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PubMed2005

Crystal structure of cyclic (APGVGV)2, an analog of elastin, and a suggested mechanism for elongation/contraction of the molecule.

Tropoelastin is a complex polymeric protein composed primarily of repeating segments of Val-Pro-Gly-Gly, Val-Pro-Gly-Val-Gly, and Ala-Pro-Gly-Val-Gly-Val that occurs in connective tissue and arteries. It has rubber-like extensible properties. A synthetic cyclic dodecapeptide, with a double repeat of the hexapeptide sequence, has been shown to undergo a reversible inverse temperature transition; that is, crystals grow at 60 degrees C and dissolve in the mother liquor upon cooling. An x-ray crystal structure analysis established that the cyclic backbone formed an elongated loop with a Pro-Gly, type II beta turn at both ends. Six internal cross strand NH...OC hydrogen bonds form between six NH donors and four O=C acceptors where two of the carbonyl O atoms are bifurcated acceptors. As a result, the molecule is pulled up into a corrugated profile. The corrugated loops form extended beta-sheets by additional intermolecular hydrogen bonds. An analysis of the dome region in a corrugated sheet suggests a reversible mechanism for extending and contracting the length of the whole molecule, akin to the motion of opening and closing an umbrella, caused by the motion of a water molecule with its associated hydrogen bonds acting as spokes. Crystal parameters: C44H72N12O12.3H2O, sp. gr. P2(1)2(1)2(1), a = 9.212 angstroms, b = 19.055 angstroms, c = 32.247 angstroms, d = 1.157 g/cm3.

Crystallography, X-Ray↗
PubMed1982

Irradiation crosslinking of the polytetrapeptide of elastin and compounding to dacron to produce a potential prosthetic material with elasticity and strength.

The poly(tetra peptide), H-(L . Val1-L . Pro2-Gly3-Gly4)n-L . Val-OMe, which is a recurring sequence in tropoelastin the precursor protein of the elastic fiber, has been irradiation crosslinked to produce an elastomeric material with limited strength. When a material such as a Dacron fabric is impregnated by the coacervate phase of the poly(tetrapeptide) prior to irradiation crosslinking at 50 Mrad, the crosslinked product exhibits stress-strain curves with good elastomeric properties and high strength. In addition to the stress-strain curves, the material is characterized by scanning electron microscopy.

Biocompatible Materials↗
PubMed2002

Basic fibroblast growth factor decreases elastin gene transcription in aortic smooth muscle cells.

The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system by imparting elasticity to blood vessel wall. In this study, we examined the effect of basic fibroblast growth factor (bFGF) on the expression of elastin in aortic smooth muscle cells (SMC) to gain insight into events associated with cardiovascular diseases. The results show that bFGF treatment of SMC causes a significant decrease in elastin mRNA and secreted tropoelastin levels. Nuclear run-on analyses demonstrate that the downregulation is due to a decrease in the level of elastin gene transcription. Transient transfections of SMC with wild-type and mutated elastin gene promoter/chloramphenicol acetyl transferase (CAT) constructs show that a previously identified activator protein-1-cAMP response element (AP1/CRE) (-564 to -558-bp) within the elastin promoter mediates the bFGF-dependent downregulation of elastin gene transcription in SMC. Addition of bFGF to SMC activates the extracellular signal-regulated kinases 1/2 (ERK1/2) resulting in their translocation into the nucleus and subsequent induction of Fra-1. The addition of PD-98059, an inhibitor of ERK1/2 kinase, abrogates the bFGF-dependent decrease of elastin mRNA in SMC. The described inhibitory effect of bFGF on elastin gene expression in SMC may significantly contribute to the inefficient repair of elastin in early stages of vascular wall injury.

Animals↗
PubMed1995

Expression of lysyl oxidase from cDNA constructs in mammalian cells: the propeptide region is not essential to the folding and secretion of the functional enzyme.

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.

Amino Acid Sequence↗
PubMed2002

Heparan sulfate depletion within pulmonary fibroblasts: implications for elastogenesis and repair.

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan-depleted fibroblasts were treated with porcine pancreatic elastase at 2-4-day intervals and the elastase-mediated release of fibroblast growth factor 2 (FGF-2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF-2 and glycosaminoglycans (GAG) from PG-depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF-2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.

