proteoglycans of the intervertebral disc.pdf

8/18/2019 Proteoglycans of the Intervertebral Disc.pdf

1/25

53I.M. Shapiro, M.V. Risbud (eds.), The Intervertebral Disc ,DOI 10.1007/978-3-7091-1535-0_4, © Springer-Verlag Wien 2014

4Proteoglycans of theIntervertebral DiscJames Melrose and Peter Roughley

4.1 Introduction

Proteoglycans are present within the extracellular matrix(ECM) of the intervertebral disc and on the surface of itscells. The disc possesses many matrix proteoglycans, withmost also being present in hyaline cartilages. The best studiedof these are aggrecan and versican (members of the hyalec-tan/lectican family) and decorin, biglycan, bromodulin,lumican, PRELP, and chondroadherin (members of the smallleucine-rich repeat protein/SLRP family). More recently, thedisc has also been shown to contain perlecan and lubricin,which were previously thought to be characteristic of base-ment membranes and the surface of articular cartilage, respec-tively. Much less is known about the disc cell-associatedproteoglycans, though it is likely that several members of thesyndecan (Tkachenko et al. 2005 ) and glypican (Franssonet al. 2004 ) families will be present, together with other unre-lated proteoglycans such as NG2 (Akeda et al. 2007 ; Stallcup2002 ). While little is known concerning the speci c functionof the cell-associated proteoglycans within the disc, it hasbeen shown that syndecan-4 expression is increased whenelevated levels of interleukin-1 (IL-1) or tumor necrosis fac-tor- a (TNF- a ) are present (Wang et al. 2011 ), and that it mayplay a role in promoting aggrecanase-mediated proteolysiswithin the disc. However, due to the scarcity of informationon disc cell-associated proteoglycans, this chapter will focuson the matrix proteoglycans, in particular aggrecan.

4.2 Glycosaminoglycan Structureand Function

Proteoglycans can be considered as specialized glycoproteinsand are a ubiquitous component of all tissues. They are distin-guished from other glycoproteins by the substitution of theircore protein with sulfated glycosaminoglycan (GAG) chains( Box 4.1 ), though they may also possess more typical O -linkedand N-linked oligosaccharides (Nilsson et al. 1982 ). The sul-fated GAGs can be divided into three families – chondroitin

J. Melrose ( * )Kolling Institute (B6), Level 10 ,Royal NorthShore Hospital , St. Leonards , New South Wales 2065 , Australiae-mail: [email protected]

P. RoughleyGenetics Unit , Shriners Hospital for Children , 1529 Cedar Avenue , Montreal , QC , Canada H3G 1A6e-mail: [email protected]

Contents4.1 Introduction ........................................................... .......... 53

4.2 Glycosaminoglycan Structure and Function ................ 53

4.3 Aggrecan ...................................................... .................... 55

4.3.1 Aggrecan Protein Structure and Function ......................... 554.3.2 Aggrecan Gene Organization, Expression,and Mutations .......................................................... .......... 57

4.3.3 Aggrecan Degradation in the Degenerate Disc ................. 59

4.4 Versican ........................................................ .................... 594.4.1 Versican Protein Structure and Function ........................... 604.4.2 Versican Gene Organization and Mutation ....................... 604.4.3 Versican Degradation in the Degenerate Disc ................... 61

4.5 Perlecan ........................................................ .................... 614.5.1 Perlecan Protein Structure ................................................. 634.5.2 Perlecan Gene Organization and Mutation ....................... 644.5.3 Perlecan Degradation ........................................................ 64

4.6 Lubricin ............................................................................ 64

4.6.1 Lubricin Protein Structure ................................................. 654.6.2 Lubricin Gene Organization and Mutation ....................... 66

4.7 The Small Leucine-Rich RepeatProteoglycans (SLRPS) ................................................... 67

4.7.1 Decorin .......................................................... .................... 684.7.2 Biglycan ........................................................ .................... 694.7.3 Asporin .......................................................... .................... 694.7.4 Fibromodulin ........................................................... .......... 694.7.5 Lumican ........................................................ .................... 704.7.6 PRELP ........................................................... .................... 704.7.7 Chondroadherin ....................................................... .......... 704.7.8 SLRP Knockout Mice and Gene Mutations ...................... 70

4.8 Summary of Critical Concepts Discussedin the Chapter ........................................................ .......... 71

References ........................................................ .............................. 72


2/25

54 J. Melrose and P. Roughley

sulfate/dermatan sulfate (CS/DS), keratan sulfate (KS), andheparan sulfate/heparin (HS/Hep) (Jackson et al. 1991 ).While many proteoglycans possess GAGs from only onefamily, some proteoglycans possess GAGs from differentfamilies.

CS is a copolymer of glucuronic acid and N -acetylgalactosamine, with the latter commonly beingsulfated at the 4 or 6 position. DS is initially synthesizedas CS, but during processing in the Golgi, some of theglucuronic acid is epimerized to iduronic acid, which maybe sulfated at the 2 position. KS is a copolymer of galac-tose and N -acetylglucosamine and may be sulfated at the 6position on either residue. HS is a copolymer of glucuronicacid or iduronic acid and N -acetylglucosamine, in whichthe iduronic acid may be sulfated at its 2 position and the

N -acetylglucosamine may be sulfated at the 3 and 6 positions.On some glucosamine residues, N-sulfation may replace theN-acetyl group. In heparin, the presence of iduronic acid andO- and N-sulfation is high. As there is no template for GAGsynthesis, GAG chain length, degree and position of sulfa-tion, and degree of epimerization can vary enormously, bothbetween different proteoglycans and on the same proteogly-can at different sites.

GAG chains have traditionally been considered structuralelectrorepulsive entities of connective tissues. This is due totheir repeating charged disaccharide and sulfated sugarmotifs or as agents which provide a high xed charge densityin the tissues. As such due to GAG-associated counterionsand the Donnan equilibrium effect, they are responsible forthe water-regain properties of tissues. With the emergingconcept of the sugar code, with the realization that dynamicstructural changes in HS produce a characteristic (nonran-dom) heparanome, these charged sugars may also be involvedin information storage and transfer (Cummings 2009 ). Thebiological importance of chondroitin sulfation during mam-malian development and growth factor signaling is poorlyunderstood (Caterson 2012 ; Caterson et al. 1990 ), althoughchondroitin 4- O -sulfation is required for proper CS localiza-tion and modulation of distinct signaling pathways duringgrowth plate morphogenesis (Kluppel et al. 2005 ). On thisbasis, chondroitin sulfation has emerging biological roles inmammalian development. The elucidation of the intimateinterplay of GAG chains with a variety of speci c bioactivebinding partners triggering cell signaling, cell proliferation,matrix production, and differentiation underscores the rangeof functionalities which may all be affected (Turnbull 2010 ).Accordingly, GAG chains are versatile tools for informationstorage and transfer and represent a new paradigm in devel-opmental biology.

Due to differences in either synthesis or degradation, eachproteoglycan does not possess a unique structure: both thecore protein and the GAG chains may vary with site, age,and pathology. Synthetic changes occurring within the cell

are most commonly associated with the GAG chains, butsplicing variations (Fulop et al. 1993 ) or the use of alterna-tive transcription start sites (Muragaki et al. 1990 ) may alsoinuence the structure of the core protein of some proteogly-cans. In contrast, degradative changes occurring within thematrix are most commonly associated with processing of thecore proteins by proteinases, although modi cation of GAGsby extracellular sulfatases or glycosidases has been reported(Vlodavsky et al. 1999 ). Irrespective of their origin, all typesof structural change have the potential to in uence proteo-glycan function.

Proteoglycans have been classi ed by the type of GAGchain that they possess or by their location within the tissue.In terms of location, the division is commonly between pro-teoglycans that reside in the ECM and those associated withthe cell. Matrix proteoglycans are often substituted with CS,DS, or KS, whereas cell-associated proteoglycans are oftenlinked with HS. Heparin is usually only de ned in relation-ship to the serglycin proteoglycan present within mast cells(Humphries and Stevens 1992 ) but can be structurally simi-lar to regions of highly sulfated and epimerized HS presenton other proteoglycans (Girardin et al. 2005 ). CS and HSmay replace one another at the same site on some proteogly-cans, as they share the same amino acid substitution motif(Ser-Gly) and linkage oligosaccharide (Xyl-Gal-Gal-GlcA).In contrast, KS has two different substitution motifs andlinkage oligosaccharides, which it shares with O -linked andN-linked oligosaccharides.

Box 4.1: A Historical Perspective of Glycosaminoglycansand ProteoglycansThe existence of glycosaminoglycans has been knownsince the 1860s when chondroitin sulfate was rstdescribed in cartilage. Discovery of the other gly-cosaminoglycans did not occur until the twentieth cen-tury, with many owing their discovery to the work ofKarl Meyer. Meyer described the existence ofhyaluronic acid in the vitreous humor of the eye in1934, dermatan sulfate in skin in 1941, and keratansulfate in the cornea in 1953. Heparin was rstdescribed in 1916 because of its anticoagulant activity,and the structurally related heparan sulfate in 1948.However, modern terminology was not in use by the1950s when the glycosaminoglycans were referred toas mucopolysaccharides which formed the groundsubstance of connective tissues. This name persiststoday with “the mucopolysaccharidoses,” a group ofheritable disorders due to gene defects in speci c lyso-somal glycosyltransferases, glycosidases, and sul-fatases responsible for glycosaminoglycan assemblyand catabolism. Similarly, the original terminology for


3/25

554 Proteoglycans of the Intervertebral Disc

4.3 Aggrecan

Aggrecan is a KS/CS proteoglycan that was originally iso-lated from hyaline cartilage and the gene was cloned fromchondrosarcoma cells (Doege et al. 1987 ). It was later shownto be present in the intervertebral disc and to be synthesizedby disc cells. On a weight basis, aggrecan is the most abun-dant proteoglycan in both the disc and cartilage and hasprobably been more extensively studied than any other pro-teoglycan. Aggrecan belongs to the family of hyaluronan(HA)-binding proteoglycans, together with versican, neuro-can, and brevican (Margolis and Margolis 1994 ). All familymembers possess an amino terminal globular domain respon-sible for interaction with HA and a carboxy terminal globu-lar domain containing a lectin homology domain. Thesecommon features give rise to the alternative family names ofhyalectins and lecticans. The interaction with HA permitsthe formation of proteoglycan aggregates (Morgelin et al.1988 ), and it was this ability to form proteoglycan aggre-gates that led to the name aggrecan.

