The extracellular matrix is a component of all mammalian tissues, a network consisting largely of the fibrous proteins collagen, elastin and associated-microfibrils, fibronectin and laminins embedded in a viscoelastic gel of anionic proteoglycan polymers. It performs many functions in addition to its structural role; as a major component of the cellular microenvironment it influences cell behaviours such as proliferation, adhesion and migration, and regulates cell differentiation and death (Hynes 2009).
ECM composition is highly heterogeneous and dynamic, being constantly remodeled (Frantz et al. 2010) and modulated, largely by matrix metalloproteinases (MMPs) and growth factors that bind to the ECM influencing the synthesis, crosslinking and degradation of ECM components (Hynes 2009). ECM remodeling is involved in the regulation of cell differentiation processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. Redundant mechanisms modulate the expression and function of ECM modifying enzymes. Abnormal ECM dynamics can lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer.
Collagen is the most abundant fibrous protein within the ECM constituting up to 30% of total protein in multicellular animals. Collagen provides tensile strength. It associates with elastic fibres, composed of elastin and fibrillin microfibrils, which give tissues the ability to recover after stretching. Other ECM proteins such as fibronectin, laminins, and matricellular proteins participate as connectors or linking proteins (Daley et al. 2008).
Chondroitin sulfate, dermatan sulfate and keratan sulfate proteoglycans are structural components associated with collagen fibrils (Scott & Haigh 1985; Scott & Orford 1981), serving to tether the fibril to the surrounding matrix. Decorin belongs to the small leucine-rich repeat proteoglycan family (SLRPs) which also includes biglycan, fibromodulin, lumican and asporin. All appear to be involved in collagen fibril formation and matrix assembly (Ameye & Young 2002).
ECM proteins such as osteonectin (SPARC), osteopontin and thrombospondins -1 and -2, collectively referred to as matricellular proteins (reviewed in Mosher & Adams 2012) appear to modulate cell-matrix interactions. In general they induce de-adhesion, characterized by disruption of focal adhesions and a reorganization of actin stress fibers (Bornstein 2009). Thrombospondin (TS)-1 and -2 bind MMP2. The resulting complex is endocytosed by the low-density lipoprotein receptor-related protein (LRP), clearing MMP2 from the ECM (Yang et al. 2001).
Osteopontin (SPP1, bone sialoprotein-1) interacts with collagen and fibronectin (Mukherjee et al. 1995). It also contains several cell adhesive domains that interact with integrins and CD44.
Aggrecan is the predominant ECM proteoglycan in cartilage (Hardingham & Fosang 1992). Its relatives include versican, neurocan and brevican (Iozzo 1998). In articular cartilage the major non-fibrous macromolecules are aggrecan, hyaluronan and hyaluronan and proteoglycan link protein 1 (HAPLN1). The high negative charge density of these molecules leads to the binding of large amounts of water (Bruckner 2006). Hyaluronan is bound by several large proteoglycans proteoglycans belonging to the hyalectan family that form high-molecular weight aggregates (Roughley 2006), accounting for the turgid nature of cartilage.
The most significant enzymes in ECM remodeling are the Matrix Metalloproteinase (MMP) and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families (Cawston & Young 2010). Other notable ECM degrading enzymes include plasmin and cathepsin G. Many ECM proteinases are initially present as precursors, activated by proteolytic processing. MMP precursors include an amino prodomain which masks the catalytic Zn-binding motif (Page-McCawet al. 2007). This can be removed by other proteinases, often other MMPs. ECM proteinases can be inactivated by degradation, or blocked by inhibitors. Some of these inhibitors, including alpha2-macroglobulin, alpha1-proteinase inhibitor, and alpha1-chymotrypsin can inhibit a large variety of proteinases (Woessner & Nagase 2000). The tissue inhibitors of metalloproteinases (TIMPs) are potent MMP inhibitors (Brew & Nagase 2010).
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Collagen is a family of at least 29 structural proteins derived from over 40 human genes (Myllyharju & Kivirikko 2004). It is the main component of connective tissue, and the most abundant protein in mammals making up about 25% to 35% of whole-body protein content. A defining feature of collagens is the formation of trimeric left-handed polyproline II-type helical collagenous regions. The packing within these regions is made possible by the presence of the smallest amino acid, glycine, at every third residue, resulting in a repeating motif Gly-X-Y where X is often proline (Pro) and Y often 4-hydroxyproline (4Hyp). Gly-Pro-Hyp is the most common triplet in collagen (Ramshaw et al. 1998). Collagen peptide chains also have non-collagenous domains, with collagen subclasses having common chain structures. Collagen fibrils are mostly found in fibrous tissues such as tendon, ligament and skin. Other forms of collagen are abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc. In muscle tissue, collagen is a major component of the endomysium, constituting up to 6% of muscle mass. Gelatin, used in food and industry, is collagen that has been irreversibly hydrolyzed.
On the basis of their fibre architecture in tissues, the genetically distinct collagens have been divided into subgroups. Group 1 collagens have uninterrupted triple-helical domains of about 300 nm, forming large extracellular fibrils. They are referred to as the fibril-forming collagens, consisting of collagens types I, II, III, V, XI, XXIV and XXVII. Group 2 collagens are types IV and VII, which have extended triple helices (>350 nm) with imperfections in the Gly-X-Y repeat sequences. Group 3 are the short-chain collagens. These have two subgroups. Group 3A have continuous triple-helical domains (type VI, VIII and X). Group 3B have interrupted triple-helical domains, referred to as the fibril-associated collagens with interrupted triple helices (FACIT collagens, Shaw & Olsen 1991). FACITs include collagen IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI plus the transmembrane collagens (XIII, XVII, XXIII and XXV) and the multiple triple helix domains and interruptions (Multiplexin) collagens XV and XVIII (Myllyharju & Kivirikko 2004). The non-collagenous domains of collagens have regulatory functions; several are biologically active when cleaved from the main peptide chain. Fibrillar collagen peptides all have a large triple helical domain (COL1) bordered by N and C terminal extensions, called the N- and C-propeptides, which are cleaved prior to formation of the collagen fibril. The intact form is referred to as a collagen propeptide, not procollagen, which is used to refer to the trimeric triple-helical precursor of collagen before the propeptides are removed. The C-propeptide, also called the NC1 domain, directs chain association during assembly of the procollagen molecule from its three constituent alpha chains (Hulmes 2002).
Fibril forming collagens are the most familiar and best studied subgroup. Collagen fibres are aggregates or bundles of collagen fibrils, which are themselves polymers of tropocollagen complexes, each consisting of three polypeptide chains known as alpha chains. Tropocollagens are considered the subunit of larger collagen structures. They are approximately 300 nm long and 1.5 nm in diameter, with a left-handed triple-helical structure, which becomes twisted into a right-handed coiled-coil 'super helix' in the collagen fibril. Tropocollagens in the extracellular space polymerize spontaneously with regularly staggered ends (Hulmes 2002). In fibrillar collagens the molecules are staggered by about 67 nm, a unit known as D that changes depending upon the hydration state. Each D-period contains slightly more than four collagen molecules so that every D-period repeat of the microfibril has a region containing five molecules in cross-section, called the 'overlap', and a region containing only four molecules, called the 'gap'. The triple-helices are arranged in a hexagonal or quasi-hexagonal array in cross-section, in both the gap and overlap regions (Orgel et al. 2006). Collagen molecules cross-link covalently to each other via lysine and hydroxylysine side chains. These cross-links are unusual, occuring only in collagen and elastin, a related protein.
The macromolecular structures of collagen are diverse. Several group 3 collagens associate with larger collagen fibers, serving as molecular bridges which stabilize the organization of the extracellular matrix. Type IV collagen is arranged in an interlacing network within the dermal-epidermal junction and vascular basement membranes. Type VI collagen forms distinct microfibrils called beaded filaments. Type VII collagen forms anchoring fibrils. Type VIII and X collagens form hexagonal networks. Type XVII collagen is a component of hemidesmosomes where it is complexed wtih alpha6Beta4 integrin, plectin, and laminin-332 (de Pereda et al. 2009). Type XXIX collagen has been recently reported to be a putative epidermal collagen with highest expression in suprabasal layers (Soderhall et al. 2007). Collagen fibrils/aggregates arranged in varying combinations and concentrations in different tissues provide specific tissue properties. In bone, collagen triple helices lie in a parallel, staggered array with 40 nm gaps between the ends of the tropocollagen subunits, which probably serve as nucleation sites for the deposition of crystals of the mineral component, hydroxyapatite (Ca10(PO4)6(OH)2) with some phosphate. Collagen structure affects cell-cell and cell-matrix communication, tissue construction in growth and repair, and is changed in development and disease (Sweeney et al. 2006, Twardowski et al. 2007). A single collagen fibril can be heterogeneous along its axis, with significantly different mechanical properties in the gap and overlap regions, correlating with the different molecular organizations in these regions (Minary-Jolandan & Yu 2009).