Animals↗
PubMed1989

Extracellular matrix: differential influence on growth and biosynthesis patterns of vascular smooth muscle cells from SHR and WKY rats.

Smooth muscle cells from spontaneously hypertensive rats (SHR) elaborated extracellular matrix (ECM) material in culture that was more stimulatory to growth of cells from normotensive (WKY) animals than their own matrix. Both cell types elaborated ECMs consisting of glycoconjugate material (proteoglycans, glycopeptides) elastin, and collagens, but there were differences in the relative proportions of the compounds synthesized. Cells from SHR produced an ECM richer in elastin than that synthesized by WKY derived cells (approximately 19% vs. 11%, respectively). However, the latter elaborated ECMs containing more (approximately 45% for WKY vs. 29% for SHR) glycoconjugate material than the former. The lysyloxidase-mediated cross-linking of elastin was more rapid in cultured cells from SHR animals than from their normotensive counterparts and may be as a consequence of increased substate (tropoelastin) availability in ECMs from SHR animals. The relative proportions and sulphate levels of the glycosaminoglycans associated with matrix material elaborated by the two cell types were similar. Radiolabelled glycoconjugate material was degraded by cells (SHR/WKY) when they were plated upon pre-formed ECMs, and their patterns of synthesis of new matrix was markedly altered under such conditions. New matrix material elaborated by cells plated upon ECM-coated dishes consisted predominantly of glycopeptide and proteoglycan matrix components. Epidermal growth factor promoted the incorporation of [3H]-thymidine into DNA by quiescent cells, and this was also markedly stimulated when cells were plated onto ECM-coated plasticware rather than onto plastic substratum.

Animals↗
PubMed2002

Regulation of growth-related genes by interleukin-6 in murine myeloma cells.

Interleukin-6 (IL-6), a pleiotropic cytokine with effects on several hematopoietic and other normal cells, is also important for the growth and survival of tumor cells such as murine plasmacytomas and human myelomas. Exploiting the 11A3 hybridoma cell line for its IL-6 requirement to proliferate in vitro, we used subtractive suppression hybridization (SSH) to identify genes whose expression is stimulated and/or repressed in response to IL-6. Northern blot analysis of 100 arbitrarily picked subtracted cDNA clones revealed that expression of 11 mRNAs were IL-6-modulated. Among these, eight were genes known to encode a variety of proteins such as enzymes (PCK, MTDNI), structural proteins (Tropoelastin), transcriptional regulators (BRG1) and proteins involved in cell division control (Cyclin A, OAZi) or cell signaling (PIX, TOPK/PBK). The recently identified MAPKK-like protein kinase TOPK/PBK gene represents a likely candidate IL-6 target gene as suggested by its significant up-regulated expression in hybridoma cells induced to grow by a brief IL-6 pulse. The diversity of growth-related genes identified in this study further emphasizes the central role of IL-6 in the regulation of myeloma cell expansion in addition to its previously demonstrated role in the inhibition of apoptosis.

Animals↗
PubMed1999

Cloning of rat fibrillin-2 cDNA and its role in branching morphogenesis of embryonic lung.

Fibrillin-2 is an extracellular matrix protein. It is associated with elastic fibers in several tissues and is believed to serve as a ligand for alphavbeta3 integrin, the latter being a known morphogen. In this study, the role of fibrillin-2 in lung development was investigated. Also, rat fibrillin-2 cDNA was isolated and sequenced and its spatiotemporal expression determined. It had approximately 88% homology with human fibrillin-2 and had Ca(2+) binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and two RGD binding sites. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-2 expression was developmentally regulated, and it paralleled that of tropoelastin. At day 13 of gestation, fibrillin-2 was expressed in the mesenchyme and at the epithelial:mesenchymal interface. From day 13 to 19 of gestation, its expression intensified and was confined around the tracheobronchial airways, while it lessened during the postnatal period. Immunoprecipitation revealed an approximately 350-kDa band by SDS-PAGE. Treatment with fibrillin-2 antisense oligodeoxynucleotide induced dysmorphogenesis of the lung explants. They were smaller and had rudimentary lung bud branches, collapsed conducting airways, and loose expanded mesenchyme. Concomitantly, fibrillin-2 mRNA, antibody reactivity in the explants, and fibrillin-2-specific radioincorporation were reduced. Anti-alphav and -laminin antibody reactivity and their respective incorporated specific radioactivities were unaltered. These data indicate that fibrillin-2 modulates organogenesis of the lung in the context of epithelial:mesenchymal interactions. Conceivably, the collapse of the conducting airways may also be related to the perturbed biology of the fibrillin-2 interacting protein, i.e., elastin, the latter being critical for the normal biophysiology of the lungs.