Aggrecan provides the disc with its ability to resist com-pressive loading on the spine, causing the disc to swell andkeep the vertebrae apart. The acquisition of a biped verticalposture resulted in loading of the spine due to gravity. Partialremoval of this load at night and the imbibition of tissue uidresult in swelling of the disc, which accounts for the diurnalvariation in disc height (Botsford et al. 1994 ). The swelling

properties of aggrecan are related to its abundance, degree ofsulfation, and ability to form proteoglycan aggregates. Theswelling is driven principally by the sulfate groups on theGAGs, which attract water into the disc by osmosis. As morewater enters the disc, the osmotic properties of the aggrecandecrease, and an equilibrium is attained in which the swell-ing of the disc is counterbalanced by the tension induced inthe collagenous framework of the tissue. On subjecting thedisc to compressive load, water is displaced, effectivelyincreasing the aggrecan concentration and its swelling poten-tial. On removal of the load, the increased swelling potentialis dissipated by re-imbibition of water into the disc and res-toration of the equilibrium state. In addition to the symmet-ric loading due to gravity, the disc experiences asymmetriccompressive loading upon bending. Under asymmetric load-ing it is essential that the aggrecan cannot diffuse from thesite of compression if optimal restoration of disc height is tooccur following straightening. The diffusion of aggrecan isrelated to its size and is minimized by the formation of largeproteoglycan aggregates. This topic is discussed further inChap. 5.

Aggrecan is located throughout the disc, though its abun-dance at different sites varies greatly with age. In the fetalhuman spine, aggrecan is prominently immunolocalized inthe cartilaginous vertebral rudiment cartilages and in thedeveloping intervertebral disc space (Fig. 4.1 ) (Smith et al.2009 ). The fetal vertebral rudiment cartilages are transientdevelopmental scaffolds which are transformed into ossi edstructures in the adult spine, while the discs remain as per-manent cartilaginous entities. In the human, aggrecan pre-dominates in the nucleus pulposus in the young juvenile. Theaggrecan content of the nucleus pulposus increases during

juvenile growth and reaches a maximum in the late adoles-cent/early adult period. During adult life the aggrecan con-tent of the nucleus pulposus declines (Fig. 4.2a ). Aging isalso associated with an increase in aggrecan abundance inthe annulus brosus, particularly the inner annulus, and inthe mature adult the aggrecan content of the annulus brosusmay surpass that of the nucleus pulposus. Thus, in the matureadult, the annulus brosus is as important as the nucleus pul-posus for resisting compression.

4.3.1 Aggrecan Protein Structure and Function

The aggrecan core protein possesses about 2,300 aminoacids, which form three disul de-bonded globular regionswith two intervening extended regions (Fig. 4.3a ) (Sandyet al. 1990 ; Watanabe et al. 1998 ). The amino terminal glob-ular region (G1) responsible for interaction with HA pos-sesses three disul de-bonded loops. The rst loop allowsinteraction with a link protein (LP) that stabilizes the proteo-glycan aggregate (Neame and Barry 1993 ), and this is

some of the mucopolysaccharides was also different,with dermatan sulfate being referred to as chondroitinsulfate B and heparan sulfate as heparitin sulfate orheparin monosulfate. Once the structure of all themucopolysaccharides was established, and it wasappreciated that they were all copolymers of a sugar

and an amino sugar, the term glycosaminoglycan cameinto use. In addition, until the 1950s it was not appreci-ated that the sulfated glycosaminoglycans wereattached to protein, and considerable effort was madeto purify them from this “contamination.” It was in1958 that Helen Muir proved that chondroitin sulfatein cartilage was covalently attached to protein via aserine residue, and the proteoglycan era was born.However, initially the term protein polysaccharide wasused. In 1966, Lennart Roden described the structureof the trisaccharide which links the chondroitin sulfatewith its serine core protein attachment point. Thisattachment region is now known to be common to allthe sulfated glycosaminoglycans, with the exception ofkeratan sulfate, irrespective of the proteoglycan core towhich the glycosaminoglycan is attached.

http://dx.doi.org/10.1007/978-3-7091-1535-0_5http://dx.doi.org/10.1007/978-3-7091-1535-0_5


4/25


Aggrecan

Versican

Aggrecan

a d g

h

ifc

b e

Versican

Toluidine blue NP NP

IAFIAF

OAFOAF

500 µm

Fig. 4.1 Immunolocalization of aggrecan ( a ), versican ( b ), and toluid-ine blue-stained proteoglycan ( c ) in a 14-week gestational age fetalhuman intervertebral disc and adjacent cartilaginous vertebral bodyrudiment cartilages. Higher-power images of the outer annulus brosus

(OAF ), inner annulus brosus ( IAF ), and nucleus pulposus ( NP ) arealso presented in selected areas of the aggrecan ( d –f ) and versican ( g –i )immunolocalizations

followed by a pair of loops responsible for the interactionwith HA (Watanabe et al. 1997 ). The second globular region(G2) possesses two disul de-bonded loops that share struc-tural homology with the HA-binding loops of the G1 region.However, they do not facilitate interaction with HA (Fosangand Hardingham 1989 ), and their function is presentlyunclear. The G1 and G2 regions are separated by a shortinterglobular domain (IGD). After the G2 region, there is along extended region to which the majority of GAG chainsare attached. There may be over 100 GAG chains attached toeach core protein, accounting for about 90 % of the molecu-lar weight of aggrecan.

The GAG-attachment region may be divided into threedomains. The domain closest to the G2 region is responsiblefor the attachment of KS (KS domain), and this is followedby two domains responsible for the attachment of CS (CS1and CS2 domains). The KS and CS chains provide the aggre-can with osmotic properties essential for its function in

resisting disc compression. While the CS chains are con nedto the CS1 and CS2 domains, KS chains may also be presenton the G1, IGD, and G2 regions (Barry et al. 1995 ). The CS2domain is followed by the carboxy terminal globular domain(G3), which possesses disul de-bonded loops having homol-ogy to epidermal growth factor (EGF), C-type lectin, andcomplement regulatory protein (CRP) sequences. The G3region facilitates transit of the aggrecan through the cell dur-ing synthesis (Zheng et al. 1998 ) and via its lectin domainalso facilitates the interaction with other components of theextracellular matrix, such as bulins and tenascins (Dayet al. 2004 ). It is not clear if these G3 region interactions areof functional signi cance in vivo, but they could potentiallylink proteoglycan aggregates to one another.

Both the abundance and structure of aggrecan changewith age, due to variations in intracellular synthesis andextracellular degradation. Apart from possible variations ingene expression, the synthesis changes are con ned to


5/25


NP AF0

10

20

30

40

50Aggregated

Large non-aggregated

Small non-aggregated

24Age, years

24 years

50 870

20

40

60

80

100

a

b

NP

AF

A g g r e c a n c o n t e n t m g / g

t i s s u e

A g g r e c a n a g g r e g a t

i o n

%

Fig. 4.2 Variation in aggrecan content ( a ) and aggregation ( b ) in thehuman intervertebral disc. Aggrecan content declines with age through-out the disc but at a faster rate in the nucleus pulposus than the annulusbrosus. Aggrecan aggregation is lower and the proportion of smallaggrecan fragments is greater in the nucleus pulposus ( NP ) than theannulus brosus ( AF )

posttranslational modi cation of the core protein, particu-larly the synthesis of KS and CS (Brown et al. 1998 ; Roughleyand White 1980 ). With age, the chain length of KS increases,while that of CS decreases. This could be viewed as a com-pensation mechanism for maintaining the sulfation of aggre-can and its swelling properties. The sulfation position of CSalso changes with age, with the level of 4-sulfation decreas-ing and 6-sulfation increasing. It is not clear whether thisvariation in sulfation position has any functional signi cance.Currently, there is no evidence for the extracellular degrada-tion of CS or KS, and degradative changes in aggrecan arecon ned to the proteolytic cleavage of its core protein(Roughley et al. 2006 ). Each proteolytic cleavage generates

one fragment possessing a G1 region that remains bound toHA (aggregated) and one fragment that is no longer bound toHA (non-aggregated) and is free to diffuse within the disc. Inarticular cartilage the latter fragments are rapidly lost intothe synovial uid, but in the disc they accumulate as theirdiffusion is impeded by the vertebral end plates and the outerbrous layers of the AF. With increasing age, the abundanceof the non-aggregated fragments may exceed that of theaggregated fragments (Fig. 4.2b ), and ongoing proteolysisfurther decreases the size of both the aggregated and non-aggregated fragments. Ultimately, the aggregated fragmentsare cleaved to their G1 region, which appears to be relativelyresistant to proteolysis. As the size of the non-aggregatedfragments declines, they are eventually lost from the tissue,and the total aggrecan content declines. The G1 fragmentsmay also be eventually lost from the tissue as the size of theaggregates decreases due to depolymerization of the HA byextracellular hyaluronidases (Durigova et al. 2011b ) or freeradicals (Roberts et al. 1987 ). The average half-life of boththe aggregated and non-aggregated aggrecan fragmentswithin the disc is about 20 years (Sivan et al. 2006 ).

Aggrecans from different species do not possess identi-cal structures, and there are differences in the structure ofboth their core proteins and GAG chains. The major coreprotein differences relate to the number of repeats in boththe KS and CS1 domains (Barry et al. 1994 ; Doege et al.1997 ). Of particular note is the absence of an extended KSdomain in the mouse and rat, though it is unclear whetherthis is of functional importance. The largest species differ-ences are in the GAG chains, which can differ enormouslyin chain length and degree and position of sulfation. Inaddition, there is variation in the abundance of aggrecanwithin the discs of different species. While changes inaggrecan structure and abundance are likely to have func-tional consequences, it is unclear whether such changesrender some species more susceptible to disc or cartilagedegeneration.

