**8. Modifications to Aggrecan Side Chain Structure Modifies Its Functional Properties in Tissues**

In adult articular cartilage, aggrecan contains ~100 CS and ~25–30 KS chains, which collectively represent ~90% of the mass of this PG [4]. CS is the predominant GAG in aggrecan and is localised on the C-terminal half of the core protein in so-called CS1 and CS2 domains (Figure 3). KS is also present in a KS rich region between the N-terminal globular domains and the CS rich region. These are *O*-linked through Serine residues to the aggrecan core-protein and have been classified as KS-II chains [2,107]. Complete sequencing of the murine core protein [108,109] shows that it does not contain the consensus sequences for attachment of KS as found in human aggrecan core protein (E-(E,K)-P-F-P-S or E-E-P-(S,F)-P-S) [8,110,111]. Humans and bovine aggrecans contain a 4–23 hexapeptide repeat segment where KS is attached, while rats and other rodents lack this region [110,111]. Rodent aggrecan is truncated in the KS rich region thus does not contain a KS rich region such as that found in human or bovine aggrecan. Rodent aggrecan does however contain small *N*- and *O*- linked KS chains in the G1, G2 and interglobular domain (IGD); IGD KS chains have been proposed to potentiate aggrecanolysis by ADAMTS4 and ADAMTS5 [112]. The lack of a KS rich region in mouse aggrecan does not appear to be detrimental to its normal properties in mouse articular cartilage.

While much still needs to be learnt of the specific roles played by KS in aggrecan, much has already been uncovered about the interactive properties of this GAG in a number of physiological processes in the last decade. Corneal KS-I is interactive with a number of cell stimulatory molecules [113] such as insulin-like growth factor binding protein-2 (IGFBP2) [114], SHH, FGF1 and FGF2 [115–118]. A proteomics and microarray screen of 8268 proteins and secondary screen of 85 extracellular nerve growth factor epitopes using surface plasmon resonance, micro-array and microsequencing has shown that KS-I interacted with 217 proteins including 75 kinases, membrane and secreted proteins, cytoskeletal proteins, nerve regulatory proteins and nerve receptor proteins [113]. In comparison, chondroitin-4-sulphate interacted with 24 proteins including 10 kinases and 2 cell surface proteins in the same microarrays. Confirmation of these interactions by surface plasmon resonance allowed binding constants to be calculated and the validity of these putative interactions to be determined. Of 85 ECM nerve-related epitopes, KS-I bound 40 proteins, including Slit, two Robos, nine Eph receptors,

eight Ephrins, eight Semaphorins and two nerve growth factor receptors. It has yet to be ascertained however if the KS-II chains of aggrecan have similar interactive properties as KS-I.

Antibodies which detect low sulphation KS motifs have now been developed (reviewed in [119]) and have demonstrated KS in a number of tissues previously thought to be KS deficient after labelling with mAbs such as 5-D-4, which is specific for highly sulphated KS epitopes [120]. Roles are emerging for low sulphation KS-epitopes in electro-sensory processes [69,115,121]. Neural tissues are the second richest source of KS in the human body after the cornea [69,115].

While aggrecan has important interactions with growth factors and morphogens which direct chondrocyte proliferation and differentiation in cartilage development and maturational processes essential in endochondral ossification and skeletogenesis, it also has important functional roles to play in weight-bearing and in the stabilisation of the cartilage ECM. Aggrecan, as its name indicates, forms massive mega Dalton aggregate ternary complexes via interaction of its N-terminal HA binding G1 domain with hyaluronan (HA) stabilised by cartilage link protein which shares homology with the G1 domain and also has HA binding properties [2,4,107]. The G3 domain of aggrecan also interacts with tenascin-C via its fibronectin type III repeats, which have lectin binding activity, and these interact with the C-type lectin motifs on the aggrecan G3 domain [19,122–124]. Tenascin-C, R, Fibulin-1 and fibulin-2 also bind to the cartilage aggrecan G3 domain through interactions with its C-type lectin and EGF domains of G3 [15]. The C-type lectin of the aggrecan G3 domain also interacts with cells and activates the Complement system [124]. Complement is a defence system against foreign pathogens and aids in the removal of dying cells, immune-complexes, misfolded proteins and invading microbes [125]. Excessive complement activation can exacerbate autoimmune disorders and pathological inflammatory conditions such as rheumatoid arthritis (RA) [126]. Complexes of matrilin-1 and -3 and biglycan or decorin also connect collagen VI microfibrils to collagen II and aggrecan [127], forming a link between the PG and fibrillar collagenous networks in cartilage and IVD [1,3,5]. Cartilage oligomeric protein (COMP and TSP-5) also binds to aggrecan, providing an extended co-operative network in cartilage [128], which helps to distribute loading stresses throughout this tissue avoiding the point loading which can be damaging to ECM components [3]. This extended collagen–aggrecan network also provides a mechanosensory biosensor system extending far from the cell through the interstitial and inter-territorial matrix, which allows the chondrocyte to perceive and respond to perturbations not only in its local mechanical microenvironment but also to more remote cartilage regions to regulate tissue homeostasis and optimal tissue functional properties [1,3].

