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PubMed1999

Sequential loss of two neighboring exons of the tropoelastin gene during primate evolution.

Previous evidence has demonstrated the absence of exons 34 and 35 within the 3' end of the human tropoelastin (ELN) gene. These exons encode conserved polypeptide domains within tropoelastin and are found in the ELN gene in vertebrate species ranging from chickens to rats to cows. We have analyzed the ELN gene in a variety of primate species to determine whether the absence of exons 34 and 35 in humans either is due to allelic variation within the human population or is a general characteristic of the Primates order. An analysis of the 3' end of the ELN gene in several nonhuman primates and in 546 chromosomes from humans of varying ethnic background demonstrated a sequential loss of exons 34 and 35 during primate evolution. The loss of exon 35 occurred at least 35-45 million years ago, when Catarrhines diverged from Platyrrhines (New World monkeys). Exon 34 loss, in contrast, occurred only about 6-8 million years ago, when Homo separated from the common ancestor shared with chimpanzees and gorillas. Loss of both exons was probably facilitated by Alu-mediated recombination events and possibly conferred a functional evolutionary advantage in elastic tissue.

Alleles↗
PubMed1986

Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers.

Solutions of tropoelastin incubated under different experimental conditions were examined by electron microscopy after negative staining and after fixation and embedding. Below 37 degrees C only polymorphous structureless elements of variable size could be found. In samples kept for a few minutes at 40 degrees C, flexible, isolated filaments of 5 nm diameter and variable length, together with a few small aggregates of filaments, were seen. No single filaments, but only bundles of filaments were detectable after incubation at 40 degrees C for longer than 5-10 min. Tropoelastin kept at 40 degrees C for longer than 10 hr formed a white precipitate, which, when fixed and embedded as in conventional electron microscopy, consisted of 0.5-2 microns thick, amorphous and branching fibers, identical to those seen in identically processed normal tissues. From these observations a model for the assembly and structure of elastic fibers is proposed.

Animals↗
PubMed2003

Rational design of tropoelastin peptide-based inhibitors of metalloproteinases.

Abnormal production of matrix metalloproteinases (MMPs) has been observed in a variety of diseases, such as emphysema, atherosclerosis, and cancer metastasis. Destruction of connective tissue ensues and elastin is often a key target. Three of the main elastolytic MMPs are the gelatinases MMP-2 and MMP-9 and the metalloelastase MMP-12. To investigate the possibility of using peptides to inhibit the elastolytic activity of these enzymes, we mapped the sites within tropoelastin recognized by MMP-9 and MMP-12. Peptides that correspond to regions overlapping these sites were then tested for their ability to inhibit these MMPs. These included an unmodified peptide directed against MMP-9 (peptide PP), cysteine-containing peptides that mimicked either the MMP-9 (peptide NCP) or the MMP-12 (peptide lin24) cleavage sites in tropoelastin and their cyclized forms (CP and cyc24, respectively), and a peptide containing a zinc-chelating hydroxamate group directed against MMP-9 (HP). The presence of a free sulfhydryl or hydroxamate group capable of chelating the zinc ion in the active site of the MMPs was generally found to increase the inhibitory activity of the peptides. The specificity of the inhibitors varied, with some of the inhibitors showing activity against all of the MMPs examined. None of the inhibitors had any significant effect on the activity of the unrelated serine protease, plasmin. K(i) values for the inhibitors were in the micromolar range. Our results suggest ways of developing other MMP inhibitors based on substrate recognition sites that may provide greater levels of inhibition.

Amino Acid Sequence↗
PubMed2003

Structural changes and facilitated association of tropoelastin.

Circular dichroism studies of tropoelastin secondary structure show 4+/-1% alpha-helix in aqueous solutions. This is in contrast to the substantially higher amounts (up to 23+/-7%) of alpha-helix predicted by computer algorithms, which propose that regions of alpha-helix are limited to the alanine-rich cross-linking domains. Through the addition of trifluoroethanol, the amount of alpha-helix increased to 17+/-1%, equivalent to that expected on the basis of primary structure. The physiological ability of the protein to coacervate and the critical concentration of monomer required for coacervation were unaffected by levels of alpha-helix. However, the temperature required for coacervation decreased linearly with increasing alpha-helical structure, which correlates with the participation of alpha-helices in association. We propose that the alanine-rich cross-linking domains exist as nascent helices in tropoelastin in aqueous solution. We further suggest a novel mechanism for coacervation whereby formation of alpha-helices and subsequent helical side chain interactions limit the conformational flexibility of the polypeptide, to facilitate associations between hydrophobic domains during elastogenesis.