Matrix metalloproteinases (MMPs), previously referred to as matrixins because of their role in degradation of the extracellular matrix (ECM), are zinc and calcium dependent proteases belonging to the metzincin family. They contain a characteristic zinc-binding motif HEXXHXXGXXH (Stocker & Bode 1995) and a conserved Methionine which forms a Met-turn. Humans have 24 MMP genes giving rise to 23 MMP proteins, as MMP23 is encoded by two identical genes. All MMPs contain an N-terminal secretory signal peptide and a prodomain with a conserved PRCGXPD motif that in the inactive enzyme is localized with the catalytic site, the cysteine acting as a fourth unpaired ligand for the catalytic zinc atom. Activation involves delocalization of the domain containing this cysteine by a conformational change or proteolytic cleavage, a mechanism referred to as the cysteine-switch (Van Wart & Birkedal-Hansen 1990). Most MMPs are secreted but the membrane type MT-MMPs are membrane anchored and some MMPs may act on intracellular proteins. Various domains determine substrate specificity, cell localization and activation (Hadler-Olsen et al. 2011). MMPs are regulated by transcription, cellular location (most are not activated until secreted), activating proteinases that can be other MMPs, and by metalloproteinase inhibitors such as the tissue inhibitors of metalloproteinases (TIMPs). MMPs are best known for their role in the degradation and removal of ECM molecules. In addition, cleavage of the ECM and other cell surface molecules can release ECM-bound growth factors, and a number of non-ECM proteins are substrates of MMPs (Nagase et al. 2006). MMPs can be divided into subgroups based on domain structure and substrate specificity but it is clear that these are somewhat artificial, many MMPs belong to more than one functional group (Vise & Nagase 2003, Somerville et al. 2003).
Elastic fibres (EF) are a major structural constituent of dynamic connective tissues such as large arteries and lung parenchyma, where they provide essential properties of elastic recoil and resilience. EF are composed of a central cross-linked core of elastin, surrounded by a mesh of microfibrils, which are composed largely of fibrillin. In addition to elastin and fibrillin-1, over 30 ancillary proteins are involved in mediating important roles in elastic fibre assembly as well as interactions with the surrounding environment. These include fibulins, elastin microfibril interface located proteins (EMILINs), microfibril-associated glycoproteins (MAGPs) and Latent TGF-beta binding proteins (LTBPs). Fibulin-5 for example, is expressed by vascular smooth muscle cells and plays an essential role in the formation of elastic fibres through mediating interactions between elastin and fibrillin (Yanigasawa et al. 2002, Freeman et al. 2005). In addition, it plays a role in cell adhesion through integrin receptors and has been shown to influence smooth muscle cell proliferation (Yanigasawa et al. 2002, Nakamura et al. 2002). EMILINs are a family of homologous glycoproteins originally identified in extracts of aortas. Found at the elastin-fibrillin interface, early studies showed that antibodies to EMILIN can affect the process of elastic fibre formation (Bressan et al. 1993). EMILIN1 has been shown to bind elastin and fibulin-5 and appears to coordinate their common interaction (Zanetti et al. 2004). MAGPs are found to co-localize with microfibrils. MAGP-1, for example, binds strongly to an N-terminal sequence of fibrillin-1. Other proteins found associated with microfibrils include vitronectin (Dahlback et al. 1990).
Fibrillin is most familiar as a component of elastic fibres but microfibrils with no elastin are found in the ciliary zonules of the eye and invertebrate circulatory systems. The addition of elastin to microfibrils is a vertebrate adaptation to high pulsatile pressures in their closed circulatory systems (Faury et al. 2003). Elastin appears to have emerged after the divergence of jawless vertebrates from other vertebrates (Sage 1982).
Fibrillin-1 is the major structural component of microfibrils. Fibrillin-2 is expressed earlier in development than fibrillin-1 and may be important for elastic fiber formation (Zhang et al. 1994). Fibrillin-3 arose as a duplication of fibrillin-2 that did not occur in the rodent lineage. It was first isolated from human brain (Corson et al. 2004).
Fibrillin assembly is not as well defined as elastin assembly. The primary structure of fibrillin is dominated by calcium binding epidermal growth factor like repeats (Kielty et al. 2002). Fibrillin may form dimers or trimers before secretion. However, multimerisation predominantly occurs outside the cell. Formation of fibrils appears to require cell surface structures suggesting an involvement of cell surface receptors. Fibrillin is assembled pericellularly (i.e. on or close to the cell surface) into microfibrillar arrays that undergo time dependent maturation into microfibrils with beaded-string appearance. Transglutaminase forms gamma glutamyl epsilon lysine isopeptide bonds within or between peptide chains. Additionally, intermolecular disulfide bond formation between fibrillins is an important contributor to fibril maturation (Reinhardt et al. 2000).
Models of fibrillin-1 microfibril structure suggest that the N-terminal half of fibrillin-1 is asymmetrically exposed in outer filaments, while the C-terminal half is buried in the interior (Kuo et al. 2007). Fibrillinopathies include Marfan syndrome, familial ectopia lentis, familial thoracic aneurysm, all due to mutations in the fibrillin-1 gene FBN1, and congenital contractural arachnodactyly which is caused by mutation of FBN2 (Maslen & Glanville 1993, Davis & Summers 2012).
In vivo assembly of fibrillin requires the presence of extracellular fibronectin fibres (Sabatier et al. 2009). Fibrillins have Arg-Gly-Asp (RGD) sequences that interact with integrins (Pfaff et al. 1996, Sakamoto et al. 1996, Bax et al., 2003, Jovanovic et al. 2008) and heparin-binding domains that interact with a cell-surface heparan sulfate proteoglycan (Tiedemann et al. 2001) possibly a syndecan (Ritty et al. 2003). Fibrillins also have a major role in binding and sequestering growth factors such as TGF beta into the ECM (Neptune et al. 2003). Proteoglycans such as versican (Isogai et al. 2002), biglycan, and decorin (Reinboth et al. 2002) can interact with the microfibrils. They confer specific properties including hydration, impact absorption, molecular sieving, regulation of cellular activities, mediation of growth factor association, and release and transport within the extracellular matrix (Buczek-Thomas et al. 2002). In addition, glycosaminoglycans have been shown to interact with tropoelastin through its lysine side chains (Wu et al. 1999), regulating tropoelastin assembly (Tu & Weiss 2008).
Elastin is synthesized as a 70kDa monomer called tropoelastin, a highly hydrophobic protein composed largely of two types of domains that alternate along the polypeptide chain. Hydrophobic domains are rich in glycine, proline, alanine, leucine and valine. These amino acids occur in characteristic short (3-9 amino acids) tandem repeats, with a flexible and highly dynamic structure (Floquet et al. 2004). Unlike collagen, glycine in elastin is not rigorously positioned every 3 residues. However, glycine is distributed frequently throughout all hydrophobic domains of elastin, and displays a strong preference for inter-glycine spacing of 0-3 residues (Rauscher et al. 2006).