4.3.2 Aggrecan Gene Organization,Expression, and Mutations

The human aggrecan gene (ACAN, AGC1, CSPG1) resideson chromosome 15 (Korenberg et al. 1993 ) and is composedof 19 exons (Valhmu et al. 1995 ). Exon 1 encodes the5¢ -untranslated region (UTR), exon 2 encodes the signal pep-tide, exons 3–6 encode the G1 region, exon 7 encodes theIGD, exons 8–10 encode the G2 region, exons 11 and 12encode the GAG-attachment region, exons 13–18 encode theG3 region, and exon 19 encodes the 3 ¢ -UTR. The G3 regiondoes not possess a unique structure, as the exons encoding itstwo EGF-like sequences and its one CRP-like sequence mayundergo alternative splicing (Doege et al. 1991 ; Fulop et al.


6/25


G1 G2 G3

G3G1

a

b

GAG- α

Key:G1, G2, G3 Globular domain

KS side chainCS side chain Core protein Disulphide stabilised loop

GAG- β

Scale50 kDa

KS CS1 CS2

Fig. 4.3 Schematic depictionof the structural organizationof aggrecan ( a ) and versican(b ) drawn to scale

1993 ). All alternatively spliced forms of aggrecan do, however,possess a G3 region with a lectin-like sequence, and hencemay participate in ECM interactions. It is not clear whetherabsence of the EGF and CRP domains in uences the functionof the G3 domain in vivo, but this has been suggested (Dayet al. 2004 ). Exon 12, which encodes the CS1 and CS2domains, exhibits a unique length polymorphism within thesequence encoding the CS1 domain in the human (Doegeet al. 1997 ). The human CS1 domain is composed of repeatsof 19 amino acids, each of which bears consensus sequencesfor the attachment of two CS chains. The number of repeatshas been reported to vary between 13 and 33, with most indi-viduals possessing 26–28 repeats. This type of polymorphismcan in uence the number of CS chains present on each aggre-can molecule, and it has therefore been suggested that thismay in uence aggrecan function; it is predicted that thoseaggrecan molecules bearing less repeats are functionally infe-rior (Roughley 2006 ). This led to the prediction that individu-als possessing aggrecan with a low number of CS1 repeatswould be more susceptible to degeneration of both the inter-vertebral disc and articular cartilage. While some evidencedoes support this conclusion (Kawaguchi et al. 1999 ), it islikely that other predisposing factors must also be present.

Aggrecan gene expression is regulated by a number offactors that relate to the unique environment within the disc.TonEBP, an osmoregulatory protein present in nucleus pul-posus cells, interacts with two conserved TonE motifs withinthe aggrecan gene promoter and promotes aggrecan synthe-sis, thereby allowing the nucleus pulposus cells to adapt totheir hyperosmotic environment (Tsai et al. 2006 ). HIF-1 a also enhances aggrecan promoter activity and increases

nucleus pulposus cell gene expression, permitting the disccells to function normally under low oxygen tension (Agrawalet al. 2007 ). In addition, both TonEBP and HIF-1 a regulatethe expression of the glucuronic acid transferase responsiblefor CS synthesis (Gogate et al. 2011 ; Hiyama et al. 2009 ).Thus, both the osmotic and hypoxic environment of the disccells participate in maintaining normal aggrecan synthesisand structure.

Mutations in the aggrecan gene and genes involved inGAG sulfation give rise to a variety of chondrodysplasias,which affect not only the hyaline cartilages but also theintervertebral disc. In humans some forms of spondyloepi-physeal dysplasia (SED) and spondyloepimetaphyseal dys-plasia (SEMD) are associated with mutations in the aggrecangene (Gleghorn et al. 2005 ; Tompson et al. 2009 ). A non-sense mutation is responsible for nanomelia in chickens (Liet al. 1993 ), and a 7bp deletion in exon 5 causing a frame-shift and a premature stop in exon 6 is responsible for carti-lage matrix de ciency (cmd) in mice (Watanabe et al. 1994 ) .Aggrecan is de cient in the extracellular matrix of themutant tissues, probably due to a combination of nonsense-mediated decay of the message and impaired secretion andintracellular degradation of any truncated product. Mutationsin the DSDST sulfate transporter gene are responsible fordiastrophic dysplasia, atelosteogenesis type II, and achon-drogenesis type 1B in humans (Karniski 2001 ; Superti-Furgaet al. 1996 ), and a mutation in the APS kinase, responsiblefor sulfate donor (PAPS) synthesis in cartilage, gives rise tobrachymorphism in mice (Kurima et al. 1998 ). Chondrocytesand disc cells require large amounts of sulfate for aggrecansynthesis, and when absent, an undersulfated product is


7/25


formed. These disorders add credence to the view that discfunction requires both a high tissue content of aggrecan anda high degree of sulfation.

4.3.3 Aggrecan Degradationin the Degenerate Disc

The interglobular domain of aggrecan is particularly suscep-tible to proteolysis and is cleaved by most proteinases in vitro(Fosang et al. 1992 ). Analysis of in vivo degradation prod-ucts indicates two predominant naturally occurring cleavagesites, which could be cleaved by aggrecanases (ADAMTS4and ADAMTS5) and matrix metalloproteinases (MMPs)(Sztrolovics et al. 1997 ). Both aggrecanases and severalMMPs have been detected in the disc (Roberts et al. 2000 ),and it is currently unclear as to which family members arepredominantly responsible for causing aggrecan damagein vivo. However, in vitro studies indicate that ADAMTS5 ismore ef cient than ADAMTS4 at cleaving within the aggre-can IGD (Gendron et al. 2007 ) and that MMP-3, MMP-7,and MMP-12 are the most ef cient MMPs (Durigova et al.2011a ). Aggrecanases are also able to cleave within the CS2region of aggrecan, and ve cleavage sites within this regionhave been identi ed (Tortorella et al. 2002 ). MMPs may alsocleave at sites outside the IGD, but at present the extent oftheir cleavage is not fully understood. One of the initialevents in aggrecan degradation, following its secretion intothe extracellular matrix, is removal of the G3 region (Flanneryet al. 1992 ), and it is unclear whether aggrecanases or MMPsare responsible.

Proteolytic cleavage of aggrecan and its loss are detri-mental to disc function and are thought to be directlyinvolved with intervertebral disc degeneration (Roughley2004 ). Not only does degradation and loss of aggrecan lessenthe ability of the disc to swell, it may also predispose it tomechanical damage. Indeed, there may be a vicious circle inwhich overloading of the disc stimulates aggrecan degrada-tion via the production of proteinases by the disc cells, whichin turn renders the disc susceptible to material damage. Suchmaterial damage may be irreversible and distinguish discdegeneration from normal aging (Adams and Roughley2006 ). Loss of aggrecan can also promote angiogenesis(Johnson et al. 2005 ) and may be a prelude to blood vesseland nerve invasion of the disc with the onset of discogenicpain (Johnson et al. 2002 ). Structural changes in aggrecanare also associated with the discs present in the scolioticspine. This may also be a consequence of abnormal loading,but this time in an asymmetric manner. Indeed, aggrecanchanges do vary between the concave and convex sides ofthe scoliotic disc. It has been suggested that dietarysupplementation with CS and glucosamine may help preventaggrecan loss in articular cartilage ( Box 4.2 ), and if true thismay also be relevant to the disc.

4.4 Versican

Versican was originally identi ed in broblasts and recog-nized to encode a CS proteoglycan (Zimmermann andRuoslahti 1989 ). Versican has a much wider tissue distribu-tion than aggrecan and together with HA has been suggested

Box 4.2: The Therapeutic Use of Chondroitin Sulfateand GlucosamineFor the past two decades, nutraceutical companies havebeen promoting oral supplements of glucosamine andchondroitin sulfate (CS) for the treatment of osteoarthri-tis. The original theory behind this treatment was that

CS was a building block for aggrecan and that glu-cosamine was a building block for CS and that theirsupplementation would therefore promote aggrecanproduction or their presence in the bloodstream wouldsomehow tolerize the body to these components, thuspreventing autoantibody production which is prevalentin some forms of immune-driven in ammatory arthri-tis. As loss of aggrecan is associated with a deteriora-tion in the functional properties of articular cartilage inosteoarthritis, it seemed reasonable that an increase inaggrecan production would be bene cial. Indeed it maybe, but the question is whether CS and glucosamine aid

in this process. Aggrecan synthesis does not involve theaddition of intact CS chains to its protein core, and CSsynthesis does not utilize glucosamine to produce its

N -acetyl galactosamine component. Commercialsources of glucosamine are derived from the chitincomponent (poly- N -acetyl glucosamine) of crustaceanshells, and while it may make more sense to administergalactosamine as a therapeutic agent, there is no readilyavailable commercial source of this material.Furthermore, it is likely that much of the CS enteringthe circulation would be degraded to its constituentmonosaccharides by the liver. It is therefore not surpris-ing that there is much skepticism concerning the abilityof CS and glucosamine to promote cartilage repair, par-ticularly given the high doses of these componentsrequired to elicit a positive response. There is howeversome evidence that CS and glucosamine therapy can aidin the relief of joint pain in arthritic patients, althoughthe mechanism for this effect is not clear. If this is true,then CS and glucosamine therapy may be an attractivealternative to more conventional nonsteroidal anti-inammatory drug (NSAID) therapy, as the former havefew side effects. It does however remain to be shownwhether all formulations of CS plus glucosamine areequally effective and whether CS plus glucosamine for-mulations are more effective than glucosamine alone.


8/25


to provide tissue hydration and viscoelasticity (Hasegawaet al. 2007 ). In the fetal intervertebral disc, versican isprominently expressed throughout the tissue, but not in theadjacent cartilaginous vertebral body rudiments, and itprominently demarcates the margins of the developing discfrom adjacent structures (Fig. 4.1 ) (Smith et al. 2009 ). Inthe mature intervertebral disc, versican is present through-out the tissues, being diffusely distributed within the nucleuspulposus, and most prominent between the lamellae of theannulus brosus (Melrose et al. 2001 ). Although versican isless abundant than aggrecan in the disc, its abundance isgreater than in articular cartilage (Sztrolovics et al. 2002 ).It is unclear whether versican serves a unique functionthroughout the disc, but it could provide viscoelastic prop-erties to the outer annulus brosus where aggrecan isdepleted. However, the function of versican may not berestricted to a structural role within the extracellular matrix,as it is also known to in uence cell function, particularly incancer (Ricciardelli et al. 2009 ). The versican G3 regionhas also been shown to in uence disc cell function (Yanget al. 2003 ).

