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).
Raghunath M, Putnam EA, Ritty T, Hamstra D, Park ES, Tschödrich-Rotter M, Peters R, Rehemtulla A, Milewicz DM.; ''Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix.''; PubMedEurope PMCScholia
Clarke AW, Arnspang EC, Mithieux SM, Korkmaz E, Braet F, Weiss AS.; ''Tropoelastin massively associates during coacervation to form quantized protein spheres.''; PubMedEurope PMCScholia
Jensen SA, Reinhardt DP, Gibson MA, Weiss AS.; ''Protein interaction studies of MAGP-1 with tropoelastin and fibrillin-1.''; PubMedEurope PMCScholia
Leduc R, Molloy SS, Thorne BA, Thomas G.; ''Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.''; PubMedEurope PMCScholia
Doi M, Nagano A, Nakamura Y.; ''Molecular cloning and characterization of a novel gene, EMILIN-5, and its possible involvement in skeletal development.''; PubMedEurope PMCScholia
Doliana R, Bot S, Mungiguerra G, Canton A, Cilli SP, Colombatti A.; ''Isolation and characterization of EMILIN-2, a new component of the growing EMILINs family and a member of the EMI domain-containing superfamily.''; PubMedEurope PMCScholia
Sherratt MJ, Wess TJ, Baldock C, Ashworth J, Purslow PP, Shuttleworth CA, Kielty CM.; ''Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.''; PubMedEurope PMCScholia
Bressan GM, Daga-Gordini D, Colombatti A, Castellani I, Marigo V, Volpin D.; ''Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.''; PubMedEurope PMCScholia
Mongiat M, Mungiguerra G, Bot S, Mucignat MT, Giacomello E, Doliana R, Colombatti A.; ''Self-assembly and supramolecular organization of EMILIN.''; PubMedEurope PMCScholia
Kozel BA, Rongish BJ, Czirok A, Zach J, Little CD, Davis EC, Knutsen RH, Wagenseil JE, Levy MA, Mecham RP.; ''Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters.''; PubMedEurope PMCScholia
Pfaff M, Reinhardt DP, Sakai LY, Timpl R.; ''Cell adhesion and integrin binding to recombinant human fibrillin-1.''; PubMedEurope PMCScholia
Elastin, the highly insoluble core protein of elastic fibers, is secreted as a soluble protein monomer referred to as tropoelastin. Under physiological conditions the monomers phase separate and coalesce into spherical packages (Clarke et al. 2006), a process known as coacervation. Packages of accumulated elastin are delivered to fibrillin-based fibres in a mechanism that is correlated with cell migration during embryonic development (Czirok et al. 2006). A transglutaminase cross-link between domain 4 of tropoelastin and domain 16 of fibrillin-1 may stabilize initial deposition (Clarke et al. 2005). Elastin is subsequently cross linked by members of the lysyl oxidase family via lysine residues, resulting in mature, insoluble fibres (Sato et al. 2007, Wise & Weiss 2009).
Fibulin-5 expressed by vascular smooth muscle cells plays an essential role in the formation of elastic fibres, mediating interactions between elastin and fibrillin (Yanigasawa et al. 2002, Freeman et al. 2005). EMILIN can affect the process of elastic fibre formation (Bressan et al. 1993). It binds elastin and fibulin-5 and appears to coordinate their common interaction (Zanetti et al. 2004).
Fibrillins can bind the prodomains of TGF-beta superfamily members bone morphogenic factor (BMP) 2, 4, 7, 10, and growth and differentiation factor (GDF) 5 (Sengle et al. 2008). Prodomain binding by ECM constituents may be a targeting mechanism for TGF family members (Sengle et al. 2011).
The core protein representing ~90% of the mass of elastic fibres is elastin, a highly insoluble protein. It is secreted as soluble protein monomers referred to as tropoelastin, which have alternating hydrophobic and cross linking domains. The self-assembly of tropoelastin into a fibrillar elastin matrix is a multi step process. The first step is the self-association of secreted monomers via hydrophobic domains, in a process known as coacervation. This process concentrates monomers and may align residues in the correct register for subsequent cross linking (Yeo et al. 2011). Under physiological conditions the ~15 nm monomers phase-separate and coalesce into spherical packages 2-6 micrometers in diameter (Clarke et al. 2006, Kozel et al. 2004). This process is represented here by the association of an arbitrary 10 tropoelastin monomers. While they grow, coacervate packages are tethered to the cell surface (Wise & Weiss 2009). The binding interactions between tropoelastin and the cell surface are not fully understood but possible partners include integrins and glycosaminoglycans (Broekelmann et al. 2005). Extracellular fibrillin microfibrils act as a scaffold for the deposition of tropoelastin globules as part of elastic fibre formation (Kozel et al. 2004).
