Banded fibers in Tropoelastin coacervates at physiological temperatures

Bressan, Giorgio M.; Castellani, I.; Giro, M.G.; Volpin, Dino; Fornieri, C.; Ronchetti, Ivonne Pasquali

doi:10.1016/s0022-5320(83)80021-5

Cited by 57 publications

(53 citation statements)

References 19 publications

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“…It has long been assumed that coacervation of tropoelastin is a crucial step in assembly of the elastic fiber (42)(43)(44). Our data suggest, however, that coacervation may not be involved in the initial steps of elastin assembly and that coacervation by itself cannot drive elastin assembly in tissues.…”

Section: Deletion Of Exon 30 Decreases But Does Not Ablate Elasticmentioning

confidence: 55%

“…Coacervation, an entropically driven, inverse temperature transition caused by the interaction of the hydrophobic domains in the molecule, occurs as tropoelastin monomers associate to form large aggregates. Several laboratories have shown that the large hydrophobic sequences in the middle of the molecule play a dominant role in the intermolecular interactions that occur during coacervation (43). If coacervation were the only requirement for assembly, then all of our deletion constructs would form fibers (or at least aggregates) because they all contain the critical middle hydrophobic sequences.…”

Section: Deletion Of Exon 30 Decreases But Does Not Ablate Elasticmentioning

confidence: 97%

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Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

Kozel

Wachi

Davis

et al. 2003

Journal of Biological Chemistry

130

138

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Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29 -36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form ␤-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.The inherent complexity of the elastic fiber, combined with the unique physical properties of its component proteins, has made understanding elastin structure and assembly one of the most difficult problems in matrix biology. Although technical advances in cell and molecular biology have given us new information about elastin gene expression and elastin synthesis, surprisingly little is known about how the elastic fiber is assembled at the molecular level. The major component of the mature fiber is a covalently cross-linked polymer of the protein elastin. Elastin is secreted from the cell as a soluble monomer called tropoelastin. In the extracellular space, lysine residues within tropoelastin are specifically modified to form covalent cross-links between tropoelastin chains. This cross-linked polymer has a high degree of reversible distensibility, including the ability to deform to large extensions with small forces.The tropoelastin molecule is characterized by a series of tandem repeats, each including a lysine-containing cross-linking region followed by a hydrophobic motif (1). Cross-linking is initiated by the extracellular enzyme(s) lysyl oxidase, which catalyzes the oxidative deamination of lysyl ⑀-amino groups. Under normal conditions, the cross-linking process is extremely efficient, with all but ϳ5 of the ϳ40 lysine residues in tropoelastin participating in covalent linkages that form the functional polymer. How the cross-linking sites within the monomer are aligned prior to cross-linking is unclear. It has long been assumed that microfibrils provide a scaffold or template for elastin assembly by binding and aligning tropoelastin monomers so that lysine-containin...

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Section: Deletion Of Exon 30 Decreases But Does Not Ablate Elasticmentioning

confidence: 55%

Section: Deletion Of Exon 30 Decreases But Does Not Ablate Elasticmentioning

confidence: 97%

Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

Kozel

Wachi

Davis

et al. 2003

Journal of Biological Chemistry

130

138

View full text Add to dashboard Cite

show abstract

“…Monomeric elastin, called tropoelastin, is secreted from the cell as a soluble protein. Isolated and purified tropoelastin has been shown to exhibit a great tendency to aggregate (coacervation) in physiological solution and at temperatures in the physiological range, giving rise to supramolecular structures very similar to those found in natural elastic fibers (4,5,11). This self-aggregation property of tropoelastin is thought to contribute to elastic fiber assembly in vivo.…”

mentioning

confidence: 94%

Targeted Disruption of Fibulin-4 Abolishes Elastogenesis and Causes Perinatal Lethality in Mice

