartilage function, as they provide the rheological properties necessary for resisting compression. The function of the G3 domain of aggrecan is unclear. Its lectin-like properties suggest the possibility of interaction with other components of the extracellular matrix , though it has also been suggested that it is involved in intracellular trafficking during aggrecan synthesis. Mutations in the aggrecan gene that prevent core protein synthesis form the basis of chondrodysplasias in mice (cartilage matrix deficiency) and chicks (nanomelia) . In addition, impaired glycosaminoglycan sulfation on aggrecan causes the chondrodysplastic phenotypes associated with the brachymorphic mouse and diastrophic dysplasia in humans.
An interesting feature of the human aggrecan gene is the existence of polymorphism in the region encoding the CS1 domain. This region is composed of repeat sequences, which may range in number from 13 to 33 . Individuals with the shortest alleles will have the lowest proportion of CS on their aggrecan molecules, and may be at risk for cartilage degeneration due to impaired aggrecan function. Irrespective of such polymorphism, the glycosaminoglycan composition of aggrecan varies considerably during juvenile development, as both the size and sulfation pattern of the CS and keratan sulfate change, though the functional consequence of this change is unclear. In addition, size heterogeneity is generated in the aggrecan core protein by the action of proteinases, with those fragments bearing a G1 domain being selectively retained in the tissue matrix. Proteolysis ultimately results in the accumulation of free G1 domains that have a long half-life in the tissue . Many proteinases are able to degrade aggrecan if they gain access to the cartilage matrix, but most physiological and pathological degradation of articular cartilage is associated with the action of matrix metalloproteinases and aggrecanases . Degradation products resulting from the action of both classes of proteinase accumulate in the synovial fluid of patients with arthritis [7,8] and have been used as markers of tissue destruction. Aggrecan synthesized in the arthritic joint has a CS sulfation pattern more akin to that in the normal juvenile than the normal adult. The appearance of this immature CS structure has also been used as a marker of the arthritic joint, and in particular of the reparative process that is being mounted. The G1-containing aggrecan fragments that accumulate with age or tissue degeneration may play a role in the induction of an autoimmune polyarthritis in susceptible individuals .
The interaction of aggrecan with hyaluronan is stabilized by the presence of link proteins. As with aggrecan, these proteins undergo proteolytic modification throughout life and can be used as an indicator of proteinase action. They provide evidence of the action of matrix metalloproteinase throughout juvenile development, and the participation of additional agents in the adult [10,11]. The link proteins are not susceptible to cleavage by the aggrecanase produced under cytokine stimulation of cartilage , and there is no evidence that any of the proteolytically modified link proteins have impaired function. Link protein can be lost from the cartilage matrix during periods of tissue degeneration, but such loss is most likely due to depolymerization of hyaluronan and involves concomitant loss of aggrecan. The importance of link protein in proteoglycan aggregate function is demonstrated by the impaired cartilage development observed in the link-protein-null mouse .
Small leucine-rich repeat proteoglycans
The small leucine-rich repeat proteoglycans (SLRPs) are characterized by a central domain composed of a series of adjacent leucine-rich repeats bordered at each end by disulfide-bonded domains . The family may be divided into two subfamilies, depending on the presence of dermatan sulfate chains or keratan sulfate chains. Human cartilage has been shown to contain three dermatan sulfate proteoglycans (also called DS-PGs)- biglycan (DS-PGI), decorin (DS-PGII), and epiphycan (DS-PGIII) - and in all of these, the dermatan sulfate chains are in the amino-terminal region of the core proteins. Only decorin and biglycan have been found in articular cartilage, and they are present throughout life. Whereas decorin remains in its intact form at all ages, biglycan exhibits age-related proteolytic processing that results in removal of the amino-terminal region bearing the dermatan sulfate chains. Such nonglycanated biglycan accumulates in the cartilage matrix with age, but it is not clear whether this is of any functional consequence . Decorin and biglycan also have short, amino-terminal propeptides that are removed in the extracellular matrix by procollagen-C proteinase, the same enzyme responsible for removing the carboxy propeptide from type II collagen. Propeptide removal is incomplete in adult cartilage , but again, the functional consequence, if any, is unclear.
Human articular cartilage contains two potential keratan sulfate proteoglycans, fibromodulin and lumican. Like decorin and biglycan, fibromodulin is present in articular cartilage throughout life, though it contains keratan sulfate chains only in the fetus and juvenile . In the adult, it exists as a glycoprotein devoid of keratan sulfate. In contrast, lumican is not present in articular cartilage of the fetus or young juvenile ; in the adult, it is present in predominantly a glycoprotein form. It is unclear whether the presence or absence of keratan sulfate influences the function of these proteoglycans in cartilage. All SLRPs have all been shown to interact with the fibrillar collagens of the extracellular matrix, though their site and strength of interaction may vary. The importance of these molecules in matrix organization is illustrated by the abnormalities associated with SLRP-null mice [18–21], though these abnormalities are perhaps less severe than might have been expected and it is possible that there is a functional redundancy between some family members. Unlike aggrecan, the SLRPs of the cartilage matrix appear relatively resistant to extensive proteolytic modification and do not show a ready sensitivity towards cytokine-induced damage . Fragments have, however, been observed in the matrix of arthritic cartilage.
The cartilage matrix also contains the proteoglycan perlecan. This is somewhat surprising, because perlecan is commonly thought of as a basement membrane proteoglycan , yet articular cartilage is devoid of basement membranes. Basement membrane perlecan is characterized by the presence of heparan sulfate chains in its amino-terminal region, though it has been reported that cartilage perlecan may exist in a nonglycanated form . The perlecan core protein is extremely large and might be expected to be a good candidate for proteolytic processing, but at present there is no information available on structural changes with either age or arthritis. The importance of perlecan to cartilage function is demonstrated by the perlecan-null mouse , in which severe chondrodysplasia is a major part of the phenotype in addition to basement membrane defects affecting heart and brain development. In the human, mutations in the perlecan gene have been associated with Schwartz-Jampel syndrome (chondrodystrophic myotonia) , and have recently been reported in dyssegmental dysplasia. At present, the function of perlecan in cartilage, and in particular in the growth plates, is unknown.
A final proteoglycan associated with cartilage has been termed superficial zone protein . It is synthesized by the superficial chondrocytes of articular cartilage and by synoviocytes, and has an attachment site for a CS chain. It is identical to the precursor protein of megakaryocyte-stimulating factor, and probably is the same as a protein originally described as lubricin, which is responsible for the lubrication and frictionless motion of the cartilage surface. While some superficial zone protein may be retained in the extracellular matrix, most is destined for secretion into the synovial cavity. The synthesis of this protein is impaired in the arthritic joint, where alternative splicing has been reported, and production is downregulated by the presence of inflammatory cytokines such as IL-1. Gene defects in this protein have been associated with camptodactyly-arthropathy-coxa vara-pericarditis syndrome . In addition to its role as a lubricant, the protein may play a role in regulating synovial cell proliferation, as this syndrome and various forms of arthritis are associated with synovial hyperplasia. In the case of camp-todactyly-arthropathy-coxa vara-pericarditis syndrome, hyperplasia occurs in the absence of inflammation.