4.4.1 Versican Protein Structure and Function

Versican is structurally related to aggrecan, possessing ter-minal domains analogous to the G1 and G3 regions ofaggrecan (Fig. 4.3b ), although there is no evidence for alter-native splicing in the versican G3 region (Grover andRoughley 1993 ). There is also no analogous IGD or G2region, and the central GAG-attachment region is com-pletely different in its amino acid sequence and GAG orga-nization. Likewise, a domain for the attachment of KS doesnot exist, although KS may be present on the G1 region, andthere are many fewer CS chains. The G1 region of versicanhas functional HA-binding and LP-binding domains, and isable to form proteoglycan aggregates, but it is uncertainwhether versican and aggrecan can reside on the sameaggregate. Interestingly, the four hyalectan genes reside intandem with an LP gene (Spicer et al. 2003 ), suggesting thatcoordinated gene expression may occur. Somewhat surpris-ingly, versican interacts best with the LP adjacent to theaggrecan gene, whereas aggrecan interacts best with the LPadjacent to the versican gene (Shi et al. 2004 ). As withaggrecan, the lectin domain within the G3 region of versi-can has the ability to interact with bulins and tenascins(Olin et al. 2001 ). The presence of multiple protein-bindingmotifs and the resulting possibility of versatility in functionled to the name versican.

The versican core protein possesses different splice vari-ants, which alter the size of its GAG-attachment region. Thecore protein of the common V1 form of versican is of a simi-lar length to that of aggrecan, whereas that of the V0 form ofversican is much larger than aggrecan, having a core protein

with about 3,400 amino acids. Versican undergoes extensive

proteolytic modi cation (Sztrolovics et al. 2002 ), which hashampered its puri cation from aggrecan. As a result, there islittle information on the structure of its CS chains andwhether they may change in structure with age in the disc.Although versican is commonly referred to as a CS proteo-glycan, the presence of DS cannot be discounted at all ages.The spectrum of versican core protein sizes within the disc isof a similar range to those of aggrecan, varying from free G1regions to intact molecules (Sztrolovics et al. 2002 ).

4.4.2 Versican Gene Organization and Mutation

The human versican gene (VCAN, CSPG2) resides onchromosome 5 (Iozzo et al. 1992 ) and is composed of 15exons (Naso et al. 1994 ). Exon 1 encodes the 5 ¢ -UTR, exon2 encodes the signal peptide, exons 3–6 encode the G1region, exons 7 and 8 encode the GAG-attachment region,exons 9–14 encode the G3 region, and exon 15 encodes the3¢ -UTR. The region encoded by exon 7 is referred to asGAG a and that encoded by exon 8 is referred to as GAG b .The exons encoding the GAG-attachment region mayundergo alternative splicing (Dours-Zimmermann andZimmermann 1994 ), giving rise to four versican mRNAs.The presence of both the GAG a and GAG b regions givesrise to the V0 form of versican, the presence of only theGAG b region gives rise to the V1 form, the presence of onlythe GAG a region gives rise to the V2 form, and the absenceof both the GAG a and GAG b regions gives rise to the V3form. The V1 form of versican appears to be the most com-mon form in most tissues, including the disc (Sztrolovicset al. 2002 ). Whether the different forms of versican serveunique functions is unknown, but it is likely that the V3form, being devoid of CS, may differ in function from theother forms. The V0 form of versican is analogous to PG-M,which was identi ed in chick limb bud mesenchyme (Itoet al. 1995 ).

Mutations in the human versican gene give rise to thedominantly inherited Wagner syndrome, and in the originalindex case, this was due to a base substitution at the exon 8/ intron 8 splice junction affecting the splicing of exon 8(Kloeckener-Gruissem et al. 2006 ). Additional mutationsaffecting the intron7/exon 8 splice junction have also beenreported in other families with Wagner syndrome(Mukhopadhyay et al. 2006 ). Defective splicing of exon 8results in a decrease in the V1 form of versican and anincrease in the V2 and V3 forms. Wagner syndrome isclassi ed as a vitreoretinopathy, and its ocular features areprobably associated with perturbation in the role played byversican in gelling of the human vitreous. However, in accordwith the widespread tissue distribution of versican, Wagnersyndrome patients also exhibit extraocular features, includ-ing skeletal defects similar to those reported in Stickler syn-drome. Accordingly, it is likely that perturbation in

intervertebral disc formation and function will also occur.


9/25


4.4.3 Versican Degradationin the Degenerate Disc

The V1 form of versican can be cleaved by ADAMTS1 andADAMTS4 to yield a product of 441 amino acids that includesthe G1 region (Sandy et al. 2001 ). Thus, aggrecanases couldpotentially play a role in versican degradation within the discin vivo. However, the size of this product appears to be largerthan that of the free G1 region present in vivo, suggesting thatother proteinases are also active. The MMPs would be themost likely candidates to ful ll this role. As with aggrecan, itis likely that premature or excessive proteolytic degradationof versican is associated with intervertebral disc degenera-tion. Peptide sequences within both the versican and aggre-can G1 regions have also been associated with the developmentof autoimmune spondyloarthropathies (Shi et al. 2003 ) .

4.5 Perlecan

Perlecan was named for its appearance when rst visualizedby rotary shadowing electron microscopy, where it appearedas multiple globular domains considered to resemble a stringof pearls on a chain. Perlecan has a widespread distributionthroughout the developing human fetal intervertebral discand vertebral cartilaginous rudiment cartilages, where it dis-plays a pericellular localization pattern. However, it is alsoprominent in the territorial and interterritorial matrix of thedeveloping disc (Fig. 4.4 ) (Smith et al. 2009 ). Perlecanexpression is elevated in hypertrophic chondrocytes sur-

rounding the ossi cation center of the developing vertebralbody and in terminally differentiated growth plate chondro-cytes (Smith et al. 2009 ). In the neonatal and adult disc, per-lecan is cell associated in the pericellular matrix of thenucleus pulposus, inner and outer annulus brosus, cartilagi-nous end plates, and vertebral growth plates (Fig. 4.5 ).However, its relative abundance diminishes with the declinein cell number evident in the aging intervertebral disc.

Perlecan interacts with a number of growth factors andmorphogens, including FGF-1, FGF-2, FGF-7, FGF-9, andFGF-18; platelet-derived growth factor (PDGF); vascularendothelial cell growth factor (VEGF); hepatocyte growthfactor; BMP-1, BMP-2, BMP-4, and BMP-7; hedgehog(Hh); and Wnt, and through these molecules in uences cellproliferation and differentiation and matrix production(Whitelock et al. 2008 ). Perlecan also interacts with a num-ber of cell attachment proteins, including laminin, bronectin,thrombospondin, and a 5b 1 and a 2b 1 integrin, and therebyplays an important role in cell attachment and recruitmentduring tissue development and remodeling. Through its abil-ity to interact with a number of matrix components, includ-ing PRELP; WARP; types IV, VI, XIII, and XVIII collagen;brillin-1 and brillin-2; nidogen-1 and nidogen-2; latenttransforming growth factor-beta binding protein-2 (LTBP-2); and tropoelastin, perlecan modulates extracellular matrixassembly and stabilization (Hayes et al. 2011c ; Iozzo 1994, 1998 ; Melrose et al. 2008b ). Via these interactions, perlecanparticipates in disc development and in conversion of thecartilaginous vertebral rudiment cartilages into bone duringspine development (Smith et al. 2009 ). This is consistent

Biglycan Fibromodulin Rabit lgG −ve Perlecan

control dcba

Fig. 4.4 Immunolocalization of biglycan ( a ), bromodulin ( b ), nonimmune rabbit IgG negative control ( c ), and perlecan ( d ) in a 14-weekgestational age fetal human intervertebral disc and adjacent cartilaginous vertebral body rudiment cartilages


10/25


VGP

NP AF

CEP

Blood vessel

100 µm50 µm

100 µm50 µm

Fig. 4.5 Immunolocalization of perlecan in the newborn ovine lumbar intervertebral disc. Perlecan is present as a pericellular proteoglycan in the verte-bral growth plate ( VGP ), cartilaginous end plate ( CEP ), a blood vessel within the CEP, and the nucleus pulposus ( NP ) and annulus brosus ( AF )

with roles recently ascribed to perlecan as an early chondro-genic marker in the development of cartilaginous tissues(Smith et al. 2010 ).

Perlecan promotes extracellular matrix productionthrough its interactions with members of the FGF family.Perlecan is a low-af nity co-receptor for several members ofthe FGF family and sequesters these molecules pericellularlyfor later presentation to FGFRs. In this way, FGF can pro-mote cell signaling events which drive proliferation andmatrix production (Chuang et al. 2010 ). This sequestrationprocess also stabilizes the FGFs, protecting them from prote-olysis in situ and extending their biological half-life. Perlecanhas been co-localized with FGF-18 in the developing spine,with an overexpression evident in terminally differentiatedhypertrophic vertebral growth plate chondrocytes and incells surrounding the ossi cation centers in the developingvertebral bodies. Perlecan associates with FGF-2 in thedeveloping intervertebral disc interspace in the fetal humanspine. Thus, FGF-18 promotes terminal differentiation of

chondrocytes, leading eventually to bone formation, whereas

FGF-2 maintains chondrocytes in the permanent cartilagesin a delayed state of differentiation, where they are respon-sible for the replenishment of matrix components to effecttissue homeostasis.

Recent studies have also shown that the HS chains of per-lecan are important in brillin and elastin assembly (Hayeset al. 2011a, c ) and support earlier observations concerningbasement membrane assembly. Perlecan is localized to anumber of elastin-associated proteins in the intervertebraldisc (Hayes et al. 2011c ). LTBP-2 interacts with the perlecanHS chains (Parsi et al. 2010 ) and in the disc co-localizes withperlecan pericellularly. The biological signi cance of thislocalization is not known; however, LTBP-2 may have someregulatory role to play in the micro brillogenesis process(Hirai et al. 2007 ; Hirani et al. 2007 ) by occupying sites onbrillin-1 that LTBP-1 normally occupies (Hirani et al. 2007 ;Vehvilainen et al. 2009 ) or by interaction with another elas-tin-associated protein. Alternatively, by acting as a competi-tive substrate for the HS chains in perlecan domain I, it may

regulate growth factor binding to perlecan.