Actins↗
PubMed2002

Genomic organization of mouse and human 65 kDa FK506-binding protein genes and evolution of the FKBP multigene family.

FK506-binding proteins (FKBPs) are peptidyl-prolyl cis/trans isomerases PPIases) that bind the immunosuppressive drug FK506. Of the many eukaryotic FKBPs that have been identified, FKBP65 is an endoplasmic reticulum-localized protein that associates with tropoelastin in the secretory pathway. Unlike any other FKBP characterized so far, FKBP65 is developmentally regulated and may be intimately involved in organogenesis. Here, we report the isolation, sequencing, and genomic organization of the mouse FKBP65 gene (Fkbp10) and provide a comparison with the human ortholog. Mouse Fkbp10 contains 10 exons and 9 introns encompassing 8.5 kb. The exon-intron organization of Fkbp10 displays a pattern of repetition that reflects the coding sequence of the four PPIase, or FK506-binding, domains present in the mature protein. The exon organization of the PPIase domains differs from that of the other FKBP family members. The evolution of the FKBP65 gene and other members of the FKBP multigene family were therefore investigated from a taxonomically diverse array of prokaryotic and eukaryotic taxa. These analyses suggest that the FKBP multigene family emerged early in the evolutionary history of eukaryotes, and during that time some members, including the FKBP65 gene, have experienced gene elongation by means of PPIase domain duplication.

Amino Acid Sequence↗
PubMed1987

Detection of elastin by immunoelectronmicroscopy. A comparison of different procedures.

Elastin components have been identified in chick aorta by different immunoelectronmicroscopic procedures (peroxidase-antiperoxidase, immunoferritin and immunogold) using affinity purified antibodies to chick tropoelastin. The PAP method used in a preembedding procedure stained the outer portion of amorphous elastin and the microfibrils very intensively. The surface of the cells was also slightly stained. On the contrary immunogold labelling on Epon or Lowicryl embedded sections produced a strong decoration only of amorphous elastin, while microfibrils remained almost completely unlabelled. The result is not due to loss of antigenicity of microfibrils during embedding, since similar data were obtained with immunoferritin in a preembedding procedure. Experiments performed under different stringency conditions showed that the products of the peroxidase reaction diffuse and redistribute in the tissue, indicating that the positive staining of microfibrils and cell surface is an artifact. The value of different immunological reagents and procedures in studying the fine mapping of elastin components is discussed.

Animals↗
PubMed1988

A synthetic polypentapeptide of elastin for initiating calcification.

A polypentapeptide (PPP) of tropoelastin having a repeating amino acid sequence of (Val-Pro-Gly-Val-Gly)n was evaluated for its potential to initiate in vivo calcification and to enhance bone formation in nonhealing calvarial wounds (8.0 mm) in 396 adult Walter Reed rats. There were four configurations of the PPP (molecular weight range of 50-100K dalton) consisting of 1-dry PPP; 2-coacervate PPP; 3-gamma irradiated, cross-linked PPP; 4-calcified, gamma irradiated, cross-linked PPP. These four iterations plus a control group of animals constituted the five treatment classes that were evaluated at days 1, 3, 7, 21, 42, and 147. Seventy two rats were used for each treatment and 36 rats for the control. Following euthanatization, specimens were placed into 70% ethanol, embedded in polymethyl methacrylate, sectioned at 3.5 micrometers, and alternating sections were stained with Masson-Goldner trichrome and von Kossa stains. Histomorphometric analysis was accomplished using a Zeiss Universal microscope (250 X) and Videoplan Image Analysis System to evaluate five random histologic fields from margin to margin of the craniotomy. Trabecular bony volume and area of calcification islands were quantitated. A Student's t test for unpaired data to determine treatment differences (within the same temporal groups) revealed that there was no significant difference between treatments and control for trabecular bony volume; however, there was a significant difference between experimentals and control for calcification islands (P less than 0.05) such that calcifications islands for the experimentals were greater than the control. There was not a significant difference between experimental treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Interactions of human tenascin-X domains with dermal extracellular matrix molecules.

