Glycosaminoglycans mediate the coacervation of human tropoelastin through dominant charge interactions involving lysine side chains.

Following cellular secretion into the extracellular matrix, tropoelastin is transported, deposited, and cross-linked to make elastin. Assembly by coacervation was examined for an isoform of tropoelastin that lacks the hydrophilic domain encoded by exon 26A. It is equivalent to a naturally secreted form of tropoelastin and shows similar coacervation performance to its partner containing 26A, thereby generalizing the concept that splice form variants are able to coacervate under comparable conditions. This is optimal under physiological conditions of temperature, salt concentration, and pH. The proteins were examined for their ability to interact with extracellular matrix glycosaminoglycans. These negatively charged molecules interacted with positively charged lysine residues and promoted coacervation of tropoelastin in a temperature- and concentration-dependent manner. A testable model for elastin-glycosaminoglycan interactions is proposed, where tropoelastin deposition during elastogenesis is encouraged by local exposure to matrix glycosaminoglycans. Unmodified proteins are retained at approximately 3 microM dissociation constant. Following lysyl oxidase modification of tropoelastin lysine residues, they are released from glycosaminoglycan interactions, thereby permitting those residues to contribute to elastin cross-links.     

Specificity in the coacervation of tropoelastin: solvent exposed lysines

Tropoelastin protein monomers associate by coacervation and are cross-linked in vivo to form elastin macro-assemblies. We provide evidence for specific protein domain contact points between tropoelastin monomers during association by coacervation. The homobifunctional cross-linker bis(sulfosuccinimidyl) suberate served as a rapid reporter of adjacent lysines and preferentially exposed domains. Intact cross-linked peptide pairs were identified after protease digestion and high-resolution electrospray mass spectrometry followed by MS/MS sequencing. Mapping of the assigned sequences indicated that the region in the monomer spanning domains 19-25 was readily accessible to solvent and enriched in cross-linking. Domains 12 and 36 were also prevalent, where these two regions were not previously thought to play a major role in the formation of mature elastin. A specificity for particular lysines allowed for the construction of a model for the first close contacts between domains and the first detailed study of the cross-linking of tropoelastin.     

Transient tropoelastin nanoparticles are early-stage intermediates in the coacervation of human tropoelastin whose aggregation is facilitated by heparan sulfate and heparin decasaccharides

Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200–300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 µm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules.     

Domain 26 of tropoelastin plays a dominant role in association by coacervation

The temperature-dependent association of tropoelastin molecules through coacervation is an essential step in their assembly leading to elastogenesis. The relative contributions of C-terminal hydrophobic domains in coacervation were assessed. Truncated tropoelastins were constructed with N termini positioned variably downstream of domain 25. The purified proteins were assessed for their ability to coacervate. Disruption to domain 26 had a substantial effect and abolished coacervation. Circular dichroism spectroscopy of an isolated peptide comprising domain 26 showed that it undergoes a structural transition to a state of increased order with increasing temperature. Protease mapping demonstrated that domain 26 is flanked by surface sites and is likely to be in an exposed position on the surface of the tropoelastin molecule. These results suggest that the hydrophobic domain 26 is positioned to play a dominant role in the intermolecular interactions that occur during coacervation.     

Connection between elastin haploinsufficiency and increased cell proliferation in patients with supravalvular aortic stenosis and Williams-Beuren syndrome

To elucidate the pathomechanism leading to obstructive vascular disease in patients with elastin deficiency, we compared both elastogenesis and proliferation rate of cultured aortic smooth-muscle cells (SMCs) and skin fibroblasts from five healthy control subjects, four patients with isolated supravalvular aortic stenosis (SVAS), and five patients with Williams-Beuren syndrome (WBS). Mutations were determined in each patient with SVAS and in each patient with WBS. Three mutations found in patients with SVAