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PubMed1995

Presence of elastin-related 45-kDa fragment in culture medium: specific cleavage product of tropoelastin in vascular smooth muscle cell culture.

Significant amount of 45-kDa polypeptide was found to be present in the cultured medium of chick aortic smooth muscle cells. The polypeptide as well as tropoelastin (65-kDa) reacted with monoclonal antibody for tropoelastin. Pulse-chase experiments revealed that the relative density of the 45-kDa polypeptide to tropoelastin increased with chase periods. Partially purified radioactive tropoelastin (65-kDa) was converted to a 45-kDa polypeptide fragment in the test tube. The processing from the 65- to the 45-kDa polypeptide in the test tube was inhibited by ethylenediaminetetraacetic acid but not by N-ethylmaleimide and aminophenylmethylsulfonyl fluoride. These results indicate that the 45-kDa fragment is a degradation product of tropoelastin and that processing is mediated by enzymatic cleavages with metal proteinase. Fully hydroxylated tropoelastin treated with ascorbic acid was more resistant to the enzymes than underhydroxylated tropoelastin with scorbutic condition, suggesting that the structural stability of tropoelastin is also involved in the processing rate.

Animals↗
PubMed1992

Elements of the rat tropoelastin gene associated with alternative splicing.

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.

Animals↗
PubMed2006

The characteristics of elastic fiber assembled with recombinant tropoelastin isoform.

OBJECTIVE: It is known that elastin mRNA is transcribed from a single gene. The variety of tropoelastin isoforms results from multiple alternative splicing of the primary transcript. The purpose of this study was to investigate the characteristics of elastic fiber assembled with tropoelastin isoform, which is full-length human tropoelastin (HTE), exon 26A missing tropoelastin (Delta26A), and exon 32 missing tropoelastin (Delta32). DESIGN AND METHODS: We demonstrated the process of elastic fiber assembly and the existence of elastic fiber resistant to pancreatic elastase with HTE, Delta26A, or Delta32 fiber using an in vitro model of elastic fiber assembly. These elastic fibers were evaluated by immunofluorescent staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS: There were no big differences getting into the matrix among these tropoelastins in immunofluorescence microscopy and semi-quantitative analysis. In the comparison with the HTE, the Delta26A and the Delta32 significantly increased and decreased, respectively, the formation of cross-linking amino acids and the binding to scaffold proteins. Furthermore, it was found that it is difficult to degrade the Delta26A assembly with pancreatic elastase as compared with HTE or Delta32 assembly. CONCLUSION: The elastic fiber assembled with the tropoelastin isoforms was characterized using an in vitro model. The present study provides important information regarding the pathology of human diseases including emphysema and atherosclerosis.

Alternative Splicing↗
PubMed1991

Alternative splicing of rat tropoelastin mRNA is tissue-specific and developmentally regulated.

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1991

Mammalian tropoelastin: multiple domains of the protein define an evolutionarily divergent amino acid sequence.

We have recently derived the complete amino acid sequence of rat tropoelastin from a series of overlapping cDNA clones. Comparison of this protein sequence to bovine and human tropoelastin has revealed significant differences in the rates of evolutionary divergence of the various domains of tropoelastin. The overall rate of divergence of the hydrophobic domains of tropoelastin was twice as fast as the cross-link domains of the protein. Certain hydrophobic domains, however, are as conserved as cross-link regions, particularly the hydrophobic sequence coded for by exon 33, the only exon subject to alternate usage in all three mammalian species and the most conserved domain in rat, bovine and human tropoelastin. This conservation of sequence strongly suggests a more complex function of the hydrophobic region encoded by exon 33, beyond the elastic recoil characteristic of all hydrophobic domains of tropoelastin. A comparison of average rates of divergence of hydrophobic and cross-link domains of tropoelastin to functionally-defined domains of other structural proteins, such as collagen, has also revealed that overall, tropoelastin is a highly divergent amino acid sequence, comparable to proteins such as globin and the fibrino-peptides.

Amino Acid Sequence↗
PubMed2000

Interaction of tropoelastin with the amino-terminal domains of fibrillin-1 and fibrillin-2 suggests a role for the fibrillins in elastic fiber assembly.