11/25


4.5.1 Perlecan Protein Structure

Perlecan is a large modular HS-proteoglycan composed ofve distinct domains with homology to growth factors and toprotein modules involved in lipid metabolism, cell adhesion,and homotypic and heterotypic interactions involved inmatrix assembly and stabilization (Melrose et al. 2008b ;Murdoch and Iozzo 1993 ) ( Box 4.3 ). The N-terminal domainI contains three HS attachment sites, through whichHS-mediated growth factor and morphogen interactionsoccur. Consensus regions for GAG attachment have alsobeen identi ed in the C-terminal domain V (Fig. 4.6a ). TheN-terminal domain is unique to perlecan, whereas domain IIexhibits homology to the low-density lipoprotein receptorand domain III bears homology to the L4 laminin-type IVdomain and LE laminin EGF domain. Domain IV, the largestdomain in perlecan, contains multiple immunoglobulinrepeats, although this domain is truncated by about 20 kDa

in mouse perlecan (Melrose et al. 2008b ; Murdoch and Iozzo1993 ). The C-terminal domain V bears homology to the LGlaminin-type G domain and contains three LG domains sepa-rated by EGF-like domains. The perlecan core protein islarge (467 kDa) and highly aggregative under associativeconditions, leading to the formation of higher molecularweight forms of about 800 kDa in free solution.

The perlecan core protein can contain three HS chains in theN-terminal domain, and additional GAG consensus attachmentpoints have been identi ed in domain V; however, it has yet tobe de nitively shown that these are occupied in the interverte-bral disc. Disc cells synthesize a hybrid HS/CS proteoglycanform of perlecan, with at least one of the HS chains replaced bya chondroitin-4-sulfate (C4S) chain. In the fetal and newborndisc, this C4S chain is capped by a unique developmental CSmotif identi ed by MAb 7D4 (Hayes et al. 2011b ). However,the abundance of this 7D4 epitope on perlecan diminishes withaging, and it is not clear if this has any functional consequence.

Box 4.3: Perlecan and Its Interactive Components

Abbreviations : FGF broblast growth factor, PDGF platelet derived growth factor, PRELP proline/arginine-rich and leucine-rich repeat protein/prolargin, BMP bonemorphogenetic protein, Ang angiopoietin, SHh sonic

hedgehog, VLDL very low density lipoprotein, LDL low

density lipoprotein, Wnt a morphogenic ligand, hybridabbreviation of Int (integration-1) and Wg (wingless),CTGF connective tissue growth factor, FGF-BP FGFbinding protein, WARP von Willebrand factor A domain-

related protein.

SEA domain

HS / CS chains

LDLreceptortype A

Laminin-1Collagen type IV

FibronectinFGF-2, 9, 18

PDFGPRELP

Fibrillin-1thrombospondin

heparanaseBMP-2, 4, 7Hedgehog

Ang-3VEGF

IL-2WARP

action Afollistatin

SHh

VLDLLDL

fibrillin-1Wnt/Ca2 +

CTGF

FGF-7,18FGF-BPPDFGWARP

nidogen-1, 2fibulin-2

fibronectincollagen type IV

heparinsulfitides

PDGF

nidogen-1fibulin-2

β1-Integrinα -dystroglycan

heparinsulfitides

FGF-7

endostatinECM1

progranulinacetylcholinesterase

α 2β1 integrin

Laminin type IV domain Immunoglobulin repeat domainLaminintype Gdomain

I II III IV V


12/25


4.5.2 Perlecan Gene Organization and Mutation

The perlecan gene ( HSPG2 ) is encoded by 94 exons locatedon chromosome 1p36–34 (Cohen et al. 1993 ; Kallunki andTryggvason 1992 ; Murdoch et al. 1992 ; Noonan et al. 1991 ),with each of the structural domains being encoded by mul-tiple exons.

The importance of perlecan in skeletogenesis (Arikawa-Hirasawa et al. 1999 ), vasculogenesis, and muscle and nervedevelopment is evident from analyses of two naturally occur-ring mutations in the human HSPG2 gene. Schwartz-Jampelsyndrome is a relatively mild skeletal condition, which arisesfrom missense, splicing, exon skipping, and deletion muta-tions. These events result in partial loss of domain IV and totalloss of domain V, complete loss of domain V only, or defectivedisul de bonding in domain III of perlecan (Arikawa-Hirasawaet al. 2002 ). As a result, there are reduced functional levels ofperlecan in cartilaginous tissues, chondrodysplasia, myotonia,impairment in the endochondral ossi cation process, and shortstature. In the more severe condition of dyssegmental dyspla-sia, Silverman-Handmaker type, perlecan is almost undetect-able in cartilaginous tissues, and this condition is characterizedby a severe chondrodysplasia; severe disruption in skeleto-genesis; profound effects on lung, heart, muscle, and cranialdevelopment; synaptogenesis; and complete absence of acetyl-cholinesterase at the neuromuscular junction leading to dysto-nia (Arikawa-Hirasawa et al. 2001a, b ). The perlecan knockoutmouse further emphasizes the essential roles of perlecan indevelopment (Arikawa-Hirasawa et al. 1999 ). Perlecan knock-out is a lethal condition with the majority of mouse pups dyingin utero, and in those few that survive to birth, there is severeimpairment in skeletal stature, cranial and long bone deve-lopment, and large vessel, heart, and lung development.

Studies with the Hspg2 exon 3 null mouse (Rossiet al. 2003 ), where perlecan domain I containing theGAG-attachment sites is ablated, are now allowing examina-tion of the speci c role of the perlecan HS chains in skeletaldevelopment. Hspg2 exon 3 null chondrocytes are poorlyresponsive to FGF-2 in cell proliferation studies. Baf-32 cellstransfected with FGFR3IIIc are also poorly responsive to kneerudiment cartilage perlecan predigested with heparitinase IIIto remove its HS chains, indicating the essential role of thedomain I HS chains for FGF-2 binding and cell signalingprocesses (Hayes et al. 2011b ). This is not the case for FGF-18, which induces cell proliferation, even in the absence ofperlecan HS chains (Hayes et al. 2011b ), but consistent witha domain III FGF-18 reactive site in perlecan. The Hspg2 exon 3 null mouse has a relatively mild phenotype with noapparent defects in cartilage assembly. However, recent stud-ies have indicated that with maturation, defects become evi-dent in cartilaginous tissues, and its reparative ability after atraumatic challenge also appears to be impaired. Fibrillin-1assembly and deposition is also impaired in the Hspg2 exon3 null mutant mouse intervertebral disc (Hayes et al. 2013 ).

4.5.3 Perlecan Degradation

Little is known about the mechanism by which perlecan is pro-cessed in the intervertebral disc. Gel electrophoresis separatesfull-length perlecan from three other perlecan species in new-born and young adult ovine discs. The latter three species aresmaller than full-length perlecan, devoid of GAG, and detect-able using a MAb to perlecan domain I. Thus, they representfragments cleaved from the domain I carboxy terminal to theHS chains. However, the cleavage sites themselves still awaitdetailed characterization. Perlecan fragmentation has also beenobserved by Western blotting of newborn and 2-year-old inter-vertebral disc extracts using a domain I-speci c MAb, withone major and four minor species evident (Fig 4.6b ). A similarrange of perlecan fragments has been observed in extracts ofhuman knee joint articular cartilage (Melrose et al. 2006 ) .

In vitro digestion of endothelial cell perlecan with MMP-1, MMP-3, and plasmin has demonstrated the susceptibilityof perlecan to cleavage in domains IV and V (Whitelocket al. 1996 ). Tolloid-like metalloprotease (BMP-1) alsocleaves perlecan between the LG2 and LG3 domains ofdomain V, within the peptide sequence HLEGSGGN- ↓-DAPGQYGA, releasing an anti-angiogenic peptide termedendorepellin (Bix et al. 2004, 2007 ; Gonzalez et al. 2005 ).Endorepellin has the ability to disrupt endothelial cell a 2b 1integrin-based basement membrane interactions, which nor-mally stabilize tube formation (Bix et al. 2004, 2007 ;Gonzalez et al. 2005 ). So far endorepellin is the only perle-can fragment that has been extensively characterized and itsfunctional properties determined.

4.6 Lubricin

Lubricin was originally identi ed as the large mucinous gly-coprotein present in synovial uid, which by providingboundary lubrication at the surface of articular cartilage wasresponsible for friction-free joint motion (Swann et al. 1977 ).This role in joint lubrication led to the name lubricin. Later,a glycoprotein was identi ed in the super cial zone of artic-ular cartilage (Schumacher et al. 1994 ) and termed super cialzone protein (SZP). It is produced by super cial zone chon-drocytes and has been shown to be analogous in structure tolubricin (Jay et al. 2001b ). Unlike lubricin, SZP has beenshown to exist in part as a CS proteoglycan, termed proteo-glycan 4 (PRG4). It is however not clear whether this dis-tinction exists at all ages or in all disease states or whetherthe CS chain contributes to SZP function. Recently, lubricinhas also been shown to reside in the intervertebral disc (Jayet al. 2001b ; Shine et al. 2009 ; Shine and Spector 2008 ).

Lubricin is present in all regions of the disc but appears tobe most abundant in the nucleus pulposus (Shine et al. 2009 ).Its core protein undergoes extensive proteolytic degradation,with accumulation of the degradation products in the tissue;


13/25


Scale50 kDa

III IV VIII

Key:

a

b

CS side chain HS side chain Core protein

2 years old

Mw(kDa)

460

268238

171

117

71

55

Std OAF IAF NP HUVEC OAF IAF NP

Newborn

Fig. 4.6 (a ) Schematic represen-tation of the structural organizationof perlecan. ( b ) SDS/PAGEanalysis of perlecan heterogeneityin newborn and 2-year-old ovineintervertebral disc samples. Notethe extensive fragmentation of discperlecan compared to that fromhuman vascular endothelial cells( HUVEC ). The multiple perlecanspecies that are discernible in thedisc are identi ed by arrows in theright hand margin. OAF outerannulus brosus, IAF innerannulus brosus, NP nucleuspulposus

it has a polydisperse size distribution comparable to othertissue sources, such as synovial uid or articular cartilage(Fig. 4.7b ). It is not known which proteinases are responsiblefor cleavage in vivo, but they are likely to be the same asthose involved in aggrecan and versican degradation. In thisrespect, MMPs have been demonstrated to degrade lubricinin vitro (Elsaid et al. 2005 ). Also unknown is the precisefunction of lubricin in the disc and how proteolysis mayaffect lubricin function. One possibility is a role in lubricat-ing motion between adjacent annulus brosus lamellae.

