*13.1. Analysis of Cartilage Aggrecan and Its GAG Side Chains*

As already shown in this review, aggrecan is a large specialised protein which provides weight-bearing or space-filling properties to cartilaginous tissues through its large solvation volume and ability to imbibe water. Cartilage aggrecan has a core protein of ~250 kDa and contains ~100 CS and 25–30 KS side chains, which collectively represent ~90% of its mass. As also shown in this review, aggrecan forms also exist in specialised tissue niches and in developmental contexts which do not contain KS or have some CS chains replaced by the HNK-1 trisaccharide which result in changes in aggrecan's interactive properties. Murine aggrecan contains a truncated core protein and is devoid of a KS rich region; however, this does not impede its functional properties in murine cartilage or the normal turn-over of this proteoglycan. Thus, the functional importance of KS in human aggrecan is unknown at present and the need for two GAG types in aggrecan is a question which has yet to be answered. While corneal KS-I has interactive properties with a range of neuron associated proteins [113,119] such as SHH, FGF-1 and FGF-2 [117], it is not known if the KS-II chains of aggrecan share this property. KS-II differs from KS-I in its capping modifications with l-fucose and *N*-acetyl neuraminic acid [119], which render KS-II resistant to total depolymerisation by keratanase-I and II and endo-β-d-galactosidase apparently through steric constraints which prevent access of these KS depolymerising enzymes to KS substrate, KS-I is totally depolymerised under the same digestion conditions thus significant differences exist between KS-I and KS-II [227]. This could also sterically

impede potential interactions of KS-II with other ligands. Furthermore, cartilage aggrecan also contains a few KS chains interspersed within its CS-1 and CS-2 rich regions. An antibody to these KS chains, (MAb 3D12/H7) identifies trisulphated fucosylated and poly-*N*-acetyllactosamine modifications in the KS linkage regions in these KS chains to aggrecan core protein [145]. These 3D12/H7 positive KS chains do not share immunological identity with the KS-II chains of the KS rich region of aggrecan however their functional properties still have to be ascertained.

To understand the properties of the aggrecan side chain GAGs and how these may contribute to the properties of aggrecan, methods have been developed to isolate aggrecan and its GAG side chains from cartilaginous tissues.