Elastic fibre formation involves the deposition of tropoelastin onto a template of fibrillin rich microfibrils. Recent results suggest that the first step of elastic fiber formation is the organization of small globules of elastin on the cell surface followed by globule aggregation into microfibres (Kozel et al. 2006). An important contribution to the initial stages assembly is thought to be made by the intrinsic ability of the protein to direct its own polymeric organization in a process termed 'coacervation' (Bressan et al. 1986). This self-assembly process appears to be determined by interactions between hydrophobic domains (Bressan et al. 1986, Vrhovski et al. 1997, Bellingham et al. 2003, Cirulis & Keeley 2010) which result in alignment of the cross-linking domains, allowing the stabilization of elastin through the formation of cross-links generated through the oxidative deamination of lysine residues, catalyzed by members of the lysyl oxidase (LOX) family (Reiser et al. 1992, Mithieux & Weiss 2005). The first step in the cross-linking reaction is the oxidative formation of the delta aldehyde, known as alpha aminoadipic semialdehyde or allysine (Partridge 1963). Subsequent reactions that are probably spontaneous lead to the formation of cross-links through dehydrolysinonorleucine and allysine aldol, a trifunctional cross-link dehydromerodesmosine and two tetrafunctional cross-links desmosine and isodesmosine (Lucero & Kagan 2006), which are unique to elastin. These cross-links confer mechanical integrity and high durability. In addition to their role in self-assembly, hydrophobic domains provide elastin with its elastomeric properties, with initial studies suggesting that the elastomeric propereties of elastin are driven through changes in entropic interactions with surrounding water molecules (Hoeve & Flory 1974).
A very specific set of proteases, broadly grouped under the name elastases, is responsible for elastin remodelling (Antonicelli et al. 2007). The matrix metalloproteinases (MMPs) are particularly important in elastin breakdown, with MMP2, 3, 9 and 12 explicitly shown to degrade elastin (Ra & Parks 2007). Nonetheless, elastin typically displays a low turnover rate under normal conditions over a lifetime (Davis 1993).
Syndecans are type I transmembrane proteins, with an N-terminal ectodomain that contains several consensus sequences for glycosaminoglycan (GAG) attachment and a short C-terminal cytoplasmic domain. Syndecan-1 and -3 GAG attachment sites occur in two distinct clusters, one near the N-terminus and the other near the membrane-attachment site, separated by a proline and threonine-rich 'spacer'. Syndecan ectodomain sequences are poorly conserved in the family and between species, but the transmembrane and cytoplasmic domains are highly conserved. Syndecan-1 and -3 form a subfamily. Syndecan core proteins form dimers (Choi et al. 2007) and at least syndecan-3 and -4 form oligomers (Asundi & Carey 1995, Shin et al. 2012). Syndecan-1 is the major syndecan of epithelial cells including vascular endothelium. Syndecan-2 is present mostly in mesenchymal, neuronal and smooth muscle cells. Syndecan-3 is the major syndecan of the nervous system, while syndecan-4 is ubiquitously expressed but at lower levels than the other syndecans (refs in Alexopoulou et al. 2007). The core syndecan protein has three to five heparan sulfate or chondroitin sulfate chains, which interact with a variety of ligands including fibroblast growth factors, vascular endothelial growth factor, transforming growth factor-beta, fibronectin, collagen, vitronectin and several integrins. Syndecans may act as integrin coreceptors. Interactions between fibronectin and syndecans are modulated by tenascin-C.
Alpha5beta1 integrin was the first integrin shown to bind fibronectin (FN1). Unlike other FN1-binding integrins it is a specialist at this task. In solution FN1 occurs as a dimer. Binding to alpha5beta1 integrin stimulates FN1 self-association; blocking the RGD-cell binding domain of FN1 blocks fibril formation (Fogerty et al. 1990). FN1 binding is believed to induce integrin clustering, which promotes FN1-FN1 interactions. Integrin clustering is mediated by association between integrins and intracellular actin stress fibers (Calderwood et al. 2000). Binding of integrins to each of the monomers in the FN1 dimer pair is thought to trigger a conformational change in FN1 that exposes 'cryptic' FN1 binding sites that allow additional fibronectin dimers to bind without the requirement for pre-association with integrins (Singh et al. 2010). This non-covalent interaction may involve interactions with fibrillin (Ohashi & Erickson 2009). I1-5 functions as a unit that is the primary FN matrix assembly domain (Sottile et al. 1991) but other units are likely to be involved (Singh et al. 2010). Other integrins able to bind FN1 include alphaIIbBeta3, which is highly expressed on platelets where it predominantly binds fibrinogen leading to thrombus formation but also binds FN1 (Savage et al. 1996). Alpha4beta1 mediates cell-cell contacts and cell-matrix contacts through the ligands VCAM-1 and FN1, respectively (Humphries et al. 1995). Integrins alpha3beta1, alpha4beta7 and alphaVbeta1, 3 and 6 are all reported to bind FN1 (Johansson et al. 1997)
Tenacious binding of free fibronectin to cells leads to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. This process is enhanced in the presence of CEACAM molecules.
The binding of integrins to each of the monomers in the FN1 dimer pair is thought to trigger a conformational change in FN1 that allows additional FN1 dimers to bind without pre-association with integrins (Singh et al. 2010). Domain I1-5 functions as the primary unit of FN1 matrix assembly (Sottile et al. 1991) but the process is not fully characterised and other FN1 units are likely to be involved (Singh et al. 2010). In this reaction an arbitrary 10 FN1 monomers are represented as being incorporated into the FN1 polymeric matrix.
Prior to matrix formation, fibronectin (FN1) exists as a protein dimer. Often the two peptide chains are differentially-spliced variants. The chains are linked by a pair of C-terminal disulfide bonds which are essential for subsequent multimerization (Schwarzbaur 1991). FN1 monomers have a molecular weight of 230-270 kDa depending on the alternative splicing and contain three types of repeating domains, type I, II, and III. Type I and II domains are stabilized by intra-chain disulfide bonds. FN1 dimer binding to alpha5beta1 integrin stimulates self-association.
The somatomedin B domain of vitronectin (VTN) binds to and stabilizes plasminogen activator inhibitor-1 (PAI1) (Declerck et al. 1988). PAI1 is the principal physiological inhibitor of both tissue (tPA) and urokinase (uPA) plasminogen activators and a key regulator of the fibrinolytic system; the stabilization of PAI1 by VTN thereby regulates proteolysis of fibrin (Zhou et al. 2003). Elevated PAI1 activity is associated with coronary thrombosis (Hamsten et al. 1987) and poor prognosis in many cancers.
Dystroglycan (DG) is a cell-surface laminin receptor. In skeletal muscle it is a central component of the dystrophin-glycoprotein (DGC) complex (Ervasti & Campbell 1991). Mutations in components of the DGC render muscle fibres more susceptible to damage and lead to various types of muscle disorder such as Duchenne muscular dystrophy and limb-girdle muscular dystrophies (Straub & Campbell, 1997, Cohn & Campbell 2000). DG is present as non-covalently associated alpha and beta subunits following cleavage at Ser654. The extracellular alpha subunit binds to laminin-2 (merosin) in the muscle basement membrane while the membrane-associated beta subunit binds dystrophin, which associates with the actin cytoskeleton (Ervasti & Campbell 1993, Yamada et al. 1994, Talts et al. 1999). Alpha-DG also binds the carboxy-terminal G domains of laminin alpha-1 (Gee et al. 1993, Zhou et al. 2012) and alpha-5 (Yu & Talts 2003). G domains are relatively well conserved in all five alpha-laminin chains, so DG is likely to bind all laminin heterotrimers.
Biglycan is a member of the small leucine-rich repeat proteoglycan family (SLRPs) which also includes decorin, fibromodulin (Hedlund et al. 1994 - binding to collagen II), lumican and asporin (Hedbom & Heinegard 1993, Ezura et al. 2000). All appear to be involved in collagen fibril formation and matrix assembly (Ameye & Young 2002).
Biglycan binds collagen types I (Schönherr et al. 1995), II (Bovine, using pig byglycan - Vynios et al. 2001, Bovine, using bovine biglycan - Douglas et al. 2008), III (Bovine, using bovine biglycan - Douglas et al. 2008), VI (Wiberg et al. 2001, 2002, human) and IX (Chen et al. 2006 - species source of collagen/biglycan unknown).
Alpha-dystroglycan (DG) binds G domain-like sequences in other extracellular matrix molecules such as AGRN (agrin) (Gee et al. 1994, Campanelli et al. 1994, Sugiyama et al. 1994, Yamada et al. 1996, Gesemann et al. 1996) and HSPG2 (perlecan) (Peng et al. 1998, Talts et al. 1999). DG knockout mice exhibit severe disruption of basement membranes and embryonic stem cells fail to deposit laminin, suggesting a role for DG in laminin matrix formation (Henry & Campbell 1998). Conditional ablation of DG expression in cultured mammary epithelial cells disrupted laminin-111-induced polarity and beta-casein production, and abolished laminin binding and assembly on the cell surface. Dystroglycan re-expression restored these deficiencies (Weir et al. 2006).
