Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: Clues to the evolutionary history of elastins

Tropoelastin is the monomeric form of elastin, a polymeric extracellular matrix protein responsible for properties of extensibility and elastic recoil in connective tissues of most vertebrates. As an approach to investigate how sequence and structural characteristics of tropoelastin assist in polymeric assembly and account for the elastomeric properties of this polymer, and to better understand the evolutionary history of elastin, we have identified and characterized tropoelastins from frog (Xenopus tropicalis) and zebrafish (Danio rerio), comparing these to their mammalian and avian counterparts. Unlike other species, two tropoelastin genes were expressed in zebrafish. All tropoelastins shared a predominant and characteristic alternating domain arrangement, as well as the fundamental crosslinking sequence motifs. However, zebrafish and frog tropoelastins had several unusual characteristics, including increased exon numbers and protein molecular weights, and decreased hydropathies. For all tropoelastins there was evidence of evolutionary expansion of the proteins by extensive replication of a hydrophobic-crosslinking exon pair. This was particularly apparent for zebrafish and frog tropoelastin genes, where remnants of sequence similarity were also seen in introns flanking the replicated exon pair. While overall alignment of mammalian, avian, frog and zebrafish tropoelastin sequences was not possible because of sequence variability, the C-terminal exon was well-conserved in all species. In addition, good sequence alignment was possible for several exons just upstream of the putative region of replication, suggesting that these conserved domains may represent 'primordial' core sequences present in the ancestral sequence common to all tropoelastins and in some way essential to the structure/function of elastin.     

Characterization of an unusual tropoelastin with truncated C-terminus in the frog

Tropoelastin is the monomeric form of elastin, a major polymeric protein of the extracellular elastic matrix of vertebrate tissues with properties of extensibility and elastic recoil. Mammalian and avian species contain a single gene for tropoelastin. A tropoelastin gene has also previously been identified in amphibians. In contrast, two tropoelastin genes with different tissue expression patterns have been described in teleosts. While general characteristics of tropoelastins, such as alternating arrangements of hydrophobic and crosslinking domains, are conserved across a wide phylogenetic range, sequences of these domains are highly variable, particularly when amphibian and teleost tropoelastins are included. For this reason exon-to-exon correspondence is not clear, and overall alignment of tropoelastin sequences across all species is not possible. An exception to this is the C-terminal exon, whose coding sequence has been very well-conserved across all species described to date. In mammalians this C-terminal domain has been shown to be important for interactions with cells and other matrix-associated proteins involved in matrix assembly. Here we identify and characterize a second tropoelastin gene in the frog with several unusual characteristics, the most striking of which is truncation of the C-terminal domain, deleting normally conserved sequence motifs. We demonstrate that, in spite of the absence of these motifs, both frog tropoelastin genes are expressed and incorporated into the elastic matrix, although with differential tissue localizations.     

Codistribution analysis of elastin and related fibrillar proteins in early vertebrate development.

Elastin is an extracellular matrix protein found in adult and neonatal vasculature, lung, skin and connective tissue. It is secreted as tropoelastin, a soluble protein that is cross-linked in the tissue space to form an insoluble elastin matrix. Cross-linked elastin can be found in association with several microfibril-associated proteins including fibrillin-1, fibrillin-2 and fibulin-1 suggesting that these proteins contribute to elastic fiber assembly, structure or function. To date, the earliest reported elastin expression was in the conotruncal region of the developing avian heart at 3.5 days of gestation. Here we report that elastin expression begins at significantly earlier developmental stages. Using a novel immunolabeling method, the deposition of elastin, fibrillin-1 and -2 and fibulin-1 was analyzed in avian embryos at several time points during the first 2 days of development. Elastin was found at the midline associated with axial structures such as the notochord and somites at 23 h of development. Fibrillin-1 and -2 and fibulin-1 were also expressed at the embryonic midline at this stage with fibrillin-1 and fibulin-1 showing a high degree of colocalization with elastin in fibers surrounding midline structures. The expression of these genes was confirmed by conventional immunoblotting and mRNA detection methods. Our results demonstrate that elastin polypeptide deposition occurs much earlier than was previously appreciated. Furthermore, the results suggest that elastin deposition at the early embryonic midline is accompanied by the deposition and organization of a number of extracellular matrix polypeptides. These filamentous extracellular matrix structures may act to transduce or otherwise stabilize dynamic forces generated during embryogenesis.     

