Aggrecan monomer

Aggrecan monomers contain two extended regions which carry the bulk of the glycosaminoglycan and three globular domains, G1 and G2 at the N-terminus and G3 at the C-terminus of the core protein. The C-terminal G3 domain comprises an alternatively spliced complement regulatory protein-like repeat at the extreme C-terminus, an adjacent repeat homologous with C-type animal lectins and an N-terminal epidermal growth factor (EGF)-like domain that is also subject to alternative splicing.

Role in the tissue
One distinct property of aggrecan is its extreme content of negatively charged polysaccharide chains. This contributes in excess of 10,000 negative charges to aggrecan and a couple of orders of magnitude more to the aggregate creating an osmotic environment that is responsible for the extremely high osmotic swelling pressure of cartilage. This swelling pressure is counteracted by the resistance of the intact collagen fibres giving cartilage its characteristic properties of being able to resist compressive forces and having a high tensile strength.

The G1 domain (HABr) mediates specific (requires a decasaccharide sequence) interactions with hyaluronan, stabilized by concomitant binding to the link protein, also binding with tight (K_D 10^-8) specificity and affinity to HA. The G2 domain is homologous, but with no known function. The ensuing keratan sulphate rich-domain binds with a K_D 10nM affinity to collagen, to a site in the gap region [Hedlund et al]. Each chondroitin sulfate glycosaminoglycan chain (GAG) is made up of 40-50 repeating disaccharide units of glucuronate and N-acetylglucosamine in the basic structure carrying a sulfate at carbon 6 or 4. In most tissues each chain contains variable stretches of one or the other sulfate variant. Linkage to the protein occurs via a specific Ser-Xylose-Galactose-Galactose-Glucuronate sequence. The other type of GAG chains on aggrecan, keratan sulfate, contains shorter repeats of galactose and N-acetyl-glucosamine with a sulfate at carbon-6. Stretches where both sugars are sulphated occur and these can be recognised by the monoclonal antibody 5D4.

The C-type lectin domain has been shown  [Aspberg et al.].to bind with high affinity to Fibulin-1 and Fibulin-2 [Aspberg et al.] and fibrillin [XX et al.]. The binding site has been mapped to the central stretch of calcium-binding EGF-like repeats [Aspberg et al.].

Molecular Markers
A prominent feature in joint disease (such as rheumatoid arthritis and osteoarthritis) is a loss of aggrecan. Two major cleavage sites are present in the IGD-domain separating the G1 and G2 domains. Cleavage occurs at DIPEN-FFG or NITEGE-ARG (aggrecanase). The enzymes (ADAM TS 3 and 4) also induce cleavage at other sites. By far the major portion of aggrecan released appears to be cleaved by aggrecanases, primarily ADAMTS-4 and 5, (which also acts during normal turnover).

The fragments of aggrecan and several of its epitopes that are released in increased amounts in pathological conditions enable measurements to be made in synovial fluid [Heinegård and Saxne 1991]. The highest levels of the CS region of the core protein are found in the synovial fluid of patients with reactive arthritis.[Saxne et al., 1993. HABr (or G1) is also released in small amounts in this condition. In rheumatoid arthritis (RA) patients, an inverse relation exists between HABr and CS epitope release, with a dominance of aggrecan release in early RA and HABr in late destructive disease [Saxne and Heinegard 1992].

Serum concentrations of cartilage (and bone) macromolecules may also be useful in delineating differences between different stages of RA and between patients with rapid and slow progressive disease. Serum levels of the 846 epitope (recognizes CS epitopes in the G3 domain) are increased in patients with slowly progressive disease [Månsson et al., 1995]. Fragments of aggrecan bearing the 846 epitope reflect degradation of newly synthesized aggrecan and in the case of RA patients is indicative of the tissue attempting a repair mechanism. However, analyses of GAG-containing fragments of aggrecan in serum should be done with extreme caution, since most of the fragments are cleared in the lymph nodes (Frazer, Saxne, Heinegård, unpublished) and do not reach the circulation.

Genetic Defects
The lethal chicken mutation (mutation at aa1523 in the CS2 domain - Li et al., 1993) nanomelia shows a severely defective skeletal phenotype in which the extracelullular aggrecan is deficient. Nanomelic chondrocytes produce truncated aggrecan molecules that lack the G3 domain and are not secreted from the cell [O'Donnell et al., 1988]. The truncated aggrecan undergoes xylosylation and GAG chain elongation but is not translocated from the Golgi to the ER [Vertel et al., 1994].

A similar phenotype is seen in the cmd (cartilage matrix deficiency) mouse, which expresses normal levels of the cartilage-specific collagen type II but fails to express aggrecan [Kimata et al., 1981]. The mutation is due to a 7bp deletion in the G1 domain causing a premature stop codon [Watanabe et a .,1994]. A recently described mutation in the human causes a truncation of the aggrecan molecule (Wallis paper XXXX). The affected individuals show a very short stature but with maintained features. This would indicate that an important role of aggrecan in the growth cartilage is to expand the matrix, an effect dependant on the negatively charged chondroitin sulfate side chains contributing an extreme fixed charge density to the matrix.

In the brachymorphic mouse defective sulphation of aggrecan results in mice with a shortened, irregular growth plate. These mice present a phenotype that is characterized by shortened limbs and a domed skull [Orkin et al., 1977]. The defect appears to lie in the channeling mechanism of APS between the ATP sulphurylase and APS kinase in the sulphation of the GAG chain, thus resulting in a decrease in the efficiency of the sulphate donor, PAPS [Schwartz et al., 1998].