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

Amino Acid Sequence↗
PubMed1999

An open reading frame element mediates posttranscriptional regulation of tropoelastin and responsiveness to transforming growth factor beta1.

Elastin, an extracellular component of arteries, lung, and skin, is produced during fetal and neonatal growth. We reported previously that the cessation of elastin production is controlled by a posttranscriptional mechanism. Although tropoelastin pre-mRNA is transcribed at the same rate in neonates and adults, marked instability of the fully processed transcript bars protein production in mature tissue. Using RNase protection, we identified a 10-nucleotide sequence in tropoelastin mRNA near the 5' end of the sequences coded by exon 30 that interacts specifically with a developmentally regulated cytosolic 50-kDa protein. Binding activity increased as tropoelastin expression dropped, being low in neonatal fibroblasts and high in adult cells, and treatment with transforming growth factor beta1 (TGF-beta1), which stimulates tropoelastin expression by stabilizing its mRNA, reduced mRNA-binding activity. No other region of tropoelastin mRNA interacted with cellular proteins, and no binding activity was detected in nuclear extracts. The ability of the exon-30 element to control mRNA decay and responsiveness to TGF-beta1 was assessed by three distinct functional assays: (i) insertion of exon 30 into a heterologous gene conferred increased reporter activity after exposure to TGF-beta1; (ii) addition of excess exon 30 RNA slowed tropoelastin mRNA decay in an in vitro polysome degradation assay; and (iii) a mutant tropoelastin cDNA lacking exon 30, compared to wild-type cDNA, produced a stable transcript whose levels were not affected by TGF-beta1. These findings demonstrate that posttranscriptional regulation of elastin production in mature tissue is conferred by a specific element within the open reading frame of tropoelastin mRNA.

Animals↗
PubMed1988

Rat tropoelastin is synthesized from a 3.5-kilobase mRNA.

A lambda gt11 cDNA library was constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats and screened with a human tropoelastin cDNA clone. DNA sequence analysis of several overlapping rat clones confirmed the presence of DNA sequences coding for murine tropoelastin and DNA sequences coding for the 3'-untranslated region of the rat tropoelastin mRNA. Northern blot analysis of total RNA from aortic tissue of neonatal rats using oligonucleotide probes derived from these rat tropoelastin cDNAs demonstrated the presence of a 3.5-kilobase tropoelastin mRNA. The size of this rat tropoelastin mRNA agrees with previous reports for the size of the mRNA coding for tropoelastin in tissue from several vertebrate species but contrasts with several reports suggesting the presence of a higher molecular weight mRNA species responsible for the synthesis of tropoelastin in rodent tissue.

Animals↗
PubMed1988

Developmental regulation of tropoelastin isoforms.

The production of tropoelastin isoforms was examined by cell-free translation of nuchal ligament RNA from cows of various ages ranging from 110 days of gestation to adult. Tropoelastin polypeptides synthesized in vitro were immunoprecipitated with a specific monoclonal antibody and separated by high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From these experiments three distinct tropoelastin isoform patterns were detected at different developmental periods. 1) The fetal period was characterized by three polypeptides of about 67, 65, and 63 kDa produced in a ratio of 1:3:2. 2) In neonatal tissue, the same molecular mass forms were detected; however, the relative isoform ratio changed to 2:3:1. 3) From adult ligament RNA, only the 67- and 65-kDa forms of tropoelastin were detected, and these were produced in equal amounts. The pattern of tropoelastin isoforms produced from RNA isolated from other fetal elastic tissues was the same as for fetal ligament. Additional studies demonstrated that the relative amount of functional tropoelastin mRNA correlated with steady state levels of mRNA consistent with transcriptional regulation of tropoelastin synthesis. Although the significance of developmental switching of individual tropoelastin isoforms is unknown, it is likely that this process is required for proper assembly and, thus, function of elastic tissue.

Aging↗
PubMed1996

Selective degradation of accumulated secretory proteins in the endoplasmic reticulum. A possible clearance pathway for abnormal tropoelastin.