Colocalization in vivo and association in vitro of perlecan and elastin

We have colocalized elastin and fibrillin-1 with perlecan in extracellular matrix of tensional and weight-bearing connective tissues. Elastin and fibrillin-1 were identified as prominent components of paraspinal blood vessels, and posterior longitudinal ligament in the human fetal spine and outer annulus fibrosus of the fetal intervertebral disc. We also colocalized perlecan with a synovial elastic basal lamina, where the attached synovial cells were observed to produce perlecan. Elastin, fibrillin-1 and perlecan were co-localized in the intima and media of small blood vessels in the synovium and in human fetal paraspinal blood vessels. Elastic fibers were observed at the insertion point of the anterior cruciate ligament to bone in the ovine stifle joint where they colocalized with perlecan. Elastin has not previously been reported to be spatially associated with perlecan in these tissues. Interactions between the tropoelastin and perlecan heparan sulfate chains were demonstrated using quartz crystal microbalance with dissipation solid phase binding studies. Electrostatic interactions through the heparan sulfate chains of perlecan and core protein mediated the interactions with tropoelastin, and were both important in the coacervation of tropoelastin and deposition of elastin onto perlecan immobilized on the chip surface. This may help us to understand the interactions which are expected to occur in vivo between the tropoelastin and perlecan to facilitate the deposition of elastin and formation of elastic microfibrils in situ and would be consistent with the observed distributions of these components in a number of connective tissues.     

Differential expression of aortic and lung elastin genes during chick embryogenesis

The ratios of tropoelastin b to a were measured in chick aorta and lung during embryogenesis. The rates of tropoelastin a and b synthesis were determined in short-term organ culture. The results demonstrated that in lung tissue the ratio of the two tropoelastins remained essentially constant. Each of the tropoelastins comprised 50% of the total elastin synthesis. In the aortic tissue, tropoelastin b represented 70% of the total elastin in the 11- to 13-day embryos and increased to 91% by Day 16. These observations seen in the organ culture system were paralleled in measurements of functional mRNAs coding for the two proteins. Measurements of functional tropoelastin mRNAs from both lung and aortic tissues were performed in a mRNA-dependent rabbit reticulocyte lysate system. Although the changes in the abundance of the tropoelastin mRNAs revealed the same trend as that seen in the organ culture data, the magnitude of the tropoelastin b to a ratio in the aortic organ culture was twice that determined in the cell-free translation of aortic mRNAs. The data obtained from both cell-free translations and organ culture experiments demonstrate that there is a differential expression of elastin genes during aorta development which is significantly different from that found in developing lung.     

Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein

FKBP65 (65-kDa FK506-binding protein) is a member of the highly conserved family of intracellular receptors called immunophilins. All have the property of peptidyl-prolyl cis-trans isomerization, and most have been implicated in folding and trafficking events. In an earlier study, we identified that FKBP65 associates with the extracellular matrix protein tropoelastin during its transport through the cell. In the present study, we have carried out a detailed investigation of the subcellular localization of FKBP65 and its relationship to tropoelastin. Using subcellular fractionation, Triton X-114 phase separation, protease protection assays, and immunofluorescence microscopy (IF), we have identified that FKBP65 is contained within the lumen of the endoplasmic reticulum (ER). Subsequent IF studies colocalized FKBP65 with tropoelastin and showed that the two proteins dissociate before reaching the Golgi apparatus. Immunohistochemical localization of FKBP65 in developing lung showed strong staining of vascular and airway smooth muscle cells. Similar areas stained positive for the presence of elastic fibers in the extracellular matrix. The expression of FKBP65 was investigated during development as tropoelastin is not expressed in adult tissues. Tissue-specific expression of FKBP65 was observed in 12-d old mouse tissues; however, the pattern of expression of FKBP65 was not restricted to those tissues expressing tropoelastin. This suggests that additional ligands for FKBP65 likely exist within the ER. Remarkably, in the adult tissues examined, FKBP65 expression was absent or barely detectable. Taken together, these results support an ER-localized FKBP65-tropoelastin interaction that occurs specifically during growth and development of tissues.     