Extracellular deposition of fibrillin requires removal of the C-terminus, which can be cleaved in vitro by several furin/PACE family convertases (Raghunath et al. 1999, Ritty et al. 1999) in a process that is inhibited by N-glycosylation and calreticulin (Ashworth et al. 1999). Furin (PACE) is a transmembrane protein, synthesized as a 100 kDa protein, which rapidly undergoes autocatalytic cleavage to a 94 kDa protein in the endoplasmic reticulum (ER). The propeptide remains bound as an auto-inhibitor. Propeptide release occurs in the acidic pH of the trans-golgi-network (TGN)/endosomal compartment, activating furin. Though furin is primarily localized to the TGN a proportion of furin molecules are found on the cell surface (Teuchert et al. 1999). Profibrillin-1 processing does not occur in the TGN, where it is bound by two ER-resident molecular chaperones, BiP and GRP94. Instead activation by furin occurs as profibrillin-1 is secreted, or immediately after secretion (Wallis et al. 2003).
Elastin microfibril interface located protein (EMILIN)-1 is localized to the microfibril-elastin interface (Bressan et al. 1993). It can bind elastin and fibulin-5 (Zanetti et al. 2004). Emilin1 knockout mice have ultrastructural alterations of the elastic fibers in aorta and skin, abnormal cell morphology and anchorage of endothelial and smooth muscle cells to elastic lamellae, and abnormal elastic fibers in cultured embryonic fibroblasts.
Transforming growth factor (TGF) beta (TGF-beta) is a family of three cytokine 'isoforms' (encoded by three separate human genes) that control proliferation, cellular differentiation and other functions. TGF-beta originally referred to the founding member TGF-beta-1, now it is often used as a collective term for all three. TGF-beta is secreted from cells in latent form as part of a complex that includes two other proteins: the cleaved propeptide of TGF beta, known as latency associated peptide (LAP), and a member of the latent TGF beta binding protein (LTBP) family. LTBPs are members of the fibrillin/LTBP superfamily, characterised by the presence of unique TGF-binding protein (TB) domains, also known as 8 cys domains as they contain eight characteristic cysteines (Ramirez & Sakai 2010). LTBPs are microfibril-associated, proteins that tether latent complexes of TGF-beta to microfibrils in the ECM (Taipale et al. 1996, Dallas et al. 2000, Isogai et al. 2003, Hyytiainen et al. 2004, Ono et al. 2009, Munger & Sheppard 2011).
LTBP1 and 3 bind all three isoforms of latent TGF-beta, while LTBP4 only weakly binds TGF-beta1 (Saharinen & Keski Oja 2000). LTBP2 does not bind TGF-beta and is a structural component of fibrillin microfibrils. The carboxyl terminus of LTBP1 binds to fibrillin-1. LTBP1 and LTBP4 incorporation into the ECM is abolished in fibrillin-1 null mice (Ono et al. 2009). The amino terminus of LTBPs binds ECM components such as collagen (Taipale et al. 1996) and fibronectin (Kantola et al. 2008). Fibulins compete for the LTBP sites in fibrillin (Ono et al. 2009).
Fibulins are a family of 7 genes encoding calcium binding glycoproteins with distinct roles in elastogenesis. They are essential for elastic fibre formation, having a role in regulating and organising tropoelastin formation (Yanagisawa & Davis 2010). Fibulins 1-5 all bind elastin (Roark et al. 1995, Sasaki et al. 1999, Kobayashi et al. 2007). Fibulins 2, 4, 5 and 7 also bind fibrillin (Reinhardt et al. 1996, El Hallous et al. 2007, de Vega et al. 2007). Fibulin 6 has a role in the formation of the cleavage furrow during cytokinesis but its binding partners are unclear (Xu & Vogel 2011).
Fibrillin-1 splice variants that include the RGD sequence located in the fourth 8-cysteine domain mediate cell adhesion, binding integrin alphaVbeta3 (Pfaff et al. 1996), alpha5beta1 (Bax et al. 2003) and alphaVbeta6 (Jovanovic et al. 2008). AlphaVbeta3 has the highest affinity for fibrillin-1.
Transforming growth factor beta (TGF-beta) is a family of three cytokine ‘isoforms’ (encoded by three separate human genes) that control proliferation, cellular differentiation and other functions. TGF-beta originally referred to the founding member TGF-beta-1, now it is often used as a collective term for all three. TGF-beta is secreted from cells in latent form as part of a complex that includes two other proteins: the cleaved propeptide of TGF beta, known as latency associated peptide (LAP), and a member of the latent TGF beta binding protein (LTBP) family. LTBPs are members of the fibrillin/LTBP superfamily, characterised by the presence of unique TGF-binding protein (TB) domains, also known as 8 cys domains as they contain eight characteristic cysteines (Ramirez & Sakai 2010). LTBPs are microfibril-associated proteins that tether latent complexes of TGF-beta to microfibrils in the ECM (Taipale et al. 1996, Dallas et al. 2000, Isogai et al. 2003, Hyytiainen et al. 2004, Ono et al. 2009, Munger & Sheppard 2011). This allows TGF-beta to be targeted to the ECM where it is maintained in an inactive, latent state (Robertson et al. 2011).
