Deficient coacervation of two forms of human tropoelastin associated with supravalvular aortic stenosis.

Human tropoelastin associates by coacervation and is subsequently cross-linked to make elastin. In Williams syndrome, defective elastin deposition is associated with hemizygous deletion of the tropoelastin gene in supravalvular aortic stenosis (SVAS). Remarkably, point-mutation forms of SVAS correspond to incomplete forms of tropoelastin which include in-frame termination by nonsense mutations, yet the resulting phenotype of these disorders is not explained because expression variably occurs from both normal and mutant alleles. Proteins corresponding to two truncated tropoelastin mutants were expressed and purified to homogeneity. Coacervation of these proteins occurred as expected with increasing temperature, but substantially contrasted with that of the performance of a normal tropoelastin. Significantly, association by coacervation of the truncated SVAS tropoelastin molecules was negligible at 37 degrees C, which contrasted with the substantial coacervation seen for normal tropoelastin. Furthermore their midpoints of coacervation increased and correlated with the extent of deletion, in accord with the loss of hydrophobic regions required for tropoelastin association. Their secondary structures are similar, as evidenced by CD studies. We propose a model for point-mutation SVAS in which aberrant tropoelastin molecules are incompetent and are mainly excluded from participation in coacervation and consequently in elastogenesis. These forms of SVAS may consequently be considered functionally similar to a hemizygous deletion, and mark point-mutation SVAS as a disorder of defective coacervation.

Retinoic acid increases elastin in neonatal rat lung fibroblast cultures.

The factors that regulate elastin synthesis during pulmonary alveolar septal formation have not been identified. Because maximal alveolar elastin synthesis occurs over a relatively brief period (postnatal days 4-14 in the rat), we hypothesized that changes in the local concentrations of factors that regulate elastin synthesis may precede or accompany this period. Because pulmonary retinoid stores decline just before the fourth postnatal day, we also hypothesized that this decline could be accompanied by the utilization of retinoic acid, one of the most biologically active retinoids, in a regulatory process that increases elastin synthesis. If these hypotheses are correct, then retinoic acid should increase elastin synthesis by pulmonary cells. Therefore, cultures of neonatal rat lung fibroblasts were exposed to retinoic acid, and elastin production was quantitated. Retinoic acid produced a two- to threefold increase in the steady-state level of elastin mRNA, in soluble elastin, and in insoluble elastin. The transcriptional initiation rate of the elastin gene was 1.8-fold higher in nuclei that were isolated from retinoic acid-treated cells than in nuclei that were isolated from control cells. This indicates that the increase in steady-state elastin mRNA results, at least partially, from an increase in elastin transcription. Lung fibroblasts that were isolated from 8-day-old rats, but not cultured, contained retinoic acid. These findings suggest that retinoic acid is a potential regulator of elastin synthesis in developing pulmonary alveoli.

The ductus arteriosus migratory smooth muscle cell phenotype processes tropoelastin to a 52-kDa product associated with impaired assembly of elastic laminae.

We established the identity of a 52-kDa protein secreted by fetal lamb ductus arteriosus (DA) smooth muscle cells (SMC) and suggest how it might be related to structural changes unique to DA development, i.e. reduced assembly of elastic laminae and associated formation of intimal cushions. We produced a monoclonal antibody (HI-20) to the 52-kDa protein and observed, by electron microscopy, immunogold labeling of elastin in both DA and aorta vessel walls. Western immunoblotting showed that HI-20, as well as antibodies to tropoelastin, reacted with the 52-kDa protein secreted by DA SMC, as well as with 68-kDa tropoelastin. The highly specific antibody to the carboxyl-terminal sequence of tropoelastin failed, however, to recognize the 52-kDa protein, although it reacted well with the 68-kDa tropoelastin. Amino acid analysis and sequencing data confirmed the identity of the affinity-purified 52-kDa protein as truncated tropoelastin with an intact amino terminus. Cell-free translation of mRNA extracted from DA and aorta SMC produced a 68-kDa, but not a 52-kDa, immunoprecipitated tropoelastin. When DA and aorta SMC were pulsed with [14C]valine, we immunoprecipitated, after only a 15-min chase, both 68-kDa and 52-kDa tropoelastin from cell extracts of DA SMC, but only the 68-kDa tropoelastin was present in aorta SMC. There was no evidence of proteolytic degradation of radiolabeled aorta 68-kDa tropoelastin to a 52-kDa species when mixed with DA SMC conditioned medium. This suggests that the 52-kDa tropoelastin is the result of cell-associated processing or degradation of an original 68-kDa product of translation. Furthermore, pulse-chase experiments showed initial secretion of equivalent amounts of 68-kDa and 52-kDa tropoelastins by cultured DA SMC with increasing accumulation of the 52-kDa species, suggesting its impaired insolubilization. The production, in high concentration, of a 52-kDa tropoelastin product that lacks the carboxyl terminus, may prevent its alignment on the microfibrillar scaffold, resulting in abnormal assembly of elastic laminae in the DA. The accumulation of this soluble tropoelastin may be associated with the previously described property of chemotaxis resulting in the increased SMC migration into the subendothelium associated with DA intimal thickening.

Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor.

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells.

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.

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.

Elastin covalent structure as determined by solid phase amino acid sequencing.

The amino acid sequences of 16 large tryptic fragments of aortic tropoelastin have been determined establishing the presence of several repeating structures: GVP, GGVP, PGVGV, PGVGVA, and AGVPGFGVG. The methodologies for achieving these results by solid phase sequencing are reviewed and also the possible biologic significance of the unusual primary structures of elastin are discussed.

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.

Banded fibers in tropoelastin coacervates at physiological temperatures.

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

Comparison of aortic and ear cartilage tropoelastins isolated from lathyritic pigs.

Tropoelastin was isolated from aortae and auricular cartilage obtained from lathyritic piglets. The two tissue-specific tropoelastins were judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on a high-pressure liquid chromatograph. Comparative studies of the tropoelastins were performed. Amino acid analysis revealed that the aortic and cartilage tropoelastins were very similar, if not identical, with the only exception that the cartilage tropoelastin contained more hydroxyproline and less lysine residues, both of which can be attributable to post-translational modifications. Both tropoelastins possess an apparent molecular weight of 70 000 and exhibit similar peptide fragments with limited trypsin cleavage. Antiserum raised to the aortic tropoelastin was used to show immunological identity between the two tissue tropoelastins.

Partial characterization of a tropoelastin precursor isolated from chick aorta.

Evidence is presented that indicates tropoelastin is derived from a soluble elastin with a molecular weight of 95000. Tropoelastin and its proposed precursor were isolated from the aortas of copper-deficient chicks. Although it is doubtful that the proposed precursor is an initial product of elastin translation, i.e., a proelastin, it is proposed to be at least a truncated form of proelastin that is converted to tropoelastin. The key to its isolation was the presence of alpha 1-antitrypsin at each step in the purification procedure. The first 11 amino acid residues at the NH2 terminal of the proposed tropoelastin precursor (GGVPGVAVPGGV) are the same as those for tropoelastin. Its amino acid composition is similar to that of tropoelastin, except for higher amounts of acidic amino acid residues. Further, the proposed precursor contains a limited number of aldehydic functions, presumably in the form of peptidyl allysine. This was taken as an indication that the proposed precursor serves as a substract for lysyl oxidase. Under the conditions used for the isolation, the precursor appeared to be in higher concentrations than tropoelastin in aorta extracts from copper-deficient chicks.

Trypsin-like neutral protease associated with soluble elastin.

Isolation of tropoelastin is complicated by the presence of a neutral protease closely associated with tropoelastin that is capable of sequentially degrading tropoelastin to small peptides. Substrate and inhibitor specificities of this neutral protease associated with purified tropoelastin were examined. The enzyme displayed proteolytic activity against casein, and esterase activity was detected when assayed against N-tosyl-L-arginine methyl ester but not against tert-butyl-oxycarbonyl-L-alanine p-nitrophenyl ester. No appreciable elastinolytic activity was detectable against either insoluble sodium dodecyl sulfate treated elastin or maleylated tropoelastin. The enzyme was not inhibited by the chymotrypsin inhibitor toluenesulfonylphenylalanine chloromethyl ketone. The enzyme was inhibited by phenylmethanesulfonyl fluoride and, to various degrees, by metal chelators. Tosyllysyl chloromethyl ketone, epsilon-aminocaproic acid, and Aprotinin (pancreatic trypsin inhibitor--Kunitz type), all inhibitors of trypsin-like enzymes, were very effective inhibitors, as were soybean trypsin inhibitor and human alpha-1-antitrypsin. The data suggest that the tropoelastin-associated enzyme is a neutral serine protease with trypsin-like specificity.