Tropoelastin

Tropoelastin is a 60-72 kDa alternatively spliced extracellular matrix protein and a key component of elastic fibres. It is found in all vertebrates except for cyclostomes. Secreted tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for cross-linking and incorporation into growing elastic fibres. Tropoelastin is characterised by alternating hydrophobic and hydrophilic domains and is highly flexible. The conserved C-terminus is an area of the molecule of particular biological importance in that it is required for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoelastin gives elastin, which confers resilience and elasticity on a diverse range of tissues. Elastin gene disruptions in disease states and knockout mice emphasise the importance of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoelastin constructs hold promise as biomaterials as they mimic many of elastin's physical and biological properties with the capacity to replace damaged elastin-rich tissue.     

Chick tropoelastin isoforms. From the gene to the extracellular matrix

Studies from several laboratories have demonstrated the existence of multiple tropoelasting mRNAs and protein isoforms. The present study was designed to examine the developmental expression of a specific tropoelastin mRNA, its encoded isoform, and the fate of that isoform in the extracellular matrix. A chick genomic DNA library was screened with a chick tropoelastin cDNA. Seven unique, overlapping clones spanning 39 kilobases were isolated. A synthetic oligonucleotide complementary to a variable tropoelastin mRNA sequence was used to identify a 1.5-kilobase PstI-BamHI genomic fragment. Nucleotide sequence data revealed that the putative exon was surrounded by intron sequences possessing canonical splice sites at the exon/intron borders. Using both immunologic and molecular probes specific to the tropoelastin isoform and mRNA, quantitative protein and RNA analyses were performed. Results demonstrate that total tropoelastin mRNAs increased significantly during aortic embryogenesis whereas the amount of mRNA containing the variable exon remained relatively constant. The amount of total tropoelastins within the same developmental period reflect the level of total tropoelastin mRNA. The amount of the tropoelastin isoform containing the variable exon essentially mirrored the corresponding mRNA with the exception that a decrease in the isoform at day 15 was not seen in the mRNA level. Immunoelectron micrographs of 13-day chick aortic tissue using both total and isoform-specific antisera showed ultrastructural localization to definable elastic fibers. Antibodies to the variable tropoelastin isoform occurred preferentially at sites where elastic fiber microfibril structures were evident.     

Fibrillins, fibulins, and matrix-associated glycoprotein modulate the kinetics and morphology of in vitro self-assembly of a recombinant elastin-like polypeptide

Elastin is the polymeric protein responsible for the properties of extensibility and elastic recoil of the extracellular matrix in a variety of tissues. Although proper assembly of the elastic matrix is crucial for its durability, the process by which this assembly takes place is not well-understood. Recent data suggest the complex interaction of tropoelastin, the monomeric form of elastin, with a number of other elastic matrix-associated proteins, including fibrillins, fibulins, and matrix-associated glycoprotein (MAGP), is important to achieve the proper architecture of the elastic matrix. At the same time, it is becoming clear that self-assembly properties intrinsic to tropoelastin itself, reflected in a temperature-induced phase separation known as coacervation, are also important in this assembly process. In this study, using a well-characterized elastin-like polypeptide that mimics the self-assembly properties of full-length tropoelastin, the process of self-assembly is deconstructed into "coacervation" and "maturation" stages that can be distinguished kinetically by different parameters. Members of the fibrillin, fibulin, and MAGP families of proteins are shown to profoundly affect both the kinetics of self-assembly and the morphology of the maturing coacervate, restricting the growth of coacervate droplets and, in some cases, causing clustering of droplets into fibrillar structures.     

Characterization of rat heart tropoelastin

Several overlapping rat tropoelastin cDNA clones were isolated from a lambda gt11 rat heart cDNA library and their nucleotide sequence was determined. The corresponding deduced amino acid sequence of rat tropoelastin revealed strong homology to bovine and human tropoelastins although possessing some unique features including greater size (18%) and composition of repetitive units. Comparison of the amino acid sequence of rat tropoelastin to four other tropoelastin species reveals that the hydrophobic peptide repeat regions in the middle of each molecule and the crosslinking areas containing three lysine residues are remarkably conserved. A possible function for the clustering of three lysine residues in providing a mechanism for the in vivo reduction of dehydrolysinonorleucine via a redox shuttle with dihydrodesmosine is proposed. In addition, the COOH-terminal sequence of the rat tropoelastin is virtually identical to tropoelastins of other species in possessing a cysteine/arginine/lysine containing segment. There are no obvious amino acid insertions or substitutions in the COOH-terminal half of the rat tropoelastin molecule which would signal unique cleavage or glycosylation sites. Examination of the steady-state levels of rat tropoelastin mRNA in 8- and 12-day neonatal lung, heart, and aortic tissues showed that the amount of tropoelastin mRNA was abundant and of similar size (3.9 kb) in all three tissues.     