Alanine↗
PubMed1993

PGAIPG, a repeated hexapeptide of bovine tropoelastin, is a ligand for the 67-kDa bovine elastin receptor.

Tropoelastin is composed of alternating hydrophobic and hydrophilic domains. A hydrophobic peptide, VGVAPG, has been shown to be a ligand for a 67-kDa elastin cell surface receptor expressed on fetal bovine auricular chondrocytes and ligamentum nuchae fibroblasts. To explore the possibility that tropoelastin contains additional peptide ligands for this elastin receptor, we have constructed two deletion proteins that are expressed in E. coli and lack the repeated VGVAPG sequence. These proteins supported bovine fibroblast attachment implying the presence of a receptor binding site. Experiments using synthetic peptides contained within these proteins identify a chemotactic peptide, PGAIPG, and a chemokinetic peptide, GAIPG, PGAIPG was identified as a ligand for the bovine elastin receptor.

Amino Acid Sequence↗
PubMed1987

Repeating structure of chick tropoelastin revealed by complementary DNA cloning.

A cDNA library was constructed from chick aorta poly(adenylic acid)-containing RNA in the expression vector pEX1. Several clones were identified by screening the library with a polyclonal antiserum raised against chick tropoelastin and confirmed by DNA sequencing. Analysis of the deduced amino acid sequence, corresponding to the mature tropoelastin and most of the signal peptide, revealed that the molecule is composed of at least 8, and possibly 13, repeating units. The common features of each unit include an N-terminal region composed largely of alanines and lysines and ending with an aromatic amino acid, followed by a GAG span and then a C-terminal region consisting mostly of valines, prolines, and glycines often present in several copies of the sequence (VPGV). This structure is discussed in terms of the functional properties of the molecule.

Amino Acid Sequence↗
PubMed1978

Elastin cross-linking in vitro. Studies on factors influencing the formation of desmosines by lysyl oxidase action on tropoelastin.

The formation of isodesmosine and desmosine in vitro by the action of lysyl oxidase on tropoelastin was studied. The synthesis of desmosines occurred in the absence of additional substances. The formation of desmosines was not affected by removal of molecular O2 from the reaction medium nor was it affected by the lack of proline hydroxylation in tropoelastin. However, there was virtually no desmosine formation at 15 degrees C, a temperature not conducive to coacervation, indicating that coacervation is an important prerequisite for cross-linking.

Amino Acid Oxidoreductases↗
PubMed2007

Domains 16 and 17 of tropoelastin in elastic fibre formation.

Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800-3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16-17 and results in a stable mRNA. Tropoelastin lacking domains 16-17 (Delta16-17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Delta16-17 with fibrillin-1 and fibulin-5. However, self-association of Delta16-17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Delta16-17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a beta-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16-17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.

Amino Acid Sequence↗
PubMed1993

Hyperoxic exposure of developing rat lung decreases tropoelastin mRNA levels that rebound postexposure.

These studies were undertaken to determine whether tropoelastin message expression in lung parenchymal tissue is altered in rats reared in an hyperoxic environment during the period of time that alveolar septation normally occurs. Rat pups were exposed to > 95% oxygen from days 4 to 14 and killed during the exposure and recovery periods. Results of in situ hybridizations indicated a delay in peak tropoelastin (TE) message levels in oxygen-exposed rats vs. controls, day 16 vs. day 11, respectively. In addition, lung parenchymal TE mRNA levels in the oxygen-exposed pups remained elevated through day 23, 1 wk after TE mRNA levels had decreased in controls. These observations suggest that the regulatory control of elastin synthesis during lung alveolar septation is altered by hyperoxic exposure. In a separate experiment, rat pups were exposed to > 95% oxygen during the period of alveolarization and followed for 4 wk postexposure. Pulmonary function measurements were conducted to determine whether lung function was altered postexposure and, if so, whether recovery occurred. We also used stereological techniques to quantitate the total length of lung parenchymal elastic fibers to determine whether elastic fiber content in the oxygen-exposed pups was restored to normal levels during the month postexposure. Although the total length of elastic fibers in lung parenchyma was found to be greater in oxygen-exposed than control pups from postnatal days 22 to 41, pressure-volume curves indicated that lungs of the oxygen-exposed pups tended to be more compliant than controls, suggesting that abnormal elastic fiber assembly might have been a contributing factor.