In articular cartilage the major non-fibrous macromolecules are aggrecan, hyaluronan (HA) and hyaluronan and proteoglycan link protein 1 (HAPLN1). The high negative charge density of these molecules leads to the binding of large amounts of water (Bruckner 2006). HA is bound by large aggregating proteoglycans (the hyalectans). Aggrecan (ACAN) is predominantly expressed in cartilage, versican is widely distributed, while brevican and neurocan are largely restricted to nervous tissues. ACAN is ~90% carbohydrate. The core protein is highly glycosylated, mostly by the glycosaminoglycan (GAG) chains chondroitin sulphate (CS) and keratan sulphate (KS). Each ACAN molecule has ~100 CS chains of around 20 kDa and ~60 KS chains of 5-15 kDa. CS is attached to an extended domain between globular domains 2 and 3, while KS is widely distributed. The core protein also contains sites for the attachment of N-linked and O-linked oligosaccharides (Nilsson et al. 1982).
The G1 N-terminal domain of ACAN has a lectin-like binding site with high affinity for HA (Watanabe et al. 1997, Hardingham 2006). HA is a long unbranched, unsulphated GAG synthesized free from protein attachment by three HA synthases (Spicer & McDonald 1998). It has an average molecular weight of several million Da. HA content steadily rises in aging cartilage and can reach 10% of the total GAG. ACAN, HA and the small glycoprotein HAPLN1, known as Link protein, are found in huge multi-molecular aggregates comprised of numerous ACAN monomers non-covalently bound to HA, stabilized by HAPLN1 which forms a ternary complex with the G1 domain of ACAN and HA (Ratcliffe & Hardingham 1983, Grover & Roughley 1994, Kiani et al. 2002).
Agrin (AGRN) is a multidomain heparan sulfate proteoglycan found in basement membranes, named for its ability to promote aggregation of AChR clusters on the muscle surface directly beneath the nerve terminal (Nitkin et al. 1987). It is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction; synaptogenesis is profoundly disrupted in its absence (Gautam et al. 1996, Daniels 2012). Two alternate N-termini exist with differential expression, tissue localization and function. The secreted and predominant longer LN form (Burgess et al. 2000) starts with a secretion signal sequence and a laminin-binding domain (Denzer et al. 1995, Kammerer et al. 1999); the shorter SN form associates with the plasma membrane (Burgess et al. 2000, Neumann et al. 2001). Following the SN or LN regions are 8 follistatin repeats, known to bind growth factors and inhibit proteases in other proteins. The central region has two repeats homologous to domain III of laminin. The C-terminal portion, which is responsible for the molecule's known signaling functions, contains four EGF repeats and three LG (G) domains homologous to those found in laminin alpha chains, neurexins and slits (Timpl et al. 2000).
The LG domains of AGRN bind alpha-dystroglycan (Yamada et al. 1996, Gee et al. 1994, Bowen et al. 1996, Campanelli et al. 1996, Gesemann et al. 1996, Hopf & Hoch 1996).
Tenascins are a family of 4 oligomeric extracellular glycoproteins, tenascin (TN) C, R, X, and W. In rotary shadowing images TNC is seen as a symmetrical structure called a hexabrachion (Erickson & Iglesias 1984). This hexamer is formed from initial trimers (Kammerer et al. 1988). All members of the family are believed able to form trimers but only C, R and W have the extra cysteine required for form hexamers. All have amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain (Hsia & Schwartzbauer 2005). TNC was the first family member to be discovered and is the best characterised (Midwood et al. 2011). Its subunits vary greatly in size (between 190 and 330 kDa of the tenascin-C monomer) due to glycosylation and splicing isoforms (Joester & Faissner 1999). During embryonic development TNC is expressed in neural, skeletal, and vascular tissues. In adults it is detectable only in tendon and tissues undergoing remodeling processes such as wound repair and neovascularization, or in pathological processes such as inflammation and tumorigenesis (Midwood & Orend, 2009).
TNC binds several integrins including alpha2beta1 (Sriramararo et al. 1993), alphaVbeta6 (Yokosaki et al. 1996), alphaVbeta3 (Sriramararo et al. 1993, Yokosaki et al. 1996), alpha9beta1 (Yokosaki et al. 1996), alphaXbeta1 (Probstmeier & Peshva 1999), alpha8beta1 (Schnapp 1995) and alpha7beta1 (Mercado et al. 2004).
Small leucine rich repeat proteoglycans (SLRPs) are a family of extracellular glycoproteins that includes decorin (DCN), biglycan (BGN), fibromodulin, lumican and asporin (Hedbom & Heinegard 1993, Ezura et al. 2000, Schaefer & Iozzo 2008, Iozzo & Schaefer 2010). DCN inhibits cellular proliferation in a TGF-Beta-dependent manner in Chinese hamster ovary (CHO) cells (Yamaguchi et al. 1990), arterial smooth muscle cells (Fischer et al. 2001), human hepatic stellate cells (Shi et al. 2006) and fibroblasts (Zhang et al. 2007). DCN, BGN and fibromodulin can all bind to TGF-Beta (Hildebrand 1994). Binding is mediated by the leucine rich repeat suggesting that all members of the SLRP family have TGF-beta binding capability (Schönherr et al. 1998). DCN has independent binding sites for collagen and TGF-Beta (Schönherr et al. 1998, Cabello-Verrugio et al. 2012). DCN binding is thought to sequester TGF-Beta extracellularly, thereby diminishing its biological activity (Markmann et al. 2000). DCN treatment has beneficial effects in fibrotic disorders involving TGF-Beta overproduction (Border et al. 1992; Kolb et al. 2001, Baghy et al. 2012). BGN attenuates the proliferative actions of TGF-beta1 on fibroblasts (Kobayashi et al. 2003). DCN and BGN appear to mediate crosstalk between Toll-like receptors (TLRs), NOD-like receptors (NLRs) and transforming growth factor Beta (TGFBeta) receptors (reviewed in Moreth et al. 2012).
Dystroglycan binds specifically to a subset of neurexin LNS domains in a tight interaction that requires the glycosylation of dystroglycan and is regulated by neurexin alternative splicing. Alpha-dystroglycan binds G domain-like sequences in neurexin-1-alpha (NRXN1) (Sugita et al. 2001).
Vitronectin (VTN) is a major plasma glycoprotein of 75 kDa, circulating at approximately 0.2 mg/ml in humans. It interacts with collagen types I, II, III, IV, V, and VI (Gebb et al. 1986). Deglycosylation enhances VTN binding to collagen and is associated with VTN multimerization (Uchibori-Iwaki et al. 2000, Sano et al. 2007).
Agrin (AGRN) is a large (>400 kDa) multi-domain heparan sulfate proteoglycan found in basement membranes. It is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction; synaptogenesis is profoundly disrupted in its absence (Gautam et al. 1996). Two alternate N-termini exist with differential expression, tissue localization and function. The predominant longer LN form (Burgess et al. 2000) starts with a secretion signal sequence and a laminin-binding domain (Denzer et al. 1995, Kammerer et al. 1999); the shorter SN form associates with the plasma membrane (Burgess et al. 2000, Neumann et al. 2001). Following the SN or LN regions are 8 follistatin repeats, known to bind growth factors and inhibit proteases in other proteins. The central region has two repeats homologous to domain III of laminin. The C-terminal portion, which is responsible for the molecule's known signalling functions, contains four EGF repeats and three LG (G) domains homologous to those found in laminin alpha chains, neurexins and slits (Timpl et al. 2000).
The N-terminus of the LN form of AGRN binds to the laminin gamma1 subunit (Denzer et al. 1997, Kammerer et al. 1999, Mascarenhas et al. 2003). This may indirectly bind AGRN to integrins on the cell surface (Bezakova & Ruegg 2003).
Several agrin (AGRN) ligands require the presence of heparan-sulfate sidechains and are probably mediated by them. Membrane-associated AGRN ligands include the neural cell adhesion molecule NCAM1 (Burg et al. 1995, Tsen et al. 1995, Cole & Halfter 1996 - represented in REACT_19071) and receptor protein tyrosine phosphatase sigma (PTPRS) (Aricescu et al. 2002).