The essential role of aggrecan to cartilage function is well illustrated in a naturally occurring Cmd (cartilage matrix deficient) mutant mouse [129], which has a single 7 bp deletion in exon 5 of the aggrecan gene which encodes the B loop of the G1 domain of aggrecan [130]. Homozygote (cmd/cmd) mice display dwarf-like features, spinal deformity, chondrodysplasia, abnormal collagen fibrillogenesis, a cleft palate [130], deafness [131] and die shortly after birth due to respiratory failure [132]. The articular cartilage of the Cmd−/Cmd<sup>−</sup> mouse displays tightly packed chondrocytes surrounded by little matrix; growth plate cartilage contains chondrocytes arranged in disorganised columns of diminished length in severely diminished proliferative, pre-hypertrophic zones consistent with the reduced proportions of these mice [132] (Figure 4a). Cultured nanomelic chick chondrocytes synthesise a truncated aggrecan core protein precursor [133] due to a premature stop codon, and this is not translocated to the Golgi apparatus for processing, which leads to an absence of aggrecan in nanomelic cartilage, chondrodysplasia, disrupted organisation of the hyaline and growth plate cartilages, severely diminishing skeletal stature [134–137] (Figure 4b).

While the role of the KS chains in the G1 and G2 domains of aggrecan is largely unknown, some G1 KS chains have been found to sterically obscure an N-terminal T cell attachment site in aggrecan and have a protective effect over autoinflammatory conditions arising from fragmentation of aggrecan (Figure 5a,b). Further T cell interactive sites in the G3 domain of aggrecan have also been identified which may contribute to auto-inflammatory arthritic conditions [138–140]. These G1 KS chains suppress a T cell mediated response initiated by free G1 when it is used as an arthritogen in

models of inflammatory arthritis [138–142]. KS chains in the IGD also potentiate aggrecanase activity in this region of the core protein [143]. A few KS chains are also interspersed within the CS rich region. KS-II chains in aggrecan from weight-bearing tissues such as articular cartilage and IVD contain 1-3 fucose and 2-6 *N*-acetyl-neuraminic acid residues [119]; however, these modifications in KS are absent in aggrecan from non-weight-bearing nasal and tracheal cartilage [144]. The significance of these KS modifications and why they only occur in aggrecan from weight-bearing tissues is unknown; antibody 3D12/H7 identifies these KS chains embedded in the CS rich region [145] but they do not share immunological identify with KS chains in the KS rich region. This KS epitope contains three sulphate groups and two fucose residues on GlcNAc residues in a branched fucosylated sialo-KS structure of unknown function.

**Figure 4.** Demonstration of the modulation of growth plate cartilage morphogenesis by aggrecan in wild type (WT) (**A**) (**a**–**h**) and nanomelic E7-E12 chick tibia (**B**) (**a**–**r**). The ISH images presented demonstrate the expression of: FGFR3 (**a**); type X collagen (**b**); and Indian Hedgehog (IHH) (**c**) in WT (**a**–**c**); and nanomelic growth plate (**d**–**f**) in E12 (**a**–**f**); E7 (**g**–**l**); and E8 chick tibia (**m**–**r**). Images modified from [134] with permission using open access.

**Figure 5.** Structural organisation of aggrecan depicting the CS, KS and HS GAG chain distributions (**a**) and T cell receptor epitopes on the G1 and G3 globular domains (**b**). The aggrecanopathies showing regions of aggrecan affected by these mutations and the diseases that result (**c**).