Tenascin-X (TNX) is a large 450 kDa extracellular matrix protein expressed in a variety of tissues including skin, joints and blood vessels. Deficiency of TNX causes a recessive form of Ehlers-Danlos syndrome characterized by joint hypermobility, skin fragility and hyperextensible skin. Skin of TNX deficient patients shows abnormal elastic fibers and reduced collagen deposition. The mechanism by which TNX deficiency leads to connective tissue alterations is unknown. Here we report that C-terminal domains of human TNX bind to major dermal fibrillar collagens and tropoelastin. We have mapped these interactions to the fibronectin type III repeat 29 (FNIII29) and the C-terminal fibrinogen domain (FbgX) of TNX. In addition we found that FNIII29 of TNX accelerates collagen fibrillogenesis in vitro. We hypothesize that TNX contributes to matrix stability and is possibly involved in collagen fibril formation.

Animals↗
PubMed2001

Supravalvular aortic stenosis: genetic and molecular dissection of a complex mutation in the elastin gene.

We have identified two elastin gene (ELN) mutations located in cis in two related families with supravalvular aortic stenosis (SVAS). These mutations included an in-frame duplication in exon 18 (1034-1057dup) and a single base substitution in exon 26 (1829G-->A) predicted to result in the amino acid substitution R610Q. Haplotype analysis in one of the families identified an individual with a recombination between exon 18 and 26 of the elastin gene. This individual was unaffected and carried the exon 18 insertion mutation but not 1829G-->A. Skin fibroblasts were established from this recombinant normal individual and from an affected individual carrying both of the mutations. Reverse transcription/polymerase chain reaction (RT-PCR) analysis indicated that the expression of the mutant allele was reduced to 12%-27% of the normal allele in the affected but not in the unaffected individual. RNA-blot hybridization and immunoprecipitation experiments revealed reduced steady-state elastin mRNA levels and tropoelastin synthesis in the affected individual. RT-PCR analysis of the mRNA rescued by cycloheximide treatment indicated that mutation 1829G-->A created a cryptic donor splice site within exon 26, resulting in the deletion of four nucleotides at the 3'-end of exon 26 and a frameshift in the mRNA. This frameshift mutation generated a premature termination codon in the domain encoded by exon 28, clearly resulting in nonsense-mediated decay (NMD) of this frameshift RNA product. Despite considerable variability in the molecular nature of mutations responsible for SVAS, the unifying mechanism appears to be the generation of null alleles by NMD leading to elastin haploinsufficiency.

Alleles↗
PubMed2000

Isolated supravalvular aortic stenosis: functional haploinsufficiency of the elastin gene as a result of nonsense-mediated decay.

We have used single-strand conformation and heteroduplex analyses of genomic amplimers to identify point mutations within the elastin gene (ELN) in patients with non-syndromic supravalvular aortic stenosis (SVAS) from a total of eight unrelated families. Six novel point mutations were identified. We have collected detailed clinical information on mutation carriers and demonstrated significant non-penetrance in some of the families. Together with the new mutations described here, 14 point mutations have been reported in SVAS patients, and 10 of these result in premature stop codons (PTCs). We have analyzed the expression of ELN alleles in skin fibroblasts from one SVAS patient and shown that PTC mutations indeed result in selective elimination of mutant transcripts. Inhibition of the nonsense-mediated decay mechanism by cycloheximide resulted in the stabilization of mutant elastin mRNA. Allelic inactivation by the ELN mutation in this patient led to an overall decrease of the steady state levels of elastin mRNA. Finally, we have demonstrated reduced synthesis and secretion of tropoelastin by skin fibroblasts from the same SVAS patient. We conclude that PTC mutations in ELN result in nonsense-mediated decay of mutant mRNA in this patient. Given the predominance of PTC mutations in SVAS, we suggest that functional haploinsufficiency may be a pathomechanism underlying most cases of non-syndromic SVAS.

