Distinct Steps of Cross-linking, Self-association, and Maturation of Tropoelastin Are Necessary for Elastic Fiber Formation

Sato, Fumiaki; Wachi, Hiroshi; Ishida, Marie; Nonaka, Risa; Onoue, Satoshi; Urban, Zsolt; Starcher, Barry; Seyama, Yousuke

doi:10.1016/j.jmb.2007.03.060

Cited by 70 publications

(73 citation statements)

References 21 publications

(23 reference statements)

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“…ELP and elastin coacervate droplets have been shown to increase in stability if they are held above the coacervation temperature for an extended period (18,48,49). This maturation process has been hypothesized to occur through increased ordering of ELPs at the surface of the droplet, resulting in a less reversible LLPS.…”

Section: Resultsmentioning

confidence: 99%

Direct observation of structure and dynamics during phase separation of an elastomeric protein

Reichheld

Muiznieks

Keeley

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

225

245

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Despite its growing importance in biology and in biomaterials development, liquid-liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical crosslinking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP 3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.phase separation | elastin | NMR | protein structure | dynamics T he liquid-liquid phase separation (LLPS) of molecules has long been exploited to concentrate and encapsulate molecules for drug delivery and food preparation (1, 2). In biology, there is increasing awareness that many proteins exhibit this type of phase behavior, allowing transient microenvironments to be quickly assembled and disassembled in response to changing solution conditions, or the availability of binding partners (3, 4). Protein phase separation occurs intracellularly to generate various membraneless organelles, such as ribonucleoprotein (RNP) bodies involved in nucleic acid processing, transport, and storage (5). A similar phenomenon is observed in the extracellular matrix as a critical step in the synthesis of elastic fibers, which provide extensibility, recoil, and resilience to tissues (6, 7). In the latter system, LLPS of monomeric elastin results in hydrated protein-rich coacervate droplets that are deposited and crosslinked to form an elastic matrix (7,8). In addition to their fundamental importance to biology, the dynamic reversibility of LLPS makes phase-separated states of proteins an attractive platform for development of responsive biomaterials with broad application, for instance as scaffolds for tissue engineering or as carriers in drug delivery systems (9-11).Despite the keen interest in proteins that undergo s...

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Section: Resultsmentioning

confidence: 99%

Direct observation of structure and dynamics during phase separation of an elastomeric protein

Reichheld

Muiznieks

Keeley

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

225

245

View full text Add to dashboard Cite

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“…Changes in the concentration of elastin crosslinks may be caused by an altered gene expression level of elastin, a variation of elastin splice variants or changes in the degree of prolyl hydroxylation of elastin. 48,49 Another potential consequence of increased fibrillin-2 expression in wound healing and fibrosis and of the co-localisation of fibrillin-2 and tenascin-C might be a change in the adhesion properties of the matrix for cells involved in wound healing. Fibrillin-2 has two RGD cell attachment sites located in the third and the fourth TB domain.…”

Section: Discussionmentioning

confidence: 99%

Enhanced fibrillin-2 expression is a general feature of wound healing and sclerosis: potential alteration of cell attachment and storage of TGF-β

Brinckmann

Hunzelmann

Kahle

et al. 2010

Laboratory Investigation

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Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-b. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-b. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-b1)) showed a marked increase of inactive LAP-bound TGF-b1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-b in the matrix.

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“…Expression of tropoelastin deletion constructs in vitro and as transgenes in mice have identified sequences encoded by exon 30 as being critical for the self-association of tropoelastin molecules (Kozel et al, 2003). Tropoelastin constructs lacking domain-36, however, were able to associate with existing elastic fibers, but crosslinking was greatly attenuated (Hsiao et al, 1999;Kozel et al, 2003;Sato et al, 2007). These studies support a multistep process for elastin assembly that involves tropoelastin self-association (mediated by domain 30) followed by crosslinking and maturation mediated by domain-36 Kozel et al, 2004;Kozel et al, 2006;Sato et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…Tropoelastin constructs lacking domain-36, however, were able to associate with existing elastic fibers, but crosslinking was greatly attenuated (Hsiao et al, 1999;Kozel et al, 2003;Sato et al, 2007). These studies support a multistep process for elastin assembly that involves tropoelastin self-association (mediated by domain 30) followed by crosslinking and maturation mediated by domain-36 Kozel et al, 2004;Kozel et al, 2006;Sato et al, 2007). The data from the current study are consistent with this model and suggest that domain-36 may function to facilitate fiber maturation by forming an initial cross-link that serves to help register the multiple tropoelastin crosslinking sites for efficient oxidation by lysyl oxidase.…”

Section: Discussionmentioning

confidence: 99%

Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

et al. 2008

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The carboxyl terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is ∼0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysinederived cross-link in this region, which would alter the extractability and antigenicity of the carboxyterminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.

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Distinct Steps of Cross-linking, Self-association, and Maturation of Tropoelastin Are Necessary for Elastic Fiber Formation

Cited by 70 publications

References 21 publications

Direct observation of structure and dynamics during phase separation of an elastomeric protein

Direct observation of structure and dynamics during phase separation of an elastomeric protein

Enhanced fibrillin-2 expression is a general feature of wound healing and sclerosis: potential alteration of cell attachment and storage of TGF-β

Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

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