Synthesis of an elastin component of molecular weight about 70,000 by polysomes from chick embryo aortas

Polysomes were isolated from aortas of 17-day-old chick embryos, and the synthesis of the nascent polypeptide chains was completed in vitro. When a mixture of a labeled amino acids found in elastin was used, the major radioactive product obtained was of molecular weight about 70,000 and was similar to elastin by several criteria. The 70,000 molecular weight product was extractable in propanol-butanol, it was not labeled with [³⁵S]methionine, and it was precipitated by antibodies against elastin. Polypeptides larger than 70,000 molecular weight were also synthesized but these larger polypeptides incorporated relatively small amounts of [¹⁴C]valine, and they appeared to represent proα chains of procollagen. The results suggest that the major gene product for elastin has a molecular weight of about 70,000.     

Characterization of a signal peptide sequence in the cell-free translation product of sheep elastin mRNA

In vitro explant cultures of near-term sheep nuchal ligament secrete tropoelastin of approximate M_r 70,000–72,000 while the elastin cell-free product of sheep nuchal ligament RNA is 2000 to 3000 M_r larger. Automated Edman degradation of immunoprecipitates of radiolabeled cell-free elastin precursor demonstrated the presence of a 26-residue signal sequence which was absent from sheep tropoelastin secreted from explant cultures. In addition, a 20-residue overlap was established between the cell-free product and the secreted protein. This overlap region, representing the N-terminal sequence of ovine tropoelastin, demonstrated complete homology with the N-terminal sequence of porcine tropoelastin and near complete homology with chick tropoelastin. These findings suggest that cotranslational removal of this hydrophobic peptide extension is likely a correlate of vectorial transport of elastin into the secretory apparatus.     

Biosynthesis and secretion of tropoelastin by chick aorta cells

Cells were isolated from the aortae of 17-day old chick embryos by digestion of the vessels with a combination of trypsin and collagenase. When these cells were incubated in suspension culture in Krebs-Ringer media containing pancreatic trypsin inhibitor and radioactive amino acids, they synthesized and secreted labeled proteins into the media. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the secreted proteins labeled with [¹⁴C]proline revealed two major components. The larger component with an approximate molecular weight of 125,000 had a [¹⁴C]hydroxyproline content consistent with a form of procollagen. The molecular weight of 70,000 and [¹⁴C]hydroxyproline content of the second component was consistent with that previously reported for tropoelastin extracted from chick aortae. By following the kinetics and secretion of tropoelastin labeled with [³H]valine, we have estimated that 17 minutes are required to synthesize and secrete the molecule under these experimental conditions.     

Isolation of soluble elastin from lathyritic chicks. Comparison to tropoelastin from copper deficient pigs.

Tropoelastin was isolated from the aortas of chicks rendered lathyritic by treatment with beta-aminopropionitrile. The soluble elastin was judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and possessed an estimated molecular weight of 70000. Automated sequential analysis revealed that the N-terminal region of the chick tropoelastin is very homologous to tropoelastin isolated from copper-deficient piglets. N-terminal analysis of a trypsin digest of chick tropoelastin showed that tyrosine frequently is found adjacent to lysine residues. This positioning of tyrosine residues may be significant in terms of a possible regulatory role in elastin cross-link formation.     

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.     

Inhibition of tropoelastin secretion by incorporation of DL-3,4-dehydroproline

Cells isolated from embryonic chick aorta were incubated in suspension culture with labeled amino acids and proline analogs. Incorporation of 4-cis-hydroxy-l-proline inhibited the secretion of labeled procollagen but not tropoelastin, while incorporation of dl-3,4-dehydroproline inhibited the secretion of both proteins and caused them to accumulate intracellularly. Protein synthesis did not appear to be significantly diminished during the 2-h incubation period. Incorporation of dl-3,4-dehydroproline may alter the conformation of tropoelastin leading to abnormal intracellular processing and a decreased rate of secretion.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Radioimmunological identification of tropoelastin.

Antiserum was prepared in sheep against insoluble elastin isolated from embryonic-chick aortae. In an indirect immunoprecipitation test, the antiserum reacted quantitatively with small amounts of radioactively labelled purified tropoelastin prepared from embryonic-chick aortae. The antiserum did not cross-react with chick procollagen, and the antiserum uas used to identify radioactively labelled tropoelastin secreted by chick aorta cells in suspension culture.     

Recycling of the 67-kDa Elastin Binding Protein in Arterial Myocytes Is Imperative for Secretion of Tropoelastin

We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab′)₂ fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A₁, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A₁ inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.     

Epidermal protein metabolism directed toward keratinization by hydrocortisone in the chick embryonic skin growing in a chemically defined medium.

The metabolism of the epidermal structural and nonstructural proteins was studied in hydrocortisone-induced in vitro keratinization of 13-day chick embryonic skin growing in a chemically defined medium. The protein metabolism of the epidermis was examined by determining the amounts of radioactivity incorporated into the fractions of reduced, S-carboxymethylated epidermal proteins (SCMEp) which were separated by polyacrylamide gel electrophoresis. A group of high molecular weight, glycine-rich derivatives of the epidermal fibrous protein called SCMEpA were found to be actively synthesized in the hydrocortisone-treated epidermis alone, while a group of undefined protein derivatives called SCMEpX was shown to be synthesized exclusively in the nontreated epidermis. Chase-culture of the prelabeled explants revealed that hydrocortisone accelerated the degradation of general proteins including SCMEpX while SCMEpA remained metabolically stable throughout the culture. Actinomycin D did not significantly affect the hydrocortisone-induced synthesis of SCMEpA but greatly inhibited that of SCMEpX of the nontreated epidermis, suggesting the induction by the steroid of relatively stable mRNA for SCMEpA. From these findings, it is concluded that hydrocortisone directed the cultured epidermis toward keratinization through acceleration of the synthesis of epidermal structural proteins and degradation of other proteins.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.     