LTBP1 and 3 bind all three isoforms of latent TGF-beta, while LTBP4 only weakly binds TGF-beta1 (Saharinen & Keski Oja 2000). LTBP2 does not bind TGF-beta and is a structural component of fibrillin microfibrils. The carboxyl termini of LTBP1 and LTBP4 binds to fibrillin. The incorporation of LTBP1 and LTBP4 into the ECM is abolished in fibrillin-1 null mice (Ono et al. 2009). The amino terminus of LTBPs binds ECM components such as collagen (Taipale et al. 1996) and fibronectin (Kantola et al. 2008). Fibulins compete for the LTBP sites in fibrillin (Ono et al. 2009).
TGF-beta is released from cells as a latent complex of three proteins: TGF-beta (which is encoded by three human genes), the processed TGF-beta propeptide (latency-associated peptide LAP), and a member of the latent TGF-beta binding protein (LTBP) family. LTBPs are microfibril (fibrillin)-associated proteins that bind LAP, tethering latent TGF-beta to microfibrils in the ECM (Taipale et al. 1996, Hyytiainen et al. 2004).
LTBP1 and LTBP4 incorporation into ECM requires fibrillin-1 (Ono et al. 2009). The protein–protein interaction sites between LTBPs and fibrillins have been determined using recombinant protein fragments and surface plasmon resonance (Ono et al. 2009). LTBP4 binds to the first hybrid domain of fibrillin-1 (Hyb1), whereas LTBP1 binds to a site involving both Hyb1 and adjacent EGF-like domains 2 and 3. Previous studies showed that the carboxyl terminus of LTBP1 binds to fibrillin-1, whereas the amino terminus of LTBPs is mainly responsible for binding ECM components made in cell culture generally, and fibronectin specifically (Kantola et al. 2008).
Other proteins found associated with elastic fibres include vitronectin (Dahlback et al. 1989,1990, Hintner et al. 1991) and and a structurally unrelated group of proteins collectively termed microfibrillar-associated proteins (MFAPs) (Gibson et al. 1996, 2000, Abrams et al. 1995, Toyashima et al. 1999). The significance of these interactions is not well understood.
Vitronectin is present in plasma, extracellular matrix, and the alpha granules of blood platelets. It has been implicated as a regulator of many processes including coagulation, fibrinolysis, pericellular proteolysis, complement dependent immune response, cell attachment and spreading (Zhuang et al. 1996). It interacts with integrins alphaVbeta1 (Marshall et al. 1995), alphaVbeta3 (Pytela et al. 1985), alphaVbeta5 (Panetti & McKeown Longo 1993) and alphaIIbBeta3 (Pytela et al. 1986) through Arg Gly Asp (RGD) cell binding sequences.
The MFAPs are not a structurally related family but grouped due to their localization with microfibrils. MFAP1 was originally called 'associated microfibril protein' (AMP). It is a 54 kDa protein, processed to 32 kDa, localizing to fibrillin-containing microfibrils in several tissues including zonule fibers (Horrigan et al. 1992). MFAP3 is a 41 kDa serine-rich protein localized to zonular microfibrils, found in extracts of developing nuchal ligament, also expressed in fetal aorta and lung (Abrams et al. 1995). MFAP4 is a 29 kDa protein localized to fibrillin-containing microfibrils surrounding elastic fibers in aorta, skin and spleen (Toyoshima et al. 1999).
The LAPs of TGF-beta1 and TGF-beta3 contain RGD sequences near the carboxyl termini that are bound by RGD-binding integrins. LAP3 binds alphaVBeta1, 3, 5 and 6 (Ludbrook et al. 2003) and 8 (Kitamura et al. 2011). Binding to integrins alphaVBeta6 and alphaVBeta8 leads to TGF-beta activation.
Fibrillin microfibril assembly is a cell regulated process, independent of tropoelastin. Distinct microfibril populations have been identified, suggesting that the cellular environment plays a role in regulating microfibril fate (Kielty et al. 2002). Fibrillin 1 may undergo limited assembly into dimers or trimers in the secretory pathway (Ashworth et al. 1999, Trask et al. 1999) but the formation of large microfibril polymers is extracellular. Microfibrils assemble close to the cell surface in a process that may require cell surface receptors. Fibrillins interact with several integrins (Sakamoto et al. 1996, Jovanovic et al. 2008) suggesting an assembly mechanism with similarities to fibronectin matrix formation. Heparan sulphate proteoglycans (HSPGs) and chondroitin sulfate containing proteoglycans (CSPGs) have also been proposed to have a role in assembly (Tiedemann et al. 2001). Fibrillin polymerization into fibres further requires the formation of disulfide bonds between fibrillins (Reinhardt et al. 2000), initially via calcium-binding epidermal growth factor domains at the C-terminus (Hubmacher et al. 2008), and transglutaminase cross-links (Kielty et al. 2002).