Decorin (DCN) belongs to the small leucine-rich repeat proteoglycan family (SLRPs) which also includes biglycan, fibromodulin (Hedlund et al. 1994 - binding to collagen II), lumican and asporin (Hedbom & Heinegard 1993, Ezura et al. 2000). Fibromodulin and lumican bind the same site while the binding site for decorin is distinct (Hedbom & Heinegard 1993). All appear to be involved in collagen fibril formation and matrix assembly (Ameye & Young 2002, Kalamajski & Oldberg 2010). DCN consists of a core protein of approximately 40 kDa attached to a single chondroitin or dermatan sulfate glycosaminoglycan (GAG) chain. It interacts with collagen types I, II (Vogel et al. 1984), III (Witos et al. 2011), V (Whinna et al. 1993), VI (Bidanset et al. 1992) and XIV (Ehnis et al. 1997). It binds collagen I and II near the N-terminus, placing it at the 'd' band gap in the fibril structure (Kalamajski et al. 2007). The binding site for DCN on collagen XIV is in the NH2-terminal fibronectin type III repeat. In addition, an auxiliary binding site located COOH-terminally to this fibronectin type III repeat interacts with the glycosaminoglycan component of DCN.
DCN binding regulates fibrillogenesis (Vogel et al. 1984, Orgel et al. 2006). One molecule of DCN interacts with four to six collagen molecules. The interaction is between collagen and the core protein, not the GAG chain, and is more likely to involve the monomeric, not dimeric form (Orgel et al. 2009). Fibronectin (Winnemoller et al. 1991) and thrombospondin-1 (Winnemoller et al. 1992) are also DCN interactors. DCN acts as a sink for all three isoforms of TGF-Beta, binding them when already bound to collagen (Markmann et al. 2000). Degradation of DCN by matrix metalloproteinases MMP-2, -3 or -7 results in release of TGF-beta (Imai et al. 1997). In addition, DCN binds to EGFR (Iozzo et al. 1999) causing prolonged down-regulation of EGFR-mediated mobilization of intracellular calcium (Csordás et al. 2000).
Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, binds Collagen type I, hydroxyapatite and Ca2+, suggesting a role in the mineralization of bone and cartilage (Termine et al. 1981). It is expressed by osteoblasts, odontoblasts, and many other cell types (Romanowski et al. 1990, Mundlos et al. 1992, Papagerakis et al. 2002). SPARC expression has been used to follow the progression of osteoblast cytodifferentiation.
Several agrin (AGRN) ligands require the presence of heparan-sulfate GAG sidechains and probably represent interactions with them. Extracellular ligands include Beta-amyloid (Donahue et al. 1999, Cotman et al. 2000). Other ligands (unconfirmed in humans) include alpha-synuclein fibrils (chicken - Liu et al. 2005), HB-GAM/pleiotropin (Dagget et al. 1996), thrombospondin and FGF2 (Cotman et al. 1999).
Tenascins are a family of 4 oligomeric extracellular glycoproteins, tenascin (TN) C, R, X, and W. In rotary shadowing images TNC is seen as a symmetrical structure called a hexabrachion (Erickson & Iglesias 1984). This hexamer is formed from initial trimers (Kammerer et al. 1988). All members of the family are believed able to form trimers but only C, R and W have the extra cysteine required for form hexamers. All have amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain (Hsia & Schwartzbauer 2005). TNC was the first to be discovered and is the best characterised. Its subunits vary greatly in size due to glycosylation and splicing isoforms (Joester & Faissner 1999). During embryonic development TNC is expressed in neural, skeletal, and vascular tissues. In adults it is detectable only in tendon and tissues undergoing remodeling processes such as wound repair and neovascularization, or in pathological processes such as inflammation and tumorigenesis. TNR forms dimers and trimers (Norenberg et al. 1992) and is expressed only in the central nervous system. TNC and TNR-null mice (single and double knock-outs) have surprisingly normal gross phenotypes, but exhibit behavioural and wound healing abnormalities (Mackie & Tucker 1999, Montag-Sallaz & Montag 2003). TNX is the largest member of the family and is widely expressed during development, but in adults is limited to musculoskeletal, cardiac, and dermal tissue. It can form trimers, though it lacks the amino-terminal cysteine residues involved in hexamer formation. It is clearly associated with a variant of a heritable connective tissue disorder known as Ehler-Danlos Syndrome, which is associated with fibrillar collagen defects (Burch et al. 1997, Mao et al. 2002). TNY is thought to be an avian orthologue of TNX (Chiquet-Ehrismann 2004). TN-W, first identified in zebrafish (Weber et al. 1998), is the least well characterized member of the tenascin family. It forms trimers and is expressed in developing skeletal tissue and neural crest cells, a pattern that partially overlaps with TNC.
TNC and TNR bind with high affinity to fibronectin (FN) (Chiquet-Ehrismann et al. 1991, Chung et al. 1995, Chung & Erickson 1997, Hauzenberger et al. 1999, Ingham et al. 2004, To & Midwood 2011, Pesheva et al. 1994), modulating the cell adhesion function of FN either by binding or restricting access of FN to integrin binding sites (Lightner & Erickson 1990) or by binding to cell receptors and altering their responsiveness to FN (Prieto et al. 1992, Fischer et al. 1997). The interaction of Tenascin and FN impacts tissue structure by controlling the assembly, maintenance, and turnover of the ECM at the cell surface (To & Midwood 2010).
Biglycan (BGN) is a member of the small leucine-rich repeat proteoglycan family (SLRPs) which also includes decorin, fibromodulin (Hedlund et al. 1994 - binding to collagen II), lumican and asporin (Hedbom & Heinegard 1993, Ezura et al. 2000). All appear to be involved in collagen fibril formation and matrix assembly (Ameye & Young 2002).
BGN-deficient mice exhibit larger and irregular fibrils leading to thin dermis and reduced bone mass (Corsi et al. 2002, Xu et al. 1998). BGN binds collagen types I (Schönherr et al. 1995), II (Bovine, using pig BGN - Vynios et al. 2001, Bovine, using bovine BGN - Douglas et al. 2008), III (Bovine, using bovine BGN - Douglas et al. 2008), VI (Wiberg et al. 2001) and IX (Chen et al. 2006 - species source of collagen/BGN unknown).
Cartilage oligomeric matrix protein (COMP, thrombospondin-5) is a 524-kDa pentameric glycoprotein expressed primarily in cartilage, tendon, ligament and synovium. In adult cartilage, COMP is located primarily in the inter-territorial matrix between chondrocytes (Murphy et al. 1999). The mature protein is pentameric with each monomer linked to its neighbour by a disulphide bond, located at the amino terminus of the protein (Hedbom et al. 1992, Morgelin et al. 1992). COMP binds directly to collagen types I, II and IX (Rosenberg et al. 1998, Thur et al. 2001) at the fibril periphery. In addition it binds fibronectin (FN1) (Di Cesare et al. 2002), matrilins 1, 3 and 4 (Mann et al. 2004), and through the glycosaminoglycans heparan sulphate and chondroitin sulphate to aggrecan (Hauser et al. 1996, Chen et al. 2007). Mutations in COMP lead to pseudoachondroplasia and multiple epiphyseal dysplasia (Jackson et al. 2012). COMP binding to FN1 and probably to other partners requires the presence of the divalent cations Ca2+, Mg2+ or Mn2+. Each COMP subunit binds approximately 10 calcium ions (Chen et al. 2000).
Agrin (AGRN) is a large multidomain heparan sulfate proteoglycan found in basement membranes, named for its ability to promote aggregation of AChR clusters on the muscle surface directly beneath the nerve terminal (Nitkin et al. 1987). It is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction; synaptogenesis is profoundly disrupted in its absence (Gautam et al. 1996). Two alternate N termini exist with differential expression, tissue localization and function. The predominant longer LN form (Burgess et al. 2000) starts with a secretion signal sequence and a laminin-binding domain (Denzer et al. 1995, Kammerer et al. 1999); the shorter SN form associates with the plasma membrane (Burgess et al. 2000, Neumann et al. 2001). Following the SN or LN regions are 8 follistatin repeats, known to bind growth factors and inhibit proteases in other proteins. The central region has two repeats homologous to domain III of laminin. The C-terminal portion, which is responsible for the molecule's known signaling functions, contains four EGF repeats and three LG (G) domains homologous to those found in laminin alpha chains, neurexins and slits (Timpl et al. 2000).