4.6.1 Lubricin Protein Structure

Intact lubricin has a molecular weight of about 240 kDa, ofwhich about 50 % is contributed by O-linked mucin-like

oligosaccharides (Swann et al. 1981b ), which reside in along central domain. This mucin-like domain is anked byN- and C-terminal cysteine-rich domains that resembledomains of vitronectin. Domains possessing somatomedinB homology and a heparin-binding domain may reside at itsN-terminus, and a hemopexin domain resides at itsC-terminus (Fig. 4.7a ). There is a single consensus sequencefor the attachment of CS near the N-terminus of the mucin-like domain. In its SZP form, lubricin has also been reportedto contain KS, but it is unclear where this resides. The lubri-cating properties of lubricin are conferred by its mucin-likedomain (Jay et al. 2001a ), but the other domains provide thepotential for extracellular interactions and cell associations.The terminal domains appear to be important, as their reduc-tion and alkylation impairs lubricin function (Swann et al.1981a ).


14/25


4.6.2 Lubricin Gene Organization and Mutation

The mRNA encoding lubricin/SZP has been shown to origi-nate by alternative splicing of the megakaryocyte stimulatingfactor (MSF) precursor gene (PRG4) (Flannery et al. 1999 ).The human PRG4 gene resides on chromosome 1 and pos-sesses 12 exons (Merberg et al. 1993 ). Exon 1 encodes the sig-nal peptide, exons 2 and 3 encode domains with somatomedinB homology, and exons 4 and 5 encode a region containing aheparin-binding domain. Exon 6 encodes the mucin-likedomain, possessing about 80 potential attachment sites forO-linked oligosaccharides and a single potential attachmentsite for CS. Exons 7–12 encode the carboxy terminus of themolecule, which contains a domain with hemopexin-likehomology. Both synovial lubricin and cartilage SZP are derivedby alternative splicing of a combination of PRG4 exons 2, 4,and 5 to yield several messages. Thus, there is no uniquestructure for the lubricin core protein, but all forms possess atleast one somatomedin B-like domain and a hemopexin-like

domain anking the central mucin-like domain. This heteroge-neity accounts for the variable size of intact lubricin.

Mutations in the human PRG4 gene give rise tothe autosomal recessive camptodactyly-arthropathy-coxavara-pericarditis syndrome (CACP) (Marcelino et al. 1999 ).Most of the originally identi ed mutations causing frame-shifts or nonsense substitution near the end of exon 6 orin subsequent exons result in truncation of the hemopexindomain. These mutations result in a lack of lubricin in syn-ovial uid and a lack of lubricin production by cultured syn-oviocytes (Rhee et al. 2005b ). Many of the features of CACPare recapitulated in the lubricin knockout mouse (Rhee et al.2005a ), supporting the concept that de cient production oflubricin is responsible for the CACP phenotype. The widerange of symptoms associated with CACP show that lubricinfunction is not restricted to joint motion but also in uencestendons and the heart. While patients with CACP have spineabnormalities (Faivre et al. 2000 ), it is not clear whether discfunction is also affected in these individuals.

CS side chain

Somatomedian B homologies

Heparin bindingdomain domains

O-linked oligosaccharidesMutin-like

Core protein

COOHNH2a

b

Key:

MWkDa

180

115

82

6449

37

2619

SF C AF NP

SF Synovial fluid

C Articular cartilageAF Annulus fibrosusNP Nucleus Pulposus

Scale

50 kDaHemopexin domain

Fig. 4.7 (a ) Schematic represen-tation of the structural organiza-tion of lubricin. ( b ) SDS/PAGEanalysis of lubricin heterogeneityin the human IVD. The lumicancore protein is more fragmented inthe intervertebral disc than in syn-ovial uid or articular cartilage


15/25


4.7 The Small Leucine-Rich RepeatProteoglycans (SLRPS)

The SLRPs are members of a large family of leucine-richrepeat (LRR) proteins, which contain multiple adjacent 24amino acid domains bearing a common leucine-rich motif(Hocking et al. 1998 ). The SLRPs have been categorized intoa number of subfamilies on the basis of their gene organiza-tion, number of LRRs, type of GAG substitutions, and gen-eral structural organization (Kalamajski and Oldberg 2010 ).Eight SLRPs have been identi ed in the intervertebral disc,

including the CS-/DS-substituted decorin and biglycan;KS-substituted lumican, bromodulin, and keratocan; andnon-glycanated proline/arginine-rich protein (PRELP, pro-largin), chondroadherin, and asporin (Fig. 4.8 ). These SLRPscontain ten LRRs, which are anked by amino and carboxyterminal disul de-bonded regions.

Decorin and biglycan possess attachment sites for theirCS/DS side chains within the extreme amino terminus oftheir core proteins. In decorin, there is one such site, whereasbiglycan contains two GAG substitution sites (Roughleyand White 1989 ). In most connective tissues, including the

DCN

BGN

FMOD

Scale10 kDa

LUM

PRELP

ASPN

CHAD

Key: RRR

RRR

Arg cluster

N-linked oligosaccharide

CS (DS) side chain KS side chain Core protein

Disulphide stabilised 4 cysteine or 2 cysteine cluster

Pro cluster Sulphated Try cluster Aspartic acid clusterPPP

PPP

DDD

DDD

DDD

Y Y

Y Y

Y

Y Y Y

Y Y Y

Y

SO

SO

SO

SO

4

4

4

4

SO SO4 4

SO

SO SO

SO4

4 4 SO 4

4SO4

Fig. 4.8 Schematic repre-sentation of the structuralorganization of SLRPs foundin the intervertebral disc


16/25


intervertebral disc, the CS chains are modi ed in the Golgiby epimerization of the b -D -glucuronic acid moieties toa -L -iduronic acid to form DS. Non-glycanated forms ofdecorin and biglycan devoid of DS chains have beenobserved in disc tissues, and as with other connective tis-sues, their relative abundance accumulates with age. Thesenon-glycanated molecules likely arise by proteolysis inthe N-terminal region. Besides the removal of a smallN-terminal signal peptide, the mature core proteins of deco-rin and biglycan are generated by removal of additionalamino acid segments of 14 and 21 amino acids, respectively(Roughley et al. 1996b ; Scott et al. 2000 ). However, thefunctional consequence of the removal of these pro-pep-tides is not known.

Lumican and bromodulin possess four N-linked oligo-saccharide chains within their central LRRs, which may bemodi ed to KS, although substitution at all sites is uncom-mon (Plaas et al. 1990 ). Non-glycanated forms of lumicanand bromodulin also occur in connective tissues, due tolack of KS substitution (Grover et al. 1995 ; Roughley et al.1996a ). Only the small N-terminal signal peptides areremoved from the lumican and bromodulin core proteins toform the mature core proteins in situ. Fibromodulin andlumican also have a number of sulfated tyrosine residuesclustered at their extreme N-termini in the mature protein,which may also contribute to the anionic nature of theseSLRPs in situ (Antonsson et al. 1991 ; Onnerfjord et al. 2004 ;Tillgren et al. 2009 ).

PRELP and asporin are non-glycanated SLRPs but con-tain clusters of arginine and proline and aspartic acid, respec-tively, at their N-termini (Bengtsson et al. 1995 ; Grover andRoughley 1998, 2001 ). PRELP is unique amongst the SLRPsin possessing a cationic N-terminal region. Chondroadherinis devoid of an N-terminal core protein region where chargedamino acids are clustered in PRELP and asporin. It has asimilar LRR core protein structure (Grover et al. 1997 ), butdiffers from the other SLRPs in the structure of its C-terminalregion.

SLRPs have important roles to play as tissue organiz-ers based on how they affect the orientation and assemblyof collagenous matrices and how they interact with growthfactors and cell surface receptors during tissue develop-ment and matrix remodeling (Iozzo et al. 2011 ). They dis-play diversi ed and overlapping functional properties witha level of redundancy between SLRP members. Asporinbinds to collagen with an af nity in the low nanomolarranges, as does decorin (Kalamajski et al. 2009 ). Asporinand decorin bind to the same region in brillar collagen andcan effectively compete with one another for this site whenapplied at equimolar concentrations, whereas biglycan can-not (Kalamajski and Oldberg 2010 ). The collagen-bindingsites on asporin and decorin differ, with the binding site for

decorin located at LRR 7 and the binding site on asporinlocated at C-terminal of this site (Kalamajski et al. 2009 ).While lumican and bromodulin both utilize leucine repeatdomains ve to seven to bind to brillar collagen and bothcan regulate early collagen bril assembly processes, onlybromodulin facilitates growth steps leading to mature brilformation (Kalamajski and Oldberg 2009 ). Decorin and big-lycan display coordinated control of collagen brillogenesisduring development and acquisition of biomechanicalproperties during tendon development (Iozzo et al. 2011 ).Alterations in the functional properties of SLRP membersthrough point mutations affecting the structure of criticalinteractive regions within the LRRs can give rise to impairedtissue assembly and function.

4.7.1 Decorin

Decorin is so named since it was found to “decorate” thesurface of collagen brils. The decorin gene (DCN) is locatedon chromosome 12q21.3–q23. It possesses 8 exons encodinga 36kDa core protein, which contains a single CS or DSchain located at position 4 in the mature human core proteinsequence (Roughley 2006 ). Decorin interacts with the “d”and “e” bands of collagen I brils, bronectin, C1q, EGFreceptor, TGF- b , and thrombospondin. Thus, it has roles inthe regulation of collagen brillogenesis in vitro and brosisin vivo and controls the bioavailability of TGF- b . Decorinalso in uences cell proliferation at certain stages of the cellcycle and has roles as a linking module in collagenous matri-ces (Iozzo and Schaefer 2010 ).