Single nucleotide polymorphisms and domain/splice variants modulate assembly and elastomeric properties of human elastin. Implications for tissue specificity and durability of elastic tissue

Polymeric elastin provides the physiologically essential properties of extensibility and elastic recoil to large arteries, heart valves, lungs, skin and other tissues. Although the detailed relationship between sequence, structure and mechanical properties of elastin remains a matter of investigation, data from both the full‐length monomer, tropoelastin, and smaller elastin‐like polypeptides have demonstrated that variations in protein sequence can affect both polymeric assembly and tensile mechanical properties. Here we model known splice variants of human tropoelastin (hTE), assessing effects on shape, polymeric assembly and mechanical properties. Additionally we investigate effects of known single nucleotide polymorphisms in hTE, some of which have been associated with later‐onset loss of structural integrity of elastic tissues and others predicted to affect material properties of elastin matrices on the basis of their location in evolutionarily conserved sites in amniote tropoelastins. Results of these studies show that such sequence variations can significantly alter both the assembly of tropoelastin monomers into a polymeric network and the tensile mechanical properties of that network. Such variations could provide a temporal‐ or tissue‐specific means to customize material properties of elastic tissues to different functional requirements. Conversely, aberrant splicing inappropriate for a tissue or developmental stage or polymorphisms affecting polymeric assembly could compromise the functionality and durability of elastic tissues. To our knowledge, this is the first example of a study that assesses the consequences of known polymorphisms and domain/splice variants in tropoelastin on assembly and detailed elastomeric properties of polymeric elastin.     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline‐poor, and forms β‐sheet amyloid‐like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of β‐sheet formation at this location. Our data support a dual role for confined β‐sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross‐linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure‐function relationships for the rational design of elastomeric biomaterials with defined mechanical properties. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 267–275, 2016.     

Methods in elastic tissue biology: elastin isolation and purification

Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.     

Elastic fiber macro-assembly is a hierarchical, cell motion-mediated process

Elastic fibers are responsible for the extensibility and resilience of many vertebrate tissues, and improperly assembled elastic fibers are implicated in a number of human diseases. It was recently demonstrated that in vitro, cells first secrete tropoelastin into a punctate pattern of globules. To study the dynamics of macroassembly, that is, the assembly of the secreted tropoelastin globules into elastic fibers, we utilized long-term time-lapse immunofluorescence imaging and a tropoelastin p Timer fusion protein, which shifts its fluorescence spectrum over time. Pulse-chase immunolabeling of the fibroblast-like RFL-6 cells demonstrates that tropoelastin globules aggregate in a hierarchical manner, creating progressively larger fibrillar structures. By analyzing the correlation between cell and extracellular matrix movements, we show that both the aggregation process and shaping the aggregates into fibrillar form is coupled to cell motion. We also show that the motion of non-adjacent cells becomes more coordinated as the physical size of elastin-containing aggregates increases. Our data imply that the formation of elastic fibers involves the concerted action and motility of multiple cells.     

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.     

Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.     

Evidence for the existence of three chick lung tropoelastins

Three tropoelastin polypeptides are identified among the cell-free translation products of chick embryo lung mRNAs and organ cultures extracts. The tropoelastins are distinguished by one and two dimensional gel electrophoretic systems and are all immunoreactive with monospecific chick tropoelastin antiserum. The ratio of the three tropoelastins does not vary significantly between 10 and 16 days of lung embryogenesis. The third tropoelastin (c) is found to co-migrate with tropoelastin b on SDS-polyacrylamide gel electrophoresis but is visible after cyanogen bromide cleavage of reticulocyte lysate proteins. Immunoprecipitates from lung organ culture also contain tropoelastins a, b and c.