Comparative studies of the cross-linked regions of elastin from bovine ligamentum nuchae and bovine, porcine and human aorta.

1. The preparative Edman degradation of desmosine-containing peptides permitted the isolation of peptides C-terminal to the desmosine cross-links in bovine, porcine and human aortic elastin as well as bovine ligamentum nuchae elastin. This identifies the lysines in the tropoelastin which give rise to the desmosine cross-links. 2. The sequences from bovine aortic elastin were identical with those obtained from bovine ligamentum nuchae elastin but differed from those obtained from the other species. The most striking difference involves the occurrence of phenylalanine in bovine elastin and tyrosine in porcine and human elastin C-terminal to the desmosine cross-links. 3. The sequences of the C-terminal peptides were found to fall into two distinct classes, one starting with hydrophobic residues, the other starting with alanine. It is proposed that thehydrophobic residue prevents the enzymic oxidative deamination of the adjacent lysine e-amino group and this then contributes the nitrogen to the pyridinium ring of the cross-links.     

Isolation of elastin from bovine auricular cartilage.

Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.24.3), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine auricular cartilage. The low level of N-terminal residues (2.8 mol per 10⁶g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. The amino acid composition of the preparation showed an overall two-fold increase in polar residues, and a 20% reduction in valine, when compared to those of aortic and ligamentum nuchae elastin, while the concentration of cross-links was almost identical in the three preparations. Analysis of peptides, isolated by gel-exclusion chromatography after digestion of auricular elastin with elastase (pancreatopeptidase E, EC 3.4.21.11) in the presence of sodium dodecyl sulfate, revealed elevated levels of polar residues in all fractions examined, with no correlation between the concentration of these amino acids and that of the lysine-derived cross-links. Comparison of auricular and ligamentum nuchae elastin by fingerprint analysis of their elastase digests also suggested that the two proteins were compositionally distinct. Finally, treatment of auricular elastin with either trypsin or chymotrypsin produced no significant reduction in the level of polar residues. It is concluded that elastin exhibits tissue-related compositional variability.     

Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin - producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine.     

Removal of adsorbed elastase from partially digested elastin.

Bovine ligamentum nuchae elastin that had been partially digested by porcine pancreatic elastase and then thoroughly washed with 0.2 M-NaC1/0.05 M-sodium borate solution continued to release peptide fragments in solution. Complete removal of elastase from the elastin surface required extraction with dilute alkali, a procedure that does not irreversibly inactivate the enzyme or hydrolyse elastin.     

Application of affinity chromatography to the purification of collagenase for the isolation of insoluble elastin.

ClostrIdium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.19) was purified by batchwise separation with DEAE-cellulose followed by affinity chromatography on a column of alkali-treated elastin. The N-terminal amino acid profile of elastin isolated from bovine ligamentum nuchae using this enzyme preparation was compared with that of a duplicate sample purified with a mixture of collagenase I and II (Yoshida, E, and Noda, H. (1965) Biochim. Biopsys. Acta 105, 562-574). An approx. three-fold decrease in the molar concentration of N-terminal residues and a considerable reduction in their number was obtained by using the former enzyme preparation.     

Identification of multiple tropoelastins secreted by bovine cells

High resolution gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cell-free translation, and elastin-specific antibodies were used to identify three tropoelastin isoforms secreted by bovine tissue and cells. Tropoelastin isolated from nuchal ligament and from conditioned culture medium or cell-matrix extracts of ligament fibroblasts and auricular chondrocytes resolved as three distinct bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of approximately 67,500 (tropoelastin I), 65,000 (tropoelastin II), and 62,000 (tropoelastin III). Three tropoelastin polypeptides with molecular mass 2-3 kDa higher than their corresponding tissue forms were also evident in cell-free translation products of ligamentum nuchae RNA, suggesting that each tropoelastin species is encoded by a unique mRNA. The presence of cysteine in all three tropoelastin isoforms was demonstrated by the incorporation of [35S]cysteine into newly synthesized tropoelastin polypeptides and by immunoreactivity with an antibody raised against a synthetic peptide that defines the cysteine-containing carboxyl-terminal region of tropoelastin. Immunological co-localization of the carboxyl-terminal antibody with insoluble elastin in lung vasculature and parenchyma suggests that intact tropoelastin and not a processed form is incorporated into the elastin fiber.     