The regions of decorin which interact with collagen arelocated within the leucine-rich repeats at dissimilar regionsto those involved in its interaction with TGF- b . Molecularmodeling predicts that decorin possesses a “horseshoe”conformation capable of accommodating a single collagenmolecule at the surface of the collagen brils within its con-cave face (Orgel et al. 2009 ; Scott 1996, 2003 ; Scott andStockwell 2006 ). However, X-ray diffraction analysis ofdecorin crystals indicates that it exists as a dimer with inter-locking concave faces (Scott et al. 2004 ), although it is notclear whether decorin dimers represent the functional formin solution and how this impacts its interactions with othermolecules. Decorin, together with biglycan, bromodulin,and lumican, also interacts with collagen VI, XII, and XIV(Nareyeck et al. 2004 ; Wiberg et al. 2002 ), bronectin andelastin, and growth factors and cytokines such as EGF, IGF,TGF- b , and TNF- a . SLRP GAG chain interactions withgrowth factors sequester them in the matrix surrounding cellsin cartilaginous matrices, with the possibility that SLRPsplay a role in the regulation of the bioavailability of thesegrowth factors.


17/25


The decorin core protein is susceptible to cleavage byMMPs in vitro (Imai et al. 1997 ; Monfort et al. 2006 ) and isfragmented in an ovine annular lesion model of intervertebraldisc degeneration (Melrose et al. 2007 ) and in an ovinemeniscectomy model of osteoarthritis in areas undergoingremodeling processes (Young et al. 2005 ). Fragmentation ofdecorin is also evident in meniscal and articular cartilagesfrom osteoarthritic knees and hips (Melrose et al. 2008a ) .Decorin is processed by three isoforms of BMP-1 (vonMarschall and Fisher 2010 ) and MT1-MMP (Mimura et al.2009 ). However, by acting as a sacri cial substrate on thesurface of collagen bers or through steric exclusion effectswhich prevent the collagenase from accessing the collagensubstrate, its presence may protect the protein from cleavageby collagenases (Geng et al. 2006 ) .

4.7.2 Biglycan

The biglycan gene (BGN) is located on chromosome Xq28(McBride et al. 1990 ). It possesses eight exons encoding the38 kDa biglycan core protein, which has two CS/DS chainslocated at amino acids ve and ten of the mature human coreprotein sequence. Non-glycanated forms of biglycan havebeen detected and appear to be the result of proteolysis withinthe amino terminal region of the core protein. Mature biglycanis cleaved by MMPs (Monfort et al. 2006 ), and probiglycan byBMP-1 (Scott et al. 2000 ). Extensive biglycan fragmentationis also evident in models of osteoarthritis induced by menis-cectomy (Young et al. 2005 ), in an annular lesion model ofexperimental disc degeneration (Melrose et al. 2007 ), and inpathological meniscal and articular cartilage from osteoar-thritic knees and hips (Melrose et al. 2008a ).

Unlike the interaction of decorin, bromodulin, and lumi-can with collagen brils, the association of biglycan withcollagen brils is sensitive to environmental conditions. Thismay explain the differences observed in the distribution ofbiglycan, which is more of a cell-associated SLRP than theother SLRP members. Biglycan interacts with the lattice-forming collagen VI to form chondron basketlike structuresaround cells in cartilaginous matrices and the intervertebraldisc. Biglycan has a widespread distribution in the develop-ing human fetal disc and prominently demarcates its marginswith the cartilaginous vertebral body rudiment cartilages,where it is also prominently localized. As such, its positiondelineates the margins of the developing intervertebral discinterspace from the presumptive cartilage end plate (Fig. 4.4 ).Biglycan also interacts with BMPs and TGF- b . Biglycanplays a role in the initiation of the in ammatory responseduring tissue stress, by binding to BMP/TGF- b , and maymodulate their activities, in uencing brosis and skeletalcell differentiation. Biglycan also has emerging roles as a

signaling molecule, with an extensive repertoire of molecu-lar interactions with growth factors and receptors, whichregulate cell growth, morphogenesis, and immunity (Iozzoand Schaefer 2010 ).

4.7.3 Asporin

The naming of asporin re ects its unique N-terminal asparticacid cluster (Henry et al. 2001 ; Lorenzo et al. 2001 ). Asporinis also known as periodontal-associated protein-1 (PLAP-1).The asporin gene (ASPN) is located at chromosome 9q21–23and possesses eight exons that encode a 43 kDa non-glycan-ated core protein. The asporin core protein exhibits polymor-phism within its N-terminal region, which contains 9–20aspartic acid repeats. An association of the ASPN D14 allelehas been observed with disc degeneration (Gruber et al.2009 ; Song et al. 2008 ). Asporin is a cell-associated SLRPand is found predominantly in the outer annulus brosus(Gruber et al. 2009 ).

4.7.4 Fibromodulin

The bromodulin gene (FMOD) is located on chromosome1q32 (Antonsson et al. 1993 ; Sztrolovics et al. 1994 ) andpossesses three exons encoding the 42 kDa bromodulincore protein. Fibromodulin is a KS-substituted SLRP whichshares signi cant amino acid sequence homology with deco-rin and biglycan (Antonsson et al. 1993 ; Oldberg et al. 1989 )but contains four N-linked oligosaccharide sites within theLRR domains, which can potentially be substituted with KS.Fibromodulin also possesses a cluster of amino terminal sul-fated tyrosine residues which impart an anionic character tothe molecule and allow this region to interact with clusters ofbasic residues in a variety of heparin-binding proteins,including a number of bioactive factors (Onnerfjord et al.2004 ; Tillgren et al. 2009 ). Non-glycanated forms ofbromodulin can accumulate in tissues (Grover et al. 1995 ;Roughley et al. 1996a ), due to an age-dependent decline inKS synthesis.

Fibromodulin interacts with collagen I and II brils andinhibits brillogenesis in vitro (Chen et al. 2010 ; Ezura et al.2000 ). Fibromodulin also interacts with TGF- b and C1q andmay have roles in TGF- b sequestration, in in ammation, inthe regulation of the assembly of collagen I and II brilsin vivo, and in the bio-regulation of TGF- b activity. The useof bromodulin knockout mice has demarcated the signi cantfunctional roles of bromodulin in cartilaginous tissues withregard to collagen assembly and in the regulation of thebrillogenesis (Chakravarti 2002 ; Goldberg et al. 2006 ;Svensson et al. 1999 ).


18/25


Fibromodulin is prominently immunolocalized in the car-tilaginous vertebral rudiment cartilages of the human fetalspine and also the developing disc, and its distribution in thefetal spine prominently demarcates the margins of the devel-opmental intervertebral disc with the vertebral rudiment car-tilages (Fig. 4.4 ). In the mature disc, bromodulin is localizedwith collagenous structures pericellularly and in the intersti-tial matrix. It occurs throughout the intervertebral disc, butpredominantly in the annulus brosus.

When bound to collagen, bromodulin is susceptible tocleavage by MMP-13 which removes the N-terminal sul-fated tyrosine cluster (Heath eld et al. 2004 ). Solublebromodulin is not, however, susceptible to cleavage byMMP-13, neither is it degraded by MMP-2, MMP-8, andMMP-9. The MMP-13 cleavage site is contained within a10 kDa N-terminal peptide, Gln19-Lys 98, which is locatedadjacent to the sulfated tyrosine cluster (Heath eld et al.2004 ). Fibromodulin is extensively fragmented in patho-logical meniscal and articular cartilage from osteoarthriticknees and hip (Melrose et al. 2008a ), in an ovine annularlesion model of intervertebral disc degeneration in areasundergoing remodeling (Melrose et al. 2007 ), and in anovine meniscectomy model of osteoarthritis (Young et al.2005 ).

4.7.5 Lumican

The lumican gene (LUM) is located at chromosome 12q21.3–q22 (Danielson et al. 1999 ; Grover et al. 1995 ) and possessesthree exons encoding the 38 kDa lumican core protein, whichhas four N-linked sites within the LRR domain that poten-tially can be substituted with KS. Lumican interacts withsimilar partners to those outlined for bromodulin and dis-plays similar roles to those of bromodulin in tissues.Lumican and bromodulin have homologous sequences inthe 5–7 LRRs which compete for collagen binding(Kalamajski and Oldberg 2009 ), and they bind to the sameregions of collagen I brils (Svensson et al. 2000 ). However,despite the signi cant similarities between lumican anddecorin, they have dissimilar binding sites on collagen I(Hedbom and Heinegard 1993 ) and modulate collagenbrillogenesis independently (Neame et al. 2000 ).

As with bromodulin and decorin, lumican is also sus-ceptible to degradation by MMPs, but apparently to a lesserextent, and it is also cleaved by MT1-MMP (Li et al. 2004 ).Fragmentation of lumican is a prominent feature in patho-logical meniscal and articular cartilages from osteoarthriticknees and hips (Melrose et al. 2008a ) and has also beenobserved in an ovine annular lesion model of disc degenera-tion in areas undergoing remodeling (Melrose et al. 2007 ).Although it does not contain KS, lumican expression is,however, upregulated in an ovine meniscectomy model of

osteoarthritis, with increased levels of lumican core proteinevident (Young et al. 2005 ).

4.7.6 PRELP

PRELP is a 55 kDa non-glycanated SLRP (Bengtsson et al.1995 ) encoded by the PRELP gene which is located on chro-mosome 1q32 and possesses three exons (Grover et al. 1996 ).Its name PRELP is based on the presence of a unique clusterof N-terminal arginine and proline residues. PRELP binds toperlecan and collagens and may act as a basement membraneanchor or as a linking molecule bringing together collage-nous tissue networks (Bengtsson et al. 2002 ). The N-terminusof PRELP binds to heparin and HS and thereby promotesinteractions with the HS-proteoglycan perlecan (Bengtssonet al. 2000 ).

4.7.7 Chondroadherin

Chondroadherin is a developmentally regulated SLRP (Shenet al. 1998 ) closely related to decorin, biglycan, bromodulin,lumican, and PRELP (Neame et al. 1994 ). The chondroad-herin gene (CHAD) is located on chromosome 17q21.33 andpossesses 4 exons encoding the 36 kDa non-glycanatedchondroadherin core protein (Grover et al. 1997 ), which con-tains 10 LRRs (Neame et al. 1994 ). Chondroadherin inter-acts with collagen II (Mansson et al. 2001 ) and a 2b 1 integrin(Camper et al. 1997 ; Haglund et al. 2011 ) and plays a role inthe attachment of connective tissue cells to matrix compo-nents; in this way it could maintain the cellular phenotypeand regulate tissue homeostasis. Depletion and proteolyticfragmentation of chondroadherin occurs in scoliotic inter-vertebral discs and appears to be associated with disc matrixremodeling (Haglund et al. 2009 ).