Fuzzy binding model of molecular interactions between tropoelastin and integrin alphaVbeta3

Tropoelastin is the highly flexible monomer subunit of elastin, required for the resilience of the extracellular matrix in elastic tissues. To elicit biological signaling, multiple sites on tropoelastin bind to cell surface integrins in a poorly understood multifactorial process. We constructed a full atomistic molecular model of the interactions between tropoelastin and integrin α_vβ₃ using ensemble-based computational methodologies. Conformational changes of integrin α_vβ₃ associated with outside-in signaling were more frequently facilitated in an ensemble in which tropoelastin bound the integrin’s α1 helix rather than the upstream canonical binding site. Our findings support a model of fuzzy binding, whereby many tropoelastin conformations and defined sites cooperatively interact with multiple α_vβ₃ regions. This model explains prior experimental binding to distinct tropoelastin regions, domains 17 and 36, and points to the cooperative participation of domain 20. Our study highlights the utility of ensemble-based approaches in helping to understand the interactive mechanisms of functionally significant flexible proteins.     

Analysis of dermal elastic fibers in the absence of fibulin-5 reveals potential roles for fibulin-5 in elastic fiber assembly

Fibulin-5 is a 66 kDa modular, extracellular matrix protein that localizes to elastic fibers. Although in vitro protein–protein binding studies have shown that fibulin-5 binds many proteins involved in elastic fiber formation, the specific role of fibulin-5 in elastogenesis remains unclear. To provide a more detailed analysis of elastic fiber assembly in the absence of fibulin-5, the dermis of wild-type and fibulin-5 gene knockout (Fbln5^?/?) mice was examined with electron microscopy (EM). Although light microscopy showed apparently normal elastic fibers near the hair follicles and the absence of elastic fibers in the intervening dermis of the Fbln5^?/? mouse, EM revealed the presence of aberrantly assembled elastic fibers in both locales. Instead of the elastin being incorporated into the microfibrillar scaffold, the elastin appeared as globules juxtaposed to the microfibrils. Desmosine analysis showed significantly lower levels of mature cross-linked elastin in the Fbln5^?/? dermis, however, gene expression levels for tropoelastin and fibrillin-1, the major elastic fiber components, were unaffected. Based on these results, the nature of tropoelastin cross-linking was investigated using domain specific antibodies to lysyl oxidase like-1 (LOXL-1). Immunolocalization with an antibody to the N-terminal pro-peptide, which is cleaved to generate the active enzyme, revealed abundant staining in the Fbln5^?/? dermis and no staining in the wild-type dermis. Overall, these results suggest two previously unrecognized functions for fibulin-5 in elastogenesis; first, to limit the extent of aggregation of tropoelastin monomers and/or coacervates and aid in the incorporation of elastin into the microfibril bundles, and second, to potentially assist in the activation of LOXL-1.     

Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells

HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular-weight heavy-metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metal-lothionein synthesis five- to ten-fold. Maximal induction is achieved with 10^–8M dexamethasone and 10^–7M hydrocortisone. Half-maximal induction is achieved at 5 ± 10^–9M dexamethasone and 5 ± 10^–8M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific ³H-dexamethasone receptors that translocate into the nucleus after one hour of incubation. ³H-dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with ³H-dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Elastin synthesis during perinatal lung development in the rat

The rate of soluble elastin synthesis was estimated in lung explants from rats of differing ages to better define periods in lung development important to the deposition of lung elastin. Lungs from rat pups at days 1, 3, 7, 9, 12, 15, and 21 post-parturition and from adult rats were incubated in a defined medium containing L-[3H]valine. Following incubation, labelled soluble elastin (tropoelastin) was separated from other soluble proteins by coacervation and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The tropoelastin synthetic rate was then estimated after correcting for differences in recovery of radioactivity as tropoelastin and lung tissue L-[3H]valine specific activity. Maximal rates of elastin synthesis were observed in lung explants from 7-12-day-old rats. The rate of elastin synthesis during this period was 5-8-times the rate observed in adult rat lung (expressed per g of fresh lung) and represented approx. 2% of the total protein synthesis. Moreover, the values derived from lung explant culture for elastin synthesis were consistent with values for lung elastin deposition in the perinatal rat (5-10 micrograms elastin/h per g lung).     

Binding of steroids to the cytoplasmic receptor of the embryonic chick retina: kinetics and structural considerations

The retina of the 12-day embryonic chick contains cortisol-binding proteins. The 3-ketone group on the steroid molecule was found to be essential for receptor binding. The 11 β-hydroxyl group, which is considered to be of primary importance in the induction of the retinal enzyme glutamyltransferase, does not affect receptor binding. The 17-group on the steroid can, in some cases, influence receptor binding as well as the enhancement of glutamyltransferase activities. These receptors exhibit a high degree of affinity for the steroid and bind rapidly to the steroid, saturating within 1 h at 0 ° to 4 °C at approximately 10^?8m with a K_d of ? 10^?9m.