**2. CS Sulphation on Aggrecan Is an Important Functional Determinant**

Studies have shown that two out of every seven non-reducing termini of normal [23] and chondrosarcoma [24] aggrecan CS chains contain 4, 6-disulphated GalNAc. Non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide. In a further study, CS from juvenile and adolescent growth plate cartilage was shown to contain non-reducing terminal GalNAc4S, whereas in adult cartilages approximately half of the non-reducing termini were disulphated GalNAc4,6S [25], representing an increase in aggrecan sulphation with tissue maturation. It is these sulphate groups which provide aggrecan's interactive properties with a number of ligands; a high density of sulphate and carboxylate groups in aggrecan confer its remarkable ability to imbibe water and to provide tissue hydration that allows some tissues to withstand compressive loading (Figure 1a,f).

While clear functional roles for HS-PGs in cell signalling transduction pathways are well established, roles for CS-PGs in such processes have often been given lower importance; nevertheless, CS can also modulate cell-signalling pathways involving hedgehog proteins, wingless-related proteins and fibroblast growth factors [26–35]. Indeed, the co-distribution of these components with aggrecan in growth plate cartilages and localisation of particular CS sulphation motifs with chondroprogenitor cell populations associated with diarthrodial joint development (Figure 2a,f) alludes to multiple interactive possibilities [36]. Studies with brachymorphic mice, nanomelic chick, dyschondroplastic chicken and Cmd mutant mice clearly show the importance of aggrecan in growth plate cartilage development and skeletogenesis. Furthermore, individuals suffering from Kashin–Beck disease, an endemic osteochondropathy that occurs in certain parts of China, is characterised by small stature and deformities of the limbs and digits, distorted growth plates, chondrocyte apoptosis and low levels of aggrecan [37–41]. The correct sulphation of the CS chains of aggrecan is essential to generate functional determinants capable of interacting with growth factors and morphogens [42–45]. Approximately one in three of aggrecan's CS chains have a non-reducing terminal chondroitin-4, 6 disulphated residue (CS-E) in articular cartilage [46]. Highly sulphated CS-E binds the HS binding growth factors midkine and pleiotrophin [47–49].

Approximately two in seven CS chains are terminated in 4, 6 disulphated GalNAc, which varies with the age and cartilage type; four in seven of CS chains are terminated by 4-sulphated GalNAc; and one in seven CS chains are terminated in a GlcUA linked to 4-sulphated GalNAc. Non-reducing terminal 4,6-disulphated GalNAc residues are 60-fold more abundant than 4,6-disulphated GalNAc in interior regions of the CS chain [24].

CS chains terminated in 4-sulphated GalNAc predominate in aggrecan from foetal to 15-year-old knee cartilage, whereas, in 22–72-year-olds, 50% of the CS chains were terminated in 4,6-disulphated GalNAc. GlcUA-4-sulphated GalNAc disaccharides terminated 7% of CS chains in foetal to 15-year-old cartilage but fell to 3% in adults, whereas GlcUA-6-sulphated GalNAc represented 9% of the CS chains in foetal to 72-year-old cartilage. This disaccharide is recognised by MAb 3-B-3 (−) [46].

**Figure 2.** Immunofluorescent localisation of the 3-B-3(−) CS sulphation motifs on aggrecan associated with rudiment cartilage of a rat intervertebral disc (**a**) and demonstration of the generation of the 3-B-3(−) epitope by digestion of aggrecan and serglycin as model proteoglycans with hyaluronidase-4 (HYAL4) (**b**). Immunolocalisation of the 3-B-3(−) and 7-D-4 CS sulphation motifs in developmental human foetal knee joint cartilage (14 weeks gestational age) (**c**,**d**). The inset of (**e**) shows foetal aggrecan samples separated by native composite agarose polyacrylamide gel electrophoresis and blotted to nitrocellulose for detection of the 3-B-3(−) and 7-D-4 proteoglycan populations. Two aggrecan populations are discernible. The 3-B-3 (−) CS sulphation epitope has a widespread distribution in the developing rudiment cartilage, whereas the 7-D-4 epitope has a more discrete distribution pattern in small stem cell niches in the cartilage surface. A schematic depicting a typical CS chain and digestion products generated by endoglycolytic cleavage by HYAL4 generating the 3-B-3(−) non-reducing terminal on the cleaved CS chain (**f**). Exhaustive digestion of CS by chondroitinase (Ch'ase) ABC also depolymerises the CS chain but generates a 3-B-3(+) stub epitope attached to the CS linkage attachment to aggrecan core protein. Inset image (**e**) modified from [50]. (**a**,**c**,**d**) Images supplied courtesy of Prof B. Caterson, University of Cardiff, UK. As already shown in this manuscript approximately ~1–2 in every seven nonreducing termini of CS chains in cartilage are terminated in the 3-B-3(−) epitope and these vary with age and cartilage type. The 3-B-3(−) epitope is a marker of tissue morphogenesis [36,51,52]. Stem cells are surrounded in proteoglycans decorated with this CS motif [8–10]. This motif is also released into synovial fluid in degenerative conditions such as OA [53–56]. Recently, Farrugia et al. [57] showed that mast cells synthesised HYAL4, a CS hydrolase that could generate the 3-B-3(−) motif in the CS chains of aggrecan and Serglycin in vitro.

The distribution of 4- and 6-sulphated CS epitopes is variable along a CS chain in aggrecan and is influenced by the maturational status of the cartilage or the extent to which the cartilage was sampled from a high or low weight-bearing cartilage region [3]. Certain trends have been observed in the sulphation patterns of CS in aggrecan chains. C-4-S is more predominant in aggrecan from foetal and young articular cartilage and occupies a central region in the CS chain, whereas non-sulphated chondroitin is more predominant towards the linkage region. C-6-S has a predominant distribution towards the non-reducing terminus and is more abundant in mature cartilage to the detriment of C-4-S sulphation [51].

Graded partial digestions of CS chains with chondroitinase ABC or ACII reveals regions along the CS chain where MAbs 6C3, 4C3 and 7D4 are most immunoreactive [51]. MAb 6C3 reacts optimally with regions of CS chains towards the non-reducing terminus where C-6-S predominates, and this reactivity is removed during early stages of chondroitinase digestion. Further digestion removes MAb 4C3 reactivity and continued digestion then removes reactivity to MAb 7-D-4. While the specific epitopes identified by MAb 4C3 and 7D4 are yet to be identified, reactivity of these antibodies in a range of tissues undergoing morphogenetic transition during development displays subtly different immunolocalisation patterns and are of functional significance [52,58–65].

MAb 3-B-3 identifies a non-reducing terminal disaccharide in CS consisting of GlcUA-GalNAc-6-sulphate, which is termed a 3-B-3(−) epitope to distinguish it from the 3-B-3(+) stub epitope disaccharide attached to the linkage region that is generated by exhaustive end-point digestion of CS chains by chondroitinase ABC [64]. As noted above, this non-reducing terminal 3-B-3(−) epitope occurs in approximately two in every seven CS chains; disulphated C-4,6-S and C-6-S GalNAc also occur as components in this non-reducing terminal disaccharide in CS chains [24,46].