Aging↗
PubMed1998

Action of tropoelastin and synthetic elastin sequences on vascular tone and on free Ca2+ level in human vascular endothelial cells.

The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to microg/mL range. Similar concentrations of an elastin peptide preparation (kappa-elastin) were previously demonstrated to induce, among other biological actions, a dose- and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10(-13) to 10(-7) mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca2+]i of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium- and dose-dependent vasorelaxation and to increase endothelial [Ca2+]i, whereas PVGV and VGVA produced these effects only at low concentration (10(-11) mol/L). A likely candidate for mediating the elastin peptide-related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.

Animals↗
PubMed1979

Degradation of tropoelastin and elastin substrates by human neutrophil elastase, free and bound to alpha2-macroglobulin in serum of the M and Z (Pi) phenotypes for alpha1-antitrypsin.

Human neutrophil elastase degraded tropoelastin approximately 9 times faster than it did solubilized elastin and approximately 19 times faster than it did lung elastin. When bound to alpha2-M, the enzyme retained approximately 6 per cent of its activity toward tropoelastin and solubilized latter observations suggest that alpha2-M--bound elastase, cleared slowly from lung extracellular tissue space, may participate normally in the turnover of soluble precursor (s) of elastin and may contribute to the development of emphysema in alpha1-antitrypsin deficiency.

Animals↗
PubMed1996

Postpneumonectomy lung growth: a model of reinitiation of tropoelastin and type I collagen production in a normal pattern in adult rat lung.

Elastic and collagen fibers confer recoil and tensile strength on the pulmonary vasculature, airways, alveolar walls, and pleura. These durable extracellular matrix components are primarily synthesized during lung development and growth, and are expressed at very low levels in healthy adult lung. However, reinitiation of elastin and collagen synthesis in diseases of adult lung, such as idiopathic pulmonary fibrosis, often leads to excessive or aberrant deposition of elastin and collagen which contribute to the pathophysiology of these diseases. We used an experimental model of postpneumonectomy lung growth to determine whether normal patterns of synthesis and deposition of these critical structural components can occur in the adult lung. Male Sprague-Dawley rats (250-300 grams) were subjected to left pneumonectomy and right lobectomy. The remaining lung tissue was harvested for analysis after 3, 7, or 14 days. Compensatory growth of the remaining right lung progressed throughout the time course. Total desmosine and hydroxyproline content increased in the postpneumonectomy lung, reflecting increased elastin and collagen accumulation, but both were normal in content per weight of lung tissue. Northern analysis demonstrated induction of tropoelastin and type I procollagen mRNA expression in lungs of pneumonectomy rats. In situ hybridization localized tropoelastin and type I procollagen mRNA expression to anatomical sites similar to those seen during lung development. These data indicate that the adult lung can reinitiate elastin and collagen production and deposit these extracellular matrix components in a normal pattern.

Animals↗
PubMed1985

Demonstration of elastin gene expression in human skin fibroblast cultures and reduced tropoelastin production by cells from a patient with atrophoderma.

Atrophoderma is a rare dermal disorder characterized by a patchy distribution of areas apparently devoid of elastic fibers. Skin fibroblast cultures were established from the normal and affected dermis of a patient with this disorder. Human tropoelastin was identified in culture medium by use of electroblotting and anti-elastin antisera. An enzyme-linked immunosorbent assay was used to establish that significantly less elastin accumulated in the media of cultured cells from lesional fibroblasts over a 3-d period. Since elastin biosynthesis in most tissues is under pretranslational control, molecular hybridization to a nick-translated genomic elastin probe was performed; however, elastin messenger RNA levels were equivalent in both cell strains. Both strains produced less elastin than did normal skin fibroblasts. Extracellular proteolysis of elastin was evaluated as a possible mechanism. Elastase activity was increased and porcine tropoelastin was degraded four times faster, on a per-cell basis, in lesional fibroblast cultures than in cells derived from an unaffected site. The two cell strains exhibited no significant differences in collagen production or collagenase activity. These results are the first demonstration of elastin production by cultured human skin fibroblasts, and they suggest that the primary defect in atrophoderma may be a result of enhanced degradation of newly synthesized elastin precursors.

Adolescent↗
PubMed1981

The biosynthesis of tropoelastin in chick and pig tissues.