The LAPs of TGF beta-1 and TGF beta-3 contain RGD sequences near the carboxyl termini that are bound by RGD binding integrins. The TGF beta-1 form of LAP (LAP1) binds the integrins alphaVBeta1 (Munger et al. 1998), alphaVBeta3 (Ludbrook et al. 2003), alphaVBeta5 (Munger et al. 1998), alphaVBeta6 (Munger et al. 1999, Araya et al. 2006), alphaVBeta8 (Mu et al. 2002, Araya et al. 2006) and alpha8Beta1 (Lu et al. 2002). Binding to integrins alphaVBeta6 and alphaVBeta8 leads to TGF beta activation.
Microfibrils are composed largely of fibrillin but also contain covalently-linked microfibril associated glycoproteins MAGP-1 (MFAP2) and MAGP-2 (MFAP5). MFAP2 is a structural component of almost all vertebrate microfibrils (Gibson et al. 1989, Trask et al. 2000, Jensen et al. 2001). It appears to be important for tissue development and/or homeostasis, including regulation of bone remodelling and deposition of tissue fat (Weinbaum et al. 2008). The C-terminal half of MFAP2 is rich in cysteines and contains a matrix-binding domain that facilitates interactions with fibrillin (Weinbaum et al. 2008). The related MFAP5 similarly binds to microfibrils (Gibson et al. 1998) but with a restricted expression profile.
Soluble monomers of tropoelastin are cross-linked by the oxidative deamination of lysine residues, catalyzed by lysyl oxidase (LOX). 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 spontaneous reactions 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.
Fibulins are a family of 7 extracellular calcium binding proteins that have developmental roles (Twal et al. 2001). Fibulins 1-5 are found in association with elastic fibers. Fibulin-1 binds fibronectin (Balbona et al. 1992, Tran et al. 1997) suppressing fibronectin-mediated inhibitory effects on cell attachment and spreading (Twal et al. 2001). Fibulin-2 also binds fibronectin (Sasaki et al. 1995).
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).
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Fibulins
EmilinsFibulin-5 expressed by vascular smooth muscle cells plays an essential role in the formation of elastic fibres, mediating interactions between elastin and fibrillin (Yanigasawa et al. 2002, Freeman et al. 2005). EMILIN can affect the process of elastic fibre formation (Bressan et al. 1993). It binds elastin and fibulin-5 and appears to coordinate their common interaction (Zanetti et al. 2004).
LTBP1 and 3 bind all three isoforms of latent TGF-beta, while LTBP4 only weakly binds TGF-beta1 (Saharinen & Keski Oja 2000). LTBP2 does not bind TGF-beta and is a structural component of fibrillin microfibrils. The carboxyl termini of LTBP1 and LTBP4 binds to fibrillin. The incorporation of LTBP1 and LTBP4 into the ECM is abolished in fibrillin-1 null mice (Ono et al. 2009). The amino terminus of LTBPs binds ECM components such as collagen (Taipale et al. 1996) and fibronectin (Kantola et al. 2008). Fibulins compete for the LTBP sites in fibrillin (Ono et al. 2009).
Vitronectin is present in plasma, extracellular matrix, and the alpha granules of blood platelets. It has been implicated as a regulator of many processes including coagulation, fibrinolysis, pericellular proteolysis, complement dependent immune response, cell attachment and spreading (Zhuang et al. 1996). It interacts with integrins alphaVbeta1 (Marshall et al. 1995), alphaVbeta3 (Pytela et al. 1985), alphaVbeta5 (Panetti & McKeown Longo 1993) and alphaIIbBeta3 (Pytela et al. 1986) through Arg Gly Asp (RGD) cell binding sequences.
The MFAPs are not a structurally related family but grouped due to their localization with microfibrils. MFAP1 was originally called 'associated microfibril protein' (AMP). It is a 54 kDa protein, processed to 32 kDa, localizing to fibrillin-containing microfibrils in several tissues including zonule fibers (Horrigan et al. 1992). MFAP3 is a 41 kDa serine-rich protein localized to zonular microfibrils, found in extracts of developing nuchal ligament, also expressed in fetal aorta and lung (Abrams et al. 1995). MFAP4 is a 29 kDa protein localized to fibrillin-containing microfibrils surrounding elastic fibers in aorta, skin and spleen (Toyoshima et al. 1999).