AGRN binds a complex of the tyrosine kinase receptor MuSK, which is responsible for mediating agrin's ability to cluster AChR (Glass et al. 1996, Sanes & Lichtman 2001, Burden et al. 2003) and the coreceptor LRP4 (Kim et al. 2008, Zhang et al. 2008, Zong et al. 2012).
Integrin alphaVbeta3 is sometimes referred to as the 'vitronectin receptor'. Vitronectin interacts with integrins alphaVbeta1 (Marshall et al. 1995), alphaVbeta3 (Pytela et al. 1985), alphaVbeta5 (Panetti & McKeown-Longo 1993) and alpha2b beta3 (Pytela et al. 1986) through Arg-Gly-Asp (RGD) cell binding sequences.
Endothelial cells lining the microvascular wall form a semi-permeable barrier to the movement of blood components. The attachment of endothelial cells to the extracellular matrix (ECM) is largely mediated by transmembrane integrins which recognize short sequence motifs such as Arg-Gly-Asp (RGD) in many ECM proteins.
Integrin alpha5beta1 and alphaVbeta3 bind to the ECM proteins fibronectin and vitronectin respectively. Both are critical for the establishment and stabilization of endothelial monolayers (Cheng & Kramer 1989). Synthetic peptides that compete with ECM proteins for the integrins or antibodies directed against alpha5beta1 and alphaVbeta3 cause endothelial cell detachment (Hayman et al. 1985, Pierschbacher & Ruoslahti 1987).
Netrins are a family of extracellular proteins that include axonal guidance factors. They have N-terminal domains that are homologous to the LN domains of laminins. Netrin-4 (NTN4), but not other forms of netrin, bind laminin gamma-1 and gamma-3 short arms in the basement membrane, suggesting a role in regulating basement membrane formation (Schneiders et al. 2007). NTN4 has been found associated in a functional complex with laminin gamma-1 chain and integrin alpha6beta1, suggesting a role in regulation of neurogenesis in the olfactory system (Staquicini et al. 2009).
Discoidin domain receptors (DDRs) are a subfamily of receptor tyrosine kinases, the only members known to respond to an ECM component. DDR2 binds the major fibrillar collagens types I, II, III, and V) and the non-fibrillar collagen X (Shrivastava et al. 1997, Vogel et al. 1997, Leitinger & Kwan 2006). DDR proteins bind collagen as dimers (Leitinger 2003, 2011). DDR2 is confined to mesenchymal cells where it controls developmental processes and regulates cell adhesion, migration, proliferation, and remodelling of the extracellular matrix by controlling the expression and activity of matrix metalloproteinases (Leitinger & Hohenester 2007).
Tenascins are a family of 4 oligomeric extracellular glycoproteins, tenascin (TN) C, R, X, and W. In rotary shadowing images TNC is seen as a symmetrical structure called a hexabrachion (Erickson & Iglesias 1984). This hexamer is formed from initial trimers (Kammerer et al. 1988). All members of the family are believed able to form trimers but only C, R and W have the extra cysteine required for form hexamers. All have amino-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain (Hsia & Schwartzbauer 2005). TNC was the first family member to be discovered and is the best characterised. Its subunits vary greatly in size (between 190 and 330 kDa of the tenascin-C monomer) due to glycosylation and splicing isoforms (Joester & Faissner 1999). During embryonic development TNC is expressed in neural, skeletal, and vascular tissues. In adults it is detectable only in tendon and tissues undergoing remodeling processes such as wound repair and neovascularization, or in pathological processes such as inflammation and tumorigenesis (Midwood & Orend 2009). TNR forms dimers and trimers (Norenberg et al. 1992) and is expressed only in the developing and adult central nervous system. TNC and TNR-null mice (single and double knock-outs) have surprisingly normal gross phenotypes, but exhibit behavioural and wound healing abnormalities (Mackie & Tucker 1999, Montag-Sallaz & Montag 2003). TNX is the largest member of the family and is widely expressed during development, but in adults is limited to musculoskeletal, cardiac, and dermal tissue. It can form trimers, though it lacks the amino-terminal cysteine residues involved in hexamer formation. It is clearly associated with a variant of a heritable connective tissue disorder known as Ehler-Danlos Syndrome, which is associated with fibrillar collagen defects (Burch et al. 1997, Mao et al. 2002). TNY is thought to be an avian orthologue of TNX (Chiquet-Ehrismann 2004). TNW, first identified in zebrafish (Weber et al. 1998), is the least well characterized member of the tenascin family. It forms trimers and is expressed in developing skeletal tissue and neural crest cells, a pattern that partially overlaps with TNC.
TNC and TNR bind to members of the lectican family, a class of extracellular chondroitin sulfate proteoglycans consisting of aggrecan, versican, brevican and neurocan. TNC binds aggrecan (Lundell et al. 2004), versican (Tsujii et al. 2006) and neurocan (Milev et al. 1994, Grumet et al. 1994, Rauch et al. 1997). TNR binds aggrecan (Aspberg et al. 1997, Lundell et al. 2004), versican (Aspberg et al. 1995, 1997), brevican Aspberg et al. 1997, Hagihara et al. 1999) and neurocan (Aspberg et al. 1997).
Discoidin domain receptors (DDRs) are a subfamily of receptor tyrosine kinases, the only members known to respond to an ECM component. DDR1 binds several of the major fibrillar collagens (types I, II, III, and V) and the non-fibrillar collagen IV (Shrivastava et al. 1997, Vogel et al. 1997, Hou et al. 2001). DDR proteins bind collagen as dimers (Leitinger 2003, 2011). DDR1 is mostly found in epithelial cells and leukocytes. It control developmental processes and regulates cell adhesion, migration, proliferation, and remodelling of the extracellular matrix by controlling the expression and activity of matrix metalloproteinases (Leitinger & Hohenester 2007).
Perlecan (HSPG2) is a modular proteoglycan primarily located in the basement membranes of vascular tissues. It is involved in several developmental processes, both during embryogenesis and in human diseases such as cancer and diabetes (Iozzo et al. 1994). HSPG2 can self-aggregate into dimeric or multimeric forms (Yurchenco et al. 1987) and is involved in heterotypic interactions with numerous extracellular macromolecules (Whitelock et al. 2008, Perlecan entry in MatrixDB). HSPG2's GAG chains mediate interactions with fibroblast growth factor-2 (Vigny et al. 1988, Knox et al. 2002), and nidogens (Entactins, represented elsewhere). The core protein binds fibronectin (Isemura et al. 1987, Heremans et al. 1990, Vlodavsky et al. 1991), transthyretin (Smeland et al. 1997) and platelet-derived growth factor A and B homodimers (Göhring et al. 1998).
Osteopontin (SPP1) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of proteins (Bellahcène et al. 2008). It is a highly phosphorylated sialoprotein and prominent component of the mineralized extracellular matrices of bones and teeth. It binds multiple integrins including alphaVbeta3, alphaVbeta1 and alphaVbeta5 (Liaw et al. 1995) alpha9beta1 (Smith et al. 1996, Yokosaki et al. 1999), alpha4beta1 (Bayliss et al. 1998) and the receptor CD44 (Weber et al. 1996, Katagiri et al. 1999). The SPP1–CD44 interaction may be important for colorectal cancer progression (Rao et al. 2013).
Vitronectin (VTN) is a major plasma glycoprotein of 75 kDa, circulating at approximately 0.2 mg/ml in humans. It interacts with collagen types I, II, III, IV, V, and VI (Gebb et al. 1986). Deglycosylation enhances VTN binding to collagen and is associated with VTN multimerization (Uchibori-Iwaki et al. 2000, Sano et al. 2007).
ECM composition is highly heterogeneous and dynamic, being constantly remodeled (Frantz et al. 2010) and modulated, largely by matrix metalloproteinases (MMPs) and growth factors that bind to the ECM influencing the synthesis, crosslinking and degradation of ECM components (Hynes 2009). ECM remodeling is involved in the regulation of cell differentiation processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. Redundant mechanisms modulate the expression and function of ECM modifying enzymes. Abnormal ECM dynamics can lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer.