The specific pathway of tropoelastin secretion was investigated in fetal calf ligamentum nuchae (FCL) cells using brefeldin A (BFA) to disrupt the secretory pathway. Electron microscopic studies of BFA-treated FCL cells showed ultrastructural changes consistent with the reported effects of BFA on intracellular organelles. When FCL cells were labeled with [3H]leucine in the presence of BFA, radiolabeled tropoelastin was not secreted, nor was there an intracellular accumulation of the protein. In contrast, fibronectin accumulated within the cells in the presence of BFA. Northern analysis of mRNA levels in FCL cells showed that the message for tropoelastin was unaffected by BFA treatment. Pulse chase experiments conducted in the presence of BFA demonstrated that the tropoelastin retained within the cells was rapidly degraded. Ammonium chloride, nocodazole, and cycloheximide had no effect on the degradation of tropoelastin, indicating that the degradation did not involve the endosome/lysosome pathway, movement via microtubules, or a short-lived protein, respectively. Incubation of FCL cells with BFA in the presence of N-acetyl-Leu-Leu-norleucinal, however, allowed tropoelastin to steadily accumulate in the cells. Cells pulsed in the presence of BFA alone showed that tropoelastin initially accumulates within the cells for approximately 1 h prior to being degraded, thus indicating that a critical threshold of tropoelastin must be reached before degradation can occur. Results from this study provide evidence for selective degradation of a soluble secreted protein by a cysteine protease following retention of the protein in the endoplasmic reticulum.

Ammonium Chloride↗
PubMed1994

Use of an intron polymorphism to localize the tropoelastin gene to mouse chromosome 5 in a region of linkage conservation with human chromosome 7.

The complete coding sequence for mouse tropoelastin was obtained from overlapping reverse transcriptase polymerase chain reaction (PCR) amplimers. These cDNA fragments were derived from mouse tropoelastin mRNA using PCR oligomers complementary to conserved domains within rat tropoelastin mRNA. A comparison of coding domains of mouse and rat tropoelastin mRNA revealed a greater than 93% homology at the nucleotide level and over 96% similarity in the predicted amino acid sequence. PCR primers complementary to regions of the mouse tropoelastin mRNA were used to define a novel intron length polymorphism (ILP) within intron 8 of the mouse tropoelastin gene (Eln). This ILP proved to be informative in an interspecific backcross in which genomic DNA samples from 75 backcross mice were used to map the tropoelastin gene to a position in the distal half of mouse chromosome 5. The linkage and genetic distances between Eln and the closest molecular markers used in this study are centromere-D5Mit95, D5Mit96-6.7 cM-Gus, Eln-4.0 cM-Zp3-telomere.

Amino Acid Sequence↗
PubMed1998

In vitro degradation of tropoelastin by reactive oxygen species.

The effects of reactive oxygen species (ROS) on elastin molecules (tropoelastin) were studied in vitro. ROS generated by ultraviolet A and hematoporphyrin rapidly degraded tropoelastin within 5 min. Their degradative activity was inhibited by the addition of NaN3. Treatment of tropoelastin with copper sulfate/ascorbic acid resulted in degradation of tropoelastin producing fragments of molecular weight 45, 30 and 10 kDa within 30 min. The degradation of tropoelastin was partially blocked by the addition of mannitol. ROS induced by the xanthine/xanthine oxidase system also degraded tropoelastin within 6 h. The degradation was blocked by catalase but not by superoxide dismutase (SOD). ROS generated by copper-ascorbate seems to be unique in that it cleaves relatively specific sites of the tropoelastin molecule. Thus ROS may play a degradative role in elastin metabolism which may cause the elastolytic changes or the deposition of fragmented elastic fibers in photoaged skin or age-related elastolytic disorders.

Ascorbic Acid↗
PubMed1987

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.

Animals↗
PubMed2005

Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro.

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to < 30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro.

Cells, Cultured↗
PubMed2004

Integrin alpha v beta 3 binds a unique non-RGD site near the C-terminus of human tropoelastin.