Glucocorticoids stimulate elastin production in differentiated bovine ligament fibroblasts but do not induce elastin synthesis in undifferentiated cells

Glucocorticoid treatment of fibroblasts from late gestation fetal bovine ligamentum nuchae resulted in a time- and dose-dependent selective increase in elastin production. Tropoelastin levels increased 2-3-fold in the presence of 10 nM dexamethasone while total protein synthesis and the rate of cell division decreased with glucocorticoid exposure. Two tropoelastin bands of molecular weights 64,500 and 61,000 were identified by immunoprecipitation and sodium dodecyl sulfate gradient-gel electrophoresis and both bands increased to an equal extent in the presence of dexamethasone. Undifferentiated cells from early-gestation animals did not synthesize elastin after hormone exposure, even though glucocorticoid receptors were demonstrated by nuclear-translocation experiments. These results indicate that glucocorticoids stimulate elastin production in elastin-producing ligament cells but do not induce elastin synthesis (differentiation) in undifferentiated cells.     

The application of collagenase, purified by affintiy chromatography, to the isolation of insoluble elastin from bovine aorta.

Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.16), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine aorta. The extremely low level of N-terminal residues (1.6 mol per 10(6) g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. The amino acid profile of the aortic elastin was found to be almost identical to that of insoluble elastin prepared from bovine ligamentum nuchae by the same method.     

Kinetics of receptor-mediated binding of tropoelastin to ligament fibroblasts

Soluble 125I-labeled tropoelastin bound to confluent cultures of bovine ligamentum nuchae fibroblasts and to fibroblast plasma membrane preparations in a time-dependent, saturable, and reversible manner. Scatchard analysis indicates that there are approximately 2 X 10(6) binding sites/cell with a binding efficiency (Kd) of 8 X 10(-9) M. Binding of tropoelastin to cells and membranes reached equilibrium by 90 min and was reversible with 50% of specifically bound material released by 40 min. Specific binding of tropoelastin to cells pre-treated with dilute trypsin solutions was reduced significantly when compared with controls. Four polypeptides of estimated molecular masses of 67, 61, 55, and 43 kDa were obtained from detergent extracts of plasma membranes by elution affinity chromatography on elastin-Affi-Gel. Our findings establish that elastin-specific binding proteins displaying characteristics of a true receptor are present on the surface of elastin-producing cells.     

Pattern of accumulation of elastin and the level of mRNA for elastin in aortic tissue of growing chickens

Synthesis and accumulation of elastin in many elastic tissues begins in the last third of fetal development, reaches a maximum shortly after birth, and then declines rapidly. For the aorta of the chick and the pig and the ligamentum nuchae and lung of the sheep, it has been shown that increased levels of elastin production with fetal development are correlated with increased levels of elastin mRNA in the tissue, measured both by cell-free translation and by hybridization to cDNA probes. In this study we examine the relationship between insoluble elastin accumulation and message levels for tropoelastin in aortic tissue of chickens during posthatching development and growth. Whether evaluated by cell-free translation or by dot blot hybridization, steady state levels of tropoelastin message increase to a maximum at 2 weeks after hatching, and then fall rapidly with further development and growth. This pattern correlates well with production of insoluble elastin by the aorta, determined either by direct measurements of synthesis or by rate of accumulation of insoluble elastin. The data indicate that the major site of regulation of elastin production is pretranslational throughout the entire period of development and growth of the chicken aorta.     

Changes in elastin composition in aorta of spontaneously hypertensive rats (SHR)

Tropoelastin and elastin preparations obtained from aortae of spontaneously hypertensive rats (SHR) show an increased proportion of polar amino acids (aspartic acid, glutamic acid, arginine and tyrosine). The content of these amino acids is 1.43-3.04 times higher in SHR rats than in similar elastin or tropoelastin preparations obtained from normotensive animals. On the other hand elastin and tropoelastin preparations obtained from SHR rats show a lower frequency of the Val-Pro sequence; this was found to be 35.93 per 1000 amino acid residues in SHR rats as compared to 51.04 per 1000 amino acids in the preparations obtained from control animals. Since similar differences were found not only in elastin preparations but also in tropoelastin, contamination of these preparations with an acidic protein seems unlikely. In general the results obtained are similar to those seen in animals kept on a long term high fat diet. It appears feasible to suggest that these differences are caused by a changed proportion of two different elastin type.     

Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Primary structures of bovine elastin a, b, and c deduced from the sequences of cDNA clones

Nucleotide sequence analysis of cDNA clones for bovine elastin revealed the occurrence of three mRNAs for elastin in fetal calf nuchal ligament, encoding three forms of elastins (a, b, and c, of 747, 733, and 713 amino acid residues, respectively). These forms arise as the result of the presence, at a single position, of 102 additional nucleotides in the mRNA for elastin a and of 60 of these nucleotides in the mRNA for elastin b as compared to the mRNA for elastin c. As expected, most lysines occur in pairs, separated by two or three small amino acid residues. However, at two places, lysines occur in groups of three. The occurrence of a group of three lysines followed by a hydrophobic residue (lysine 400, 404, and 407) offers an explanation for the formation of lysinonorleucine. The alignment of amino acid sequences of porcine tropoelastin tryptic peptides with the sequence for bovine elastin a results in the ordering of these tryptic peptides. The analysis of the complete primary structures of elastin a, b, and c provides further insight into the structure-function relations of elastin.     

High-performance liquid chromatographic quantification of allysine as bis-p-cresol derivative in elastin

Summary. The first step in normal cross-linking in elastin is the formation of α-aminoadipic-δ-semialdehyde, allysine, through oxidative deamination of specific peptidyl lysine by the enzyme lysyl oxidase (EC 1.4.3.13). For the analysis of allysine, allysine was derivatized with p-cresol. The derivatization was carried out by acid hydrolysis (6N HCl containing 5% (w/v) p-cresol at 110°C for 48 h) accompanied with the hydrolysis of elastin. A bis-p-cresol derivative of allysine was isolated from bovine ligamentum nuchae elastin hydrolysates, and was characterized by UV, FAB-MS and NMR. This derivative was identified as 2-amino-6,6-bis(2-hydroxy-5-methylphenyl)hexanoic acid. A rapid, sensitive reverse-phase high-performance liquid chromatographic method with UV detection was developed for the quantitative determination of allysine as its bis-p-cresol derivative. The lower limit of detection of the bis-p-cresol derivative was 58 pmol in the standard sample with a 20-μl injection at a signal-to-noise ratio of 3. This method was applied to the determination of allysine in bovine ligamentum nuchae, aorta, lung, and rat aorta elastin. The allysine content in rat aorta elastin dramatically increased from 1 week to 2 weeks of age. Received June 30, 2000 Accepted September 22, 2000     

Characterization of a structural glycoprotein from bovine ligamentum nuchae exhibiting dual amine oxidase activity

A structural glycoprotein has been extracted from bovine ligamentum nuchae by using 5 M guanidine hydrochloride containing a disulfide bond reducing agent and purified by preparative gel electrophoresis. The isolated material appeared to be monodisperse, with a molecular weight of approximately 34000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical ultracentrifugation. It contains 10% carbohydrate comprising mannose, N-acetylglucosamine, galactose, and sialic acid in a 6:5:3:3 molar ratio. The glycoprotein has been assayed for peptidyl-lysine oxidase activity by using [3H]lysine-aortic elastin, prepared from 15- to 17-day-old chick embryos, as a substrate. In the absence of free lysine, the specific activity of the preparation over a 2-h incubation was approximately 60 X 10(4) dpm/mg of purified protein. Addition of 10 mM lysine resulted in an approximately 50% decrease in the specific activity. Free lysine was shown to act as a substrate for the glycoprotein preparation as indicated by control experiments using [3H]lysine in place of the aortic substrate. These results demonstrate that the glycoprotein exhibits a dual amine oxidase activity. In the presence of 0.27 mM beta-aminopropionitrile fumarate, a concentration which completely inhibits peptidyl-lysine oxidase activity in other lysyl oxidases, the glycoprotein preparation was inhibited by approximately 14%. In the absence of 5 M guanidine hydrochloride and reducing agent, the glycoprotein undergoes aggregation which in the presences of copper ions results in the formation of cylindrical tactoids, the diameter of which (11 nm) corresponds closely to that of the fibrils which in the majority of connective tissue matrices constitute the microfibrillar component mainly associated with elastic fibers.