Collagen is the most abundant fibrous protein within the ECM constituting up to 30% of total protein in multicellular animals. Collagen provides tensile strength. It associates with elastic fibres, composed of elastin and fibrillin microfibrils, which give tissues the ability to recover after stretching. Other ECM proteins such as fibronectin, laminins, and matricellular proteins participate as connectors or linking proteins (Daley et al. 2008).
Chondroitin sulfate, dermatan sulfate and keratan sulfate proteoglycans are structural components associated with collagen fibrils (Scott & Haigh 1985; Scott & Orford 1981), serving to tether the fibril to the surrounding matrix. Decorin belongs to the small leucine-rich repeat proteoglycan family (SLRPs) which also includes biglycan, fibromodulin, lumican and asporin. All appear to be involved in collagen fibril formation and matrix assembly (Ameye & Young 2002).
ECM proteins such as osteonectin (SPARC), osteopontin and thrombospondins -1 and -2, collectively referred to as matricellular proteins (reviewed in Mosher & Adams 2012) appear to modulate cell-matrix interactions. In general they induce de-adhesion, characterized by disruption of focal adhesions and a reorganization of actin stress fibers (Bornstein 2009). Thrombospondin (TS)-1 and -2 bind MMP2. The resulting complex is endocytosed by the low-density lipoprotein receptor-related protein (LRP), clearing MMP2 from the ECM (Yang et al. 2001).
Osteopontin (SPP1, bone sialoprotein-1) interacts with collagen and fibronectin (Mukherjee et al. 1995). It also contains several cell adhesive domains that interact with integrins and CD44.
Aggrecan is the predominant ECM proteoglycan in cartilage (Hardingham & Fosang 1992). Its relatives include versican, neurocan and brevican (Iozzo 1998). In articular cartilage the major non-fibrous macromolecules are aggrecan, hyaluronan and hyaluronan and proteoglycan link protein 1 (HAPLN1). The high negative charge density of these molecules leads to the binding of large amounts of water (Bruckner 2006). Hyaluronan is bound by several large proteoglycans proteoglycans belonging to the hyalectan family that form high-molecular weight aggregates (Roughley 2006), accounting for the turgid nature of cartilage.
The most significant enzymes in ECM remodeling are the Matrix Metalloproteinase (MMP) and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families (Cawston & Young 2010). Other notable ECM degrading enzymes include plasmin and cathepsin G. Many ECM proteinases are initially present as precursors, activated by proteolytic processing. MMP precursors include an amino prodomain which masks the catalytic Zn-binding motif (Page-McCawet al. 2007). This can be removed by other proteinases, often other MMPs. ECM proteinases can be inactivated by degradation, or blocked by inhibitors. Some of these inhibitors, including alpha2-macroglobulin, alpha1-proteinase inhibitor, and alpha1-chymotrypsin can inhibit a large variety of proteinases (Woessner & Nagase 2000). The tissue inhibitors of metalloproteinases (TIMPs) are potent MMP inhibitors (Brew & Nagase 2010).
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HAPLN1Fibril forming collagens are the most familiar and best studied subgroup. Collagen fibres are aggregates or bundles of collagen fibrils, which are themselves polymers of tropocollagen complexes, each consisting of three polypeptide chains known as alpha chains. Tropocollagens are considered the subunit of larger collagen structures. They are approximately 300 nm long and 1.5 nm in diameter, with a left-handed triple-helical structure, which becomes twisted into a right-handed coiled-coil 'super helix' in the collagen fibril. Tropocollagens in the extracellular space polymerize spontaneously with regularly staggered ends (Hulmes 2002). In fibrillar collagens the molecules are staggered by about 67 nm, a unit known as D that changes depending upon the hydration state. Each D-period contains slightly more than four collagen molecules so that every D-period repeat of the microfibril has a region containing five molecules in cross-section, called the 'overlap', and a region containing only four molecules, called the 'gap'. The triple-helices are arranged in a hexagonal or quasi-hexagonal array in cross-section, in both the gap and overlap regions (Orgel et al. 2006). Collagen molecules cross-link covalently to each other via lysine and hydroxylysine side chains. These cross-links are unusual, occuring only in collagen and elastin, a related protein.
The macromolecular structures of collagen are diverse. Several group 3 collagens associate with larger collagen fibers, serving as molecular bridges which stabilize the organization of the extracellular matrix. Type IV collagen is arranged in an interlacing network within the dermal-epidermal junction and vascular basement membranes. Type VI collagen forms distinct microfibrils called beaded filaments. Type VII collagen forms anchoring fibrils. Type VIII and X collagens form hexagonal networks. Type XVII collagen is a component of hemidesmosomes where it is complexed wtih alpha6Beta4 integrin, plectin, and laminin-332 (de Pereda et al. 2009). Type XXIX collagen has been recently reported to be a putative epidermal collagen with highest expression in suprabasal layers (Soderhall et al. 2007). Collagen fibrils/aggregates arranged in varying combinations and concentrations in different tissues provide specific tissue properties. In bone, collagen triple helices lie in a parallel, staggered array with 40 nm gaps between the ends of the tropocollagen subunits, which probably serve as nucleation sites for the deposition of crystals of the mineral component, hydroxyapatite (Ca10(PO4)6(OH)2) with some phosphate. Collagen structure affects cell-cell and cell-matrix communication, tissue construction in growth and repair, and is changed in development and disease (Sweeney et al. 2006, Twardowski et al. 2007). A single collagen fibril can be heterogeneous along its axis, with significantly different mechanical properties in the gap and overlap regions, correlating with the different molecular organizations in these regions (Minary-Jolandan & Yu 2009).
SPARC Hydroxylapatitie
Ca2+AGRN
HSPG2Dystrophin
LamininsFibrillin is most familiar as a component of elastic fibres but microfibrils with no elastin are found in the ciliary zonules of the eye and invertebrate circulatory systems. The addition of elastin to microfibrils is a vertebrate adaptation to high pulsatile pressures in their closed circulatory systems (Faury et al. 2003). Elastin appears to have emerged after the divergence of jawless vertebrates from other vertebrates (Sage 1982).
Fibrillin-1 is the major structural component of microfibrils. Fibrillin-2 is expressed earlier in development than fibrillin-1 and may be important for elastic fiber formation (Zhang et al. 1994). Fibrillin-3 arose as a duplication of fibrillin-2 that did not occur in the rodent lineage. It was first isolated from human brain (Corson et al. 2004).
Fibrillin assembly is not as well defined as elastin assembly. The primary structure of fibrillin is dominated by calcium binding epidermal growth factor like repeats (Kielty et al. 2002). Fibrillin may form dimers or trimers before secretion. However, multimerisation predominantly occurs outside the cell. Formation of fibrils appears to require cell surface structures suggesting an involvement of cell surface receptors. Fibrillin is assembled pericellularly (i.e. on or close to the cell surface) into microfibrillar arrays that undergo time dependent maturation into microfibrils with beaded-string appearance. Transglutaminase forms gamma glutamyl epsilon lysine isopeptide bonds within or between peptide chains. Additionally, intermolecular disulfide bond formation between fibrillins is an important contributor to fibril maturation (Reinhardt et al. 2000).
Models of fibrillin-1 microfibril structure suggest that the N-terminal half of fibrillin-1 is asymmetrically exposed in outer filaments, while the C-terminal half is buried in the interior (Kuo et al. 2007). Fibrillinopathies include Marfan syndrome, familial ectopia lentis, familial thoracic aneurysm, all due to mutations in the fibrillin-1 gene FBN1, and congenital contractural arachnodactyly which is caused by mutation of FBN2 (Maslen & Glanville 1993, Davis & Summers 2012).
In vivo assembly of fibrillin requires the presence of extracellular fibronectin fibres (Sabatier et al. 2009). Fibrillins have Arg-Gly-Asp (RGD) sequences that interact with integrins (Pfaff et al. 1996, Sakamoto et al. 1996, Bax et al., 2003, Jovanovic et al. 2008) and heparin-binding domains that interact with a cell-surface heparan sulfate proteoglycan (Tiedemann et al. 2001) possibly a syndecan (Ritty et al. 2003). Fibrillins also have a major role in binding and sequestering growth factors such as TGF beta into the ECM (Neptune et al. 2003). Proteoglycans such as versican (Isogai et al. 2002), biglycan, and decorin (Reinboth et al. 2002) can interact with the microfibrils. They confer specific properties including hydration, impact absorption, molecular sieving, regulation of cellular activities, mediation of growth factor association, and release and transport within the extracellular matrix (Buczek-Thomas et al. 2002). In addition, glycosaminoglycans have been shown to interact with tropoelastin through its lysine side chains (Wu et al. 1999), regulating tropoelastin assembly (Tu & Weiss 2008).