Chen

Horiguchi

et al. 2006

Molecular and Cellular Biology

186

189

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Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4 ؊/؊ mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4 ؊/؊ embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4 ؊/؊ mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4 ؊/؊ tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4 ؊/؊ mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.Elastic fibers with morphologically distinct architectures are present in the extracellular matrix (ECM) to accommodate elastic requirements and mechanical stresses imposed on different tissues. They are particularly abundant in elastic tissues such as large blood vessels, lung, and skin. Loss of elasticity is a major contributing factor in aging and a myriad of pathological conditions including emphysema, artery diseases, and cutis laxa (39,41,44). Elastic fibers undergo irreversible structural and compositional changes with age and in some pathological conditions (41). Regardless of morphology, all elastic fibers consist of cross-linked elastin, fibrillin-rich microfibrils, and several associated molecules (23,37,38,46). Elastin endows the fiber with the characteristic property of elastic recoil. It is chemically inert, extremely hydrophobic, and insoluble under most conditions. Monomeric elastin, called tropoelastin, is secreted from the cell as a soluble protein. Isolated and purified tropoelastin has been shown to exhibit a great tendency to aggregate (coacervation) in physiological solution and at temperatures in the physiological range, giving rise to supramolecular structures very similar to those found in natural elastic fibers (4,5,11). This self-aggregation property of tropoelastin is thought to contribute to elastic fiber assembly in vivo. However, self-aggregation alone is insufficient to explain the efficiency of the assembly process and the variable form of elastic fibers ...

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“…Through coacervation, tropoelastin molecules were thought to be converted from molecules largely lacking secondary and tertiary structure to a more ordered state (2,7,9,10). Electron micrographs show parallel arrays of 5-nm-wide filaments of tropoelastin coacervates that are similar to the fibrous structure of mature elastin (3,4,11). The importance of coacervation in the formation of lysine cross-links has been shown in smooth muscle cells where culturing at temperatures below 37°C hampers elastin formation (12,13).…”

mentioning

confidence: 99%

Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

Toonkool¹,

Regan²,

Kuchel³

et al. 2001

Journal of Biological Chemistry

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Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20°C in phosphate-buffered saline, pH 7.4, but at 37°C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20°C) and after (37°C) coacervation showed that the soluble component was strictly monomeric. Sedimentation velocity experiments revealed that at both temperatures soluble tropoelastin exists as two independently sedimenting monomeric species present in approximately equal concentrations. Species 1 had a frictional ratio at both temperatures of ϳ2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20°C of 1.4 that increased to 1.7 at 37°C, indicating a compact and symmetrical conformation that expanded or became asymmetric at the higher temperature. The slow interconversion of the two monomeric species contrasts with the rapid and reversible process of coacervation suggesting both efficiently incorporate into the coacervate. A hydrated protein of equivalent molecular weight modeled as a sphere and a flexible chain was predicted to have a frictional ratio of 1.2 and 1.6, respectively. Tropoelastin appeared as a single species when studied by pulsed field-gradient spinecho NMR, but computer modeling showed that the method was insensitive to the presence of two species of equal concentration having similar diffusion coefficients. Scintillation proximity assays using radiolabeled tropoelastin and sedimentation analysis showed that the coacervation at 37°C was a highly cooperative monomer-n-mer self-association. A critical concentration of 3.4 g/liter was obtained when the coacervate was modeled as a helical polymer formed from the monomers via oligomeric intermediates.Elastin forms a highly insoluble cross-linked extracellular matrix that is predominantly responsible for the elasticity of vertebrate tissue. The precursor of elastin, tropoelastin, is devoid of cross-links. Following secretion from the cell surface, tropoelastin undergoes coacervation, which is a process of selfassociation characterized by an inverse temperature transition (1). Tropoelastin is soluble in aqueous solution at room temperature in vitro, but upon raising the temperature to 37°C the solution becomes turbid as tropoelastin molecules associate to form large aggregates (2, 3). This process of coacervation results from multiple intermolecular interactions of the hydrophobic domains (3, 4). The tropoelastin coacervate is a thick viscoelastic phase that is not miscible with the overlying solution (5). On cooling to 20°C, the aggregates dissociate reversibly, and the solution turns clear. Alternating between hydrophobic domains in the protein are short sequences of amino acid residues that form the cross-linking domains (6). Coacervation may concentrate and align tropoelastin molecules prior to elastin formation via lysyl oxidase-mediated cross-linkage of the lysine residues that leads to a grow...

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Banded fibers in Tropoelastin coacervates at physiological temperatures

Cited by 57 publications

References 19 publications

Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

Targeted Disruption of Fibulin-4 Abolishes Elastogenesis and Causes Perinatal Lethality in Mice

Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

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