4.7.8 SLRP Knockout Mice and Gene Mutations

A number of human diseases are associated with SLRPmutations, and mouse knockout models have identi edpathological consequences resulting from the ablation ofSLRP genes. Examples of pathological features induced bySLRP gene ablation and human SLRP gene mutations aredescribed in Tables 4.1 and 4.2 . Thus far, asporin is the onlySLRP speci cally associated with human disc disease andthe disc phenotype. The intervertebral disc neverthelessexhibits similarities in collagenous organization to tensionaland weight-bearing connective tissues which display pheno-types in SLRP knockout mouse models. It is highly probablethat SLRPs have speci c roles to play in disc degenerationwhich await detailed elucidation.


19/25


4.8 Summary of Critical Concepts Discussedin the Chapter

Role of Proteoglycans in Disc FunctionAggrecan provides the disc with its ability to swell and•resist compressive loads. This property is dependent ontwo features of aggrecan: rst, its ability to form largeproteoglycan aggregates in association with HA, whichlimit its diffusion within the matrix, and second, theosmotic properties and water binding are due to its sub-stitution with numerous CS and KS chains. Aggrecan

swelling maintains disc height underload and is respon-sible for the diurnal variation in disc height associatedwith daily life.Versican and lubricin are present throughout the interverte-•bral disc, but their precise functions are unclear. The highabundance of versican in the immature disc may suggest arole in disc development during fetal life, and it has beensuggested that lubricin plays a role in enhancing motionbetween adjacent lamellae within the annulus brosus.Perlecan promotes and stabilizes matrix assembly through•its interaction with a diverse repertoire of matrix compo-nents. Roles are now also emerging for perlecan inbrillin-1 and elastin assembly in the disc, with the HSchains of perlecan being important. The HS chains of per-lecan also sequester a number of bioactive growth factorsand morphogens of relevance to developmental andremodeling processes in the disc through their abilities toregulate cell proliferation and differentiation. Furthermore,the ability of perlecan to interact with a number of cellattachment proteins may regulate cellular recruitmentduring development and remodeling of the disc.The SLRPs have important regulatory roles to play in col-•lagen brillogenesis and are important for disc extracellu-lar matrix assembly and repair. Emerging interactive rolesof SLRPs with cytokines and bioactive growth factorsimplicate them in cell signaling, affecting a diverse rangeof biological processes, including brosis, in ammation,and the immune response.

Role of Proteoglycans in Disc DiseaseAggrecan degradation is associated with disc degenera-•tion in the adult and probably in the juvenile with scolio-sis. The proteolytic degradation of aggrecan results in

Table 4.1 Pathological consequences of targeted ablation of SLRPgenes in mouse models

Ablated gene Pathology Phenotype ReferencesDecorin Collagen bril

structure in skinand tendonabnormal

Skin fragility Danielsonet al.( 1997 )

Reduced tendonfunction

Biglycan Decreased bonemass, structuralcollagen brilabnormalities inmedial aorta

Osteoporosis Heegaardet al.( 2007 ) andXu et al.( 1998 )

Spontaneousaortic dissection/ rupture

Lumican Abnormalcollagen brilorganization incornea and dermis

Skin fragility Chakravartiet al.( 1998 )

Corneal opacity

Fibromodulin Abnormalcollagen brilorganization intendon

Lax tendons withreducedbiomechanicalfunction

Svenssonet al.( 1999 )

Biglycan/ decorin

Abnormalcollagen bril

formation inbone, tendon,dermis

Similar toprogeroid form

of Ehlers-Danlossyndrome

Corsi et al.( 2002 )

Biglycan/ bromodulin

Maturationalstructural/ biomechanicalabnormalities incollagen brils intendon

Gait impairment,ectopiccalci cation,premature OA

Ameye andYoung( 2002 )

Lumican/ bromodulin

Abnormalcollagenmaturation andstructureof tendons

Joint laxity andimpaired tendonfunction

Jepsenet al.( 2002 )

Asporin Reducedinhibition ofperiodontalligamentmineralization

Diminishednegativeregulation ofperiodontalligament cellcytodifferentia-tion by pointmutationsdisrupting asporinLRR5-mediatedinteractions withBMP-2

Tomoedaet al.( 2008 ) andYamadaet al.( 2007 )

Table 4.2 Human diseases linked to mutations in SLRP genes

GeneType ofmutation

Pathology/clinicalphenotype References

Decorin Frameshiftmutationgenerating aC-terminaltruncated coreprotein

Corneal opacity,congenital stromaldystrophy ofcornea

Bredrup et al.( 2005 )

Lumican,bromodulin,PRELP

Intronicvariations,single-nucle-otidepolymor-phisms inpromoter

Corneal detach-ment, choroidalneovasculariza-tion, high myopia

Chen et al.( 2009 ), Majavaet al. ( 2007 ),andWang et al.( 2006 )

Asporin Asporin D4allelepolymor-phismsaffectingN-terminal of

core protein

Early onset of OAand discdegeneration

Gruber et al.( 2009 ) andSong et al.( 2008 )


20/25


fragments that are no longer able to interact with HA;these are slowly lost as their size is further decreased bycontinuing proteolysis. This decline in aggrecan contentof the degenerate disc may facilitate blood vessel andnerve ingrowth and contribute to discogenic pain.Mutations in the aggrecan gene result in osteochondrodys-plasia, the features of which include developmental

abnormalities in the disc.Versican and lubricin undergo extensive proteolytic frag-•mentation throughout life, which is likely caused by thesame proteinases responsible for aggrecan degradation. Itis, however, unclear whether this contributes to discpathology.Perlecan also undergoes extensive fragmentation within•the disc, though little is known as to how this occurs orwhether the released perlecan fragments provide newfunctional biological properties. An exception to this isthe C-terminal peptide, endorepellin, which has anti-angiogenic properties due to its ability to destabilize tubeformation by endothelial cells. In degenerate discs, if thefragments are not lost or further degraded, these proper-ties might be expected to maintain the avascular nature ofthe disc.A number of the SLRPs are also fragmented in degenerate•discs, and the loss of the CS/DS chains in decorin andbiglycan may adversely affect their abilities to interactwith cytokines and growth factors and thus their partici-pation in cell regulatory processes. Fragmentation oflumican and bromodulin is also evident in the degener-ate disc, but it is not known how this impacts tissuefunction.

Acknowledgements We would like to thank the Shriners of NorthAmerica and the Canadian Institutes of Health Research for support ofthe work described in this chapter. Support from the National Healthand Medical Research Council of Australia is also gratefullyacknowledged.

References

Adams MA, Roughley PJ (2006) What is intervertebral disc degenera-tion, and what causes it? Spine (Phila Pa 1976) 31:2151–2161

Agrawal A, Guttapalli A, Narayan S, Albert TJ, Shapiro IM, RisbudMV (2007) Normoxic stabilization of HIF-1alpha drives glycolyticmetabolism and regulates aggrecan gene expression in nucleus pul-posus cells of the rat intervertebral disk. Am J Physiol Cell Physiol293:C621–C631

Akeda K, An HS, Pichika R, Patel K, Muehleman C, Nakagawa K,Uchida A, Masuda K (2007) The expression of NG2 proteoglycanin the human intervertebral disc. Spine (Phila Pa 1976) 32:306–314

Ameye L, Young MF (2002) Mice de cient in small leucine-rich pro-teoglycans: novel in vivo models for osteoporosis, osteoarthritis,Ehlers-Danlos syndrome, muscular dystrophy, and corneal diseases.Glycobiology 12:107R–116R

Antonsson P, Heinegard D, Oldberg A (1991) Posttranslationalmodi cations of bromodulin. J Biol Chem 266:16859–16861

Antonsson P, Heinegard D, Oldberg A (1993) Structure and deducedamino acid sequence of the human bromodulin gene. BiochimBiophys Acta 1174:204–206

Arikawa-Hirasawa E, Watanabe H, Takami H, Hassell JR, Yamada Y(1999) Perlecan is essential for cartilage and cephalic development.Nat Genet 23:354–358

Arikawa-Hirasawa E, Wilcox WR, Le AH, Silverman N, Govindraj P,Hassell JR, Yamada Y (2001a) Dyssegmental dysplasia, Silverman-Handmaker type, is caused by functional null mutations of the per-lecan gene. Nat Genet 27:431–434

Arikawa-Hirasawa E, Wilcox WR, Yamada Y (2001b) Dyssegmentaldysplasia, Silverman-Handmaker type: unexpected role of perlecanin cartilage development. Am J Med Genet 106:254–257

Arikawa-Hirasawa E, Le AH, Nishino I, Nonaka I, Ho NC, FrancomanoCA, Govindraj P, Hassell JR, Devaney JM, Spranger J, StevensonRE, Iannaccone S, Dalakas MC, Yamada Y (2002) Structural andfunctional mutations of the perlecan gene cause Schwartz-Jampelsyndrome, with myotonic myopathy and chondrodysplasia. AmJ Hum Genet 70:1368–1375

Barry FP, Neame PJ, Sasse J, Pearson D (1994) Length variation in thekeratan sulfate domain of mammalian aggrecan. Matrix Biol14:323–328

Barry FP, Rosenberg LC, Gaw JU, Koob TJ, Neame PJ (1995) N- andO-linked keratan sulfate on the hyaluronan binding region of aggre-can from mature and immature bovine cartilage. J Biol Chem270:20516–20524

Bengtsson E, Neame PJ, Heinegard D, Sommarin Y (1995) The pri-mary structure of a basic leucine-rich repeat protein, PRELP, foundin connective tissues. J Biol Chem 270:25639–25644

Bengtsson E, Aspberg A, Heinegard D, Sommarin Y, Spillmann D(2000) The amino-terminal part of PRELP binds to heparin andheparan sulfate. J Biol Chem 275:40695–40702

Bengtsson E, Morgelin M, Sasaki T, Timpl R, Heinegard D, Aspberg A(2002) The leucine-rich repeat protein PRELP binds perlecan andcollagens and may function as a basement membrane anchor. J BiolChem 277:15061–15068

Bix G, Fu J, Gonzalez EM, Macro L, Barker A, Campbell S, Zu

proteoglycans of the intervertebral disc.pdf

Documents