The cell-free translation of elastin mRNA's from different tissues as well as different animal sources was investigated. In all cases elastin mRNA's coding for both tropoelastin a and b were found. Of special significance was the finding that the ratio of the two tropoelastins varied between lung and aortic tissues and this observation was consistent for two animal species.

Animals↗
PubMed1994

Persistence, re-expression, and induction of pulmonary arterial fibronectin, tropoelastin, and type I procollagen mRNA expression in neonatal hypoxic pulmonary hypertension.

Changes in the structure and function of muscular pulmonary arteries are crucial for normal adaptation to extrauterine life and rapid changes in matrix protein gene expression are likely necessary for this adaptation. We hypothesized that pathological stimuli imposed at the time of birth would alter developmental changes in matrix protein gene expression thereby affecting the normal post-uterine changes in pulmonary hemodynamics. We used in situ hybridization to examine the normal developmental expression of three extracellular matrix protein mRNAs, fibronectin, tropoelastin, and alpha 1 (I) procollagen, in muscular pulmonary arteries of both fetal and neonatal calves and assessed the impact of severe hypoxia-induced pulmonary hypertension on their expression. Morphometric techniques were used to assess whether changes in matrix protein mRNA levels were related to changes in matrix fiber accumulation. Exposure to chronic hypoxia postnatally resulted in the persistence, reexpression, and induction of fibronectin, tropoelastin, and alpha 1 (I) procollagen mRNAs, respectively, in muscular pulmonary arteries. In each case the hybridization signal was localized primarily to the adventitial layer of the vessel. Morphometric analysis showed that the increased hybridization signals seen correlated with an increase in both vascular elastin and collagen fiber volumes in the adventitial layer. We conclude that the change in expression of matrix genes in the pulmonary artery wall during exposure to chronic hypoxia is an important adaptive response to changes in hemodynamic factors and/or oxygen tension. The unchecked increase in matrix protein expression seen likely contributes to the pathological pulmonary arterial structural remodeling and loss of vasoreactivity that occurs during the development of severe neonatal pulmonary hypertension.

Animals↗
PubMed1993

Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.

The use of recombinant human tropoelastin (rTE) and selected variants thereof as substrates for the assay of lysyl oxidase activity in vitro was explored. The possibility was also assessed that an insoluble elastin-like product could be generated from this elastin precursor in the absence of other macromolecules found associated with elastin in vivo. rTE was more efficiently oxidized by lysyl oxidase than the insoluble chick aorta elastin substrate conventionally used. Anionic amphiphilic elastin ligands strongly inhibited rTE oxidation consistent with the importance of electrostatic enzyme-substrate interactions previously noted with the insoluble elastin substrate. An rTE variant, rTE delta 26A, lacking the hydrophilic sequence coded by exon 26A, was a less effective substrate than rTE, largely due to an increase in Km, while the kinetic parameters for the oxidation of rTE delta 36, lacking the C-terminal polybasic sequence coded by exon 36, were quite similar to those for rTE. Incubation of rTE delta 26A with lysyl oxidase not only resulted in the generation of peptidyl alpha-aminoadipic-delta-semialdehyde and lysine-derived cross-linkages, but also yielded a product insoluble in hot 0.1 N NaOH, consistent with the properties of insoluble elastin. Thus, oxidation, cross-linking and insolubilization of elastin substrates by lysyl oxidase can occur in the absence of other macromolecules implicated as being involved in this process in vivo, although such macromolecules may be essential to obtain the proper alignment between tropoelastin units for specifically placed cross-linkages and optimally functional elastic fibers.

Amino Acid Sequence↗
PubMed1976

Fibrillation of tropoelastin induced by proteoglycan.

Electrostatic interaction between tropoelastin, the native precursor of elastin, and proteoglycan results in tropoelastin fibrillation. The finding suggests a possible involvement of proteoglycans in elastogenesis.

Chemical Phenomena↗
PubMed1976

Optical diffraction of tropoelastin and alpha-elastin coacervates.

Optical diffraction applied to micrographs of coacervated tropoelastin and alpha-elastin show an equatorial repeat around 50 A. This confirms a 50 A center-to-center distance of parallel aligned filaments to be a fundamental property of the tropoelastin and alpha-elastin coacervates. This periodicity is similar to that of mature cross-linked elastin. These results allow the conclusion that hydrophobic association is the predominant driving force for formation of filamentous elastin in vitro. It is suggested that the coacervate is a model for relaxed fibrous elastin.

Elastin↗