Elastin is synthesized as a 70kDa monomer called tropoelastin, a highly hydrophobic protein composed largely of two types of domains that alternate along the polypeptide chain. Hydrophobic domains are rich in glycine, proline, alanine, leucine and valine. These amino acids occur in characteristic short (3-9 amino acids) tandem repeats, with a flexible and highly dynamic structure (Floquet et al. 2004). Unlike collagen, glycine in elastin is not rigorously positioned every 3 residues. However, glycine is distributed frequently throughout all hydrophobic domains of elastin, and displays a strong preference for inter-glycine spacing of 0-3 residues (Rauscher et al. 2006).
Elastic fibre formation involves the deposition of tropoelastin onto a template of fibrillin rich microfibrils. Recent results suggest that the first step of elastic fiber formation is the organization of small globules of elastin on the cell surface followed by globule aggregation into microfibres (Kozel et al. 2006). An important contribution to the initial stages assembly is thought to be made by the intrinsic ability of the protein to direct its own polymeric organization in a process termed 'coacervation' (Bressan et al. 1986). This self-assembly process appears to be determined by interactions between hydrophobic domains (Bressan et al. 1986, Vrhovski et al. 1997, Bellingham et al. 2003, Cirulis & Keeley 2010) which result in alignment of the cross-linking domains, allowing the stabilization of elastin through the formation of cross-links generated through the oxidative deamination of lysine residues, catalyzed by members of the lysyl oxidase (LOX) family (Reiser et al. 1992, Mithieux & Weiss 2005). The first step in the cross-linking reaction is the oxidative formation of the delta aldehyde, known as alpha aminoadipic semialdehyde or allysine (Partridge 1963). Subsequent reactions that are probably spontaneous lead to the formation of cross-links through dehydrolysinonorleucine and allysine aldol, a trifunctional cross-link dehydromerodesmosine and two tetrafunctional cross-links desmosine and isodesmosine (Lucero & Kagan 2006), which are unique to elastin. These cross-links confer mechanical integrity and high durability. In addition to their role in self-assembly, hydrophobic domains provide elastin with its elastomeric properties, with initial studies suggesting that the elastomeric propereties of elastin are driven through changes in entropic interactions with surrounding water molecules (Hoeve & Flory 1974).
A very specific set of proteases, broadly grouped under the name elastases, is responsible for elastin remodelling (Antonicelli et al. 2007). The matrix metalloproteinases (MMPs) are particularly important in elastin breakdown, with MMP2, 3, 9 and 12 explicitly shown to degrade elastin (Ra & Parks 2007). Nonetheless, elastin typically displays a low turnover rate under normal conditions over a lifetime (Davis 1993).
Annotated Interactions
Tenacious binding of free fibronectin to cells leads to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. This process is enhanced in the presence of CEACAM molecules.Biglycan binds collagen types I (Schönherr et al. 1995), II (Bovine, using pig byglycan - Vynios et al. 2001, Bovine, using bovine biglycan - Douglas et al. 2008), III (Bovine, using bovine biglycan - Douglas et al. 2008), VI (Wiberg et al. 2001, 2002, human) and IX (Chen et al. 2006 - species source of collagen/biglycan unknown).
The G1 N-terminal domain of ACAN has a lectin-like binding site with high affinity for HA (Watanabe et al. 1997, Hardingham 2006). HA is a long unbranched, unsulphated GAG synthesized free from protein attachment by three HA synthases (Spicer & McDonald 1998). It has an average molecular weight of several million Da. HA content steadily rises in aging cartilage and can reach 10% of the total GAG. ACAN, HA and the small glycoprotein HAPLN1, known as Link protein, are found in huge multi-molecular aggregates comprised of numerous ACAN monomers non-covalently bound to HA, stabilized by HAPLN1 which forms a ternary complex with the G1 domain of ACAN and HA (Ratcliffe & Hardingham 1983, Grover & Roughley 1994, Kiani et al. 2002).
The LG domains of AGRN bind alpha-dystroglycan (Yamada et al. 1996, Gee et al. 1994, Bowen et al. 1996, Campanelli et al. 1996, Gesemann et al. 1996, Hopf & Hoch 1996).
TNC binds several integrins including alpha2beta1 (Sriramararo et al. 1993), alphaVbeta6 (Yokosaki et al. 1996), alphaVbeta3 (Sriramararo et al. 1993, Yokosaki et al. 1996), alpha9beta1 (Yokosaki et al. 1996), alphaXbeta1 (Probstmeier & Peshva 1999), alpha8beta1 (Schnapp 1995) and alpha7beta1 (Mercado et al. 2004).
The N-terminus of the LN form of AGRN binds to the laminin gamma1 subunit (Denzer et al. 1997, Kammerer et al. 1999, Mascarenhas et al. 2003). This may indirectly bind AGRN to integrins on the cell surface (Bezakova & Ruegg 2003).
TNC and TNR bind with high affinity to fibronectin (FN) (Chiquet-Ehrismann et al. 1991, Chung et al. 1995, Chung & Erickson 1997, Hauzenberger et al. 1999, Ingham et al. 2004, To & Midwood 2011, Pesheva et al. 1994), modulating the cell adhesion function of FN either by binding or restricting access of FN to integrin binding sites (Lightner & Erickson 1990) or by binding to cell receptors and altering their responsiveness to FN (Prieto et al. 1992, Fischer et al. 1997). The interaction of Tenascin and FN impacts tissue structure by controlling the assembly, maintenance, and turnover of the ECM at the cell surface (To & Midwood 2010).
BGN-deficient mice exhibit larger and irregular fibrils leading to thin dermis and reduced bone mass (Corsi et al. 2002, Xu et al. 1998). BGN binds collagen types I (Schönherr et al. 1995), II (Bovine, using pig BGN - Vynios et al. 2001, Bovine, using bovine BGN - Douglas et al. 2008), III (Bovine, using bovine BGN - Douglas et al. 2008), VI (Wiberg et al. 2001) and IX (Chen et al. 2006 - species source of collagen/BGN unknown).
Mutations in COMP lead to pseudoachondroplasia and multiple epiphyseal dysplasia (Jackson et al. 2012). COMP binding to FN1 and probably to other partners requires the presence of the divalent cations Ca2+, Mg2+ or Mn2+. Each COMP subunit binds approximately 10 calcium ions (Chen et al. 2000).
AGRN binds a complex of the tyrosine kinase receptor MuSK, which is responsible for mediating agrin's ability to cluster AChR (Glass et al. 1996, Sanes & Lichtman 2001, Burden et al. 2003) and the coreceptor LRP4 (Kim et al. 2008, Zhang et al. 2008, Zong et al. 2012).
Endothelial cells lining the microvascular wall form a semi-permeable barrier to the movement of blood components. The attachment of endothelial cells to the extracellular matrix (ECM) is largely mediated by transmembrane integrins which recognize short sequence motifs such as Arg-Gly-Asp (RGD) in many ECM proteins.
Integrin alpha5beta1 and alphaVbeta3 bind to the ECM proteins fibronectin and vitronectin respectively. Both are critical for the establishment and stabilization of endothelial monolayers (Cheng & Kramer 1989). Synthetic peptides that compete with ECM proteins for the integrins or antibodies directed against alpha5beta1 and alphaVbeta3 cause endothelial cell detachment (Hayman et al. 1985, Pierschbacher & Ruoslahti 1987).
TNC and TNR bind to members of the lectican family, a class of extracellular chondroitin sulfate proteoglycans consisting of aggrecan, versican, brevican and neurocan. TNC binds aggrecan (Lundell et al. 2004), versican (Tsujii et al. 2006) and neurocan (Milev et al. 1994, Grumet et al. 1994, Rauch et al. 1997). TNR binds aggrecan (Aspberg et al. 1997, Lundell et al. 2004), versican (Aspberg et al. 1995, 1997), brevican Aspberg et al. 1997, Hagihara et al. 1999) and neurocan (Aspberg et al. 1997).