Adolescent↗
PubMed1990

The elastic system fibres in healthy human gingiva.

In human gingiva, the elastic system fibres, namely oxytalan, elaunin and elastic fibres, are distributed in the upper, medium and deep layers of gingival connective tissue, respectively. They are formed by a microfibrillar and an amorphous component characterized as elastin. In the gingival connective tissue fibroblastic cells are likely to be the main source of production of elastin in the extracellular matrix. Elastin is secreted as a soluble precursor (tropoelastin), which spontaneously forms insoluble aggregates of elastin. Elastin is then laid down at the surface of the microfibrillar component, which could serve as a site for deposition of elastin during elastogenesis, and subsequently be incorporated in an amorphous area to form elaunin and elastic fibres.

Actin Cytoskeleton↗
PubMed1976

Arterial elastin synthesis in the young chick.

In the growing chick a marked stimulation in soluble and mature arterial elastin synthesis occurs 2 and 5 weeks after hatching. Measurement of [3H]valine and [3H]proline incorporation into arterial soluble protein during this period indicated that most of the label is found in a 70 000 dalton protein subunit. The labeled soluble subunit had the characteristics of native soluble elastin or tropoelastin. During the period in which the greatest percentage increase in mature elastin occurs, the highest specific activities of soluble [3H]valine-labeled protein were observed. These changes were striking and suggest a developmental period for the growing chick in which factors related to elastin metabolism may be more easily studied.

Aging↗
PubMed1981

The microfibrillar components of porcine lung elastic fiber.

Lung elastic fiber was isolated from anatomically defined porcine parenchymal tissue. Constituent microfibrillar acidic structural glycoproteins (external and meso) were obtained by mild dissociative quantitative procedures, leaving the elastin-rich fraction as the insoluble residue. The presumptive cross-link lysylnorleucine was identified in both of the highly aggregated acidic structural glycoprotein fractions. The external-acidic structural glycoproteins require dithiothreitol but not sodium dodecyl sulfate for solubility. Both sodium dodecyl sulfate and dithiothreitol at 55 degrees C are required to release meso-acidic structural glycoproteins from the elastic fiber. On the basis of physical characteristics, I propose a new fraction from elastic fiber, the meso-acidic structural glycoprotein. The meso-acidic structural glycoproteins may have been in longest and closest contact with the elastin core of the elastic fiber. Alkali-soluble peptides from the elastin-rich fraction contained desmosine and high concentrations of hydroxyproline, polar amino acids and lysylnorleucine. The cross-link, lysylnorleucine, may be evidence of covalent bonding of glycoprotein to elastin, explaining my inability to obtain the biopolymer elastin with the composition of tropoelastin corrected for the desmosine-lysine content.

Amino Acids↗
PubMed1984

Characterization of a sheep elastin cDNA clone containing translated sequences.

mRNA, isolated from the ligamentum nuchae of fetal sheep by guanidine HCl extraction and oligo(dT) cellulose chromatography, was used to synthesize blunt-ended cDNA molecules by the successive application of AMV reverse transcriptase, DNA polymerase and S1 nuclease. The cDNA was centrifuged on a 15-30% sucrose gradient and molecules greater than 700 bp were tailed with dCTP and cloned into the PstI site of pBR322 which had been tailed with dGTP. Ampicillin-sensitive and tetracycline-resistant colonies were screened by in situ hybridization with elastin-enriched mRNA that had been terminally labeled with 32p. Recombinant plasmids prepared from strongly hybridizing colonies were characterized by restriction mapping and the plasmid with the largest insert (1300 bp) thought to contain elastin sequences was characterized in more detail. The nick-translated cDNA hybridized to a single 3.5 kb mRNA species upon blot hybridization, a size identical to that previously identified for chick elastin mRNA (Burnett et al. (1982) J. Biol. Chem. 259, 1569-1572). Nucleotide sequencing of the 5' end of the cDNA demonstrated a sequence which was extremely GC rich and which corresponded to an amino acid sequence partially homologous to that previously identified in porcine tropoelastin (Foster et al. (1973) J. Biol. Chem. 248, 2876-2879). This is the first report of the identification of a plasmid containing sequences complementary to a translated region of elastin mRNA.

Amino Acid Sequence↗