Several mutations in the aggrecan gene have been documented, which affect variable regions in the aggrecan core protein leading to a number of conditions collectively termed the aggrecanopathies (Figure 5c) [146,147]. The aggrecanopathies are a spectrum of non-lethal skeletal dysplasias including spondyloepimetaphyseal (SEMD) and spondyloepiphyseal dysplasia (SED), osteochondritis dissecans (OCD) and a number of accelerated bone maturation disorders that result in short stature and idiopathic short stature (ISS) [146,148]. Skeletal abnormalities are also prominent features of animal models which display deficient levels of cartilage aggrecan such as the Cmd mouse [129,130] or nanomelic chick [137,149]. Brachymorphism [150] also results in reduced PAPS levels, and the aggrecan synthesised is deficiently sulphated and functionally impaired, resulting in abnormal skull development and short squat skeletal frames [151–153]. Manipulation of the diastrophic dysplasia sulphate-transporter gene (*DDST*) also results in the synthesis of aggrecan with deficient sulphation levels and a variety of skeletal abnormalities such as short stature and joint dysplasia in diastrophic dysplasia [151], micromelia in atelosteogenesis Type II [152] and short skeletal proportions due to aberrant trunk and extremity development in achondrogenesis Type II. Heterozygous ACAN mutations result in a phenotypic spectrum of skeletal abnormalities including short stature, accelerated bone maturation, early growth cessation, poor responsiveness to human growth hormone, brachydactyly, early-onset OA and susceptibility to the development of degenerative disc disease due to dysfunctional articular cartilage and IVD tissues [147,154–156]. Osteochondritis dissecans (OCD) is a disabling condition characterised by abnormal deposition of aggrecan in cartilage and the appearance of cracks in the cartilage and subchondral bone. This condition effects juveniles and adults but its aetiology is unknown. Trauma has been suggested as a predisposing factor in juveniles and recent genomic wide studies have identified a cluster of genes associated with this condition suggesting that it may also have a genetic basis [157–159]. Several skeletal dysplasias have been shown to be due to a constitutively activated mutation in a transient receptor potential vanilloid 4 (TRPV4) cation channel protein [160–164]. This results in abnormal cation mediated cell signalling by chondrocytes and altered regulation by BMP2 and TGFβ1 activity [164].

Aggrecan is required for correct growth plate cytoarchitecture and differentiation, endochondral ossification and skeletogenesis [165]. The CS side chains of aggrecan make important contributions to this process and their sulphation status is an important functional determinant [60]. Six CS *N*-acetylgalactosaminyltransferases (t1–t6) have been described. Initial stages of CS sulphation is undertaken by t1 and t2; t1 and t2 double knockout mice display shortened growth plates, distorted hypertrophic growth plate regions, reduced growth plate chondrocyte proliferation, type X expression, dwarfism, disruption in the postnatal formation of the secondary ossification centres and chondrodysplasia; this is lethal postnatally [166] (Figure 8). Aggrecan aggregates are also formed in the CNS and PNS stabilised by interaction with tenascin-C and tenascin-R, HA and a brain specific link protein variant Bral-1 (HAPLN2) to form perineuronal nets (PNNs), which are structures assembled around neurons (Figure 7) that scavenge oxygen free radicals in neural tissues thus preventing oxidative stress [19,20,93,167–169]. Brain tissue is fatty acid rich and prone to oxidative damage, which produces reactive species that detrimentally affect mitochondrial activity in neurons. Brain tissue is metabolically demanding and requires optimal mitochondrial activity to ensure energy production to power neuronal signalling.

**Figure 6.** Chondroitin sulphate *N*-acetylgalactosaminyltransferase-1 and -2 (T1/2) knockout induces

dwarfism in mice and altered cartilage structural organisation of the femoral condyle, its ossification centre and growth plate in wild type mice (**a**,**c**,**e**) and T1/2 knockout mice (**b**,**d**,**f**). The boxed area in (**c**,**d**) is depicted at higher magnification in (**e**,**f**). Safarin O-Fast green stain depicting aggrecan GAG distribution. Arrows depict normal ossification centre in (**c**) and abnormal structural organisation in T1/2 knockout mice in (**d**,**f**). Figure modified from [166]. Figure reproduced under the terms of the Creative Commons Attribution Licence Copyright: 2017 Shimbo et al. [166].

**Figure 7.** Visualisation of: perineuronal net structures (**a**,**b**); vascular features (**c**); and ganglion cells (**d**,**e**) in cerebellum and dorsal root ganglia using MAb 1B5 in confocal images. Immunolocalisation of CS Isomer 1B5 in paraformaldehyde fixed 20-µm cryo-sections of 24-month-old Wistar rat brain and lumbar dorsal root ganglia. Confocal z-stacked images of IB5 CS stub epitope generated by chondroitinase ABC digestion using Alexa 488 secondary antibody for detection and propidium iodide nuclear counterstain, mounted under coverslips using Vectasheld mountant. Images courtesy of Prof B. Caterson, University of Cardiff. Copyright Caterson, Hayes 2012 (**a**–**f**). Immunolocalisation of perineuronal nets surrounding isolated neurons in murine visual cortex using: antibody to parvalbumin (**g**,**i**); Wisteria floribunda lectin (**h**); and in a merged image (**j**). A schematic model of the perineuronal net structure in the boxed area in (**k**) showing its constituent lectican proteoglycans (aggrecan, versican, neurocan and brevican) interacting with tenascin hexabrachion and hyaluronan to form an aggregate structure stabilised by link protein. A key is provided to explain items in (**k**,**l**). Figure modified from [170] under Open Access under the auspices of a Creative Commons Attribution License.