Tropoelastin is the soluble precursor of the essential resilient connective tissue protein elastin. We examined the binding of integrin alpha(v)beta(3) to tropoelastin. In quantitative colorimetric solid-phase assays, purified alpha(v)beta(3) demonstrated saturable, divalent cation-dependent, single-site binding behavior on tropoelastin with a dissociation constant of 3.8 +/- 0.9 nM in the presence of 1 mM Mn(2+) which increased to 23 +/- 5 nM in the presence of 1 mM Ca(2+). Association with alpha(v)beta(3) was localized to the C-terminal 16 residues of tropoelastin, encompassing the region encoded by exon 36. This region comprises a unique disulfide loop in tropoelastin that is not essential for the interaction. This is the first identification of a specific, single binding site on tropoelastin and the first observation of direct binding of an integrin to a tropoelastin domain.

Binding Sites↗
PubMed2006

Domains 17-27 of tropoelastin contain key regions of contact for coacervation and contain an unusual turn-containing crosslinking domain.

The central region of tropoelastin including domains 19-25 of human tropoelastin forms a hot-spot for contacts during the inter-molecular association of tropoelastin by coacervation [Wise, S.G., Mithieux, S.M., Raftery, M.J. and Weiss, A.S (2005). "Specificity in the coacervation of tropoelastin: solvent exposed lysines." Journal of Structural Biology 149: 273-81.]. We explored the physical properties of this central region using a sub-fragment bordered by domains 17-27 of human tropoelastin (SHEL 17-27) and identified the intra- and inter-molecular contacts it forms during coacervation. A homobifunctional amine reactive crosslinker (with a maximum reach of 11 A, corresponding to approximately 7 residues in an extended polypeptide chain) was used to capture these contacts and crosslinked regions were identified after protease cleavage and mass spectrometry (MS) with MS/MS verification. An intermolecular crosslink formed between the lysines at positions 353 of each strand of tropoelastin at the lowest of crosslinker concentrations and was observed in all samples tested, suggesting that this residue forms an important initial contact during coacervation. At higher crosslinker concentrations, residues K425 and K437 showed the highest levels of involvement in crosslinks. An intramolecular crosslink between these K425 and K437, separated by 11 residues, indicated that a structural bend must serve to bring these residues into close proximity. These studies were complemented by small angle X-ray scattering studies that confirmed a bend in this important subfragment of the tropoelastin molecule.

Amino Acid Sequence↗
PubMed1986

Tropoelastin synthesis in fetal bovine tissues.

Tropoelastin was examined in bovine lung, aortic and ligament tissues using both organ culture and cell-free translation systems. The bovine tissues synthesized two tropoelastin polypeptides of approximately 70,000 and 68,000 daltons. Two polypeptides were also seen amongst the translation products directed by mRNAs isolated from each of the individual tissues. Both proteins were shown to be tropoelastins directly by immunoprecipitation with specific antibody and limited NH2-terminal sequence analyses and indirectly by two-dimensional gel electrophoresis. The finding of two forms of tropoelastin is similar to that previously reported in chick tissues although the apparent molecular weights of the tropoelastins differ between the two species. Another interesting observation is that the proportion of the two tropoelastins differs amongst the three fetal tissues examined. This situation is similar to the differences seen in the ratio of tropoelastin a and be between embryonic chick lung and aortic tissues.

Animals↗
PubMed1992

Cellular expression of tropoelastin mRNA splice variants.

The primary transcript of tropoelastin is alternatively spliced into multiple mRNAs. The pattern and frequency of exon splicing is developmentally regulated, but the cellular profile of isoform expression within and among elastic tissues is not known. We used splice-variant specific antisense oligomeric deoxyribonucleotide probes in an in situ hybridization assay to assess the distribution of cells undergoing specific alternative splicing of tropoelastin pre-mRNA in developing bovine elastic tissues. Antisense oligomers were synthesized to exon sequences that are not alternatively spliced (exon 36) and to sequences that become abutted after high frequency (exon 33) and low frequency (exons 13 and 14) alternative splicing. The specificity of these probes for tropoelastin splice variants was verified by Southern hybridization to tropoelastin cDNAs with known exon deletions, and their specificity for tropoelastin mRNA was demonstrated by Northern hybridization. In situ hybridization with [35S]-labeled oligomers on sections of bovine lobar pulmonary artery and other elastic tissues showed that all elastogenic cells produce multiple forms of tropoelastin mRNA. These observations suggest that the production of tropoelastin isoforms is common to all cells within an elastin tissue and that this multiplicity may not be involved in regional differences in elastic tissue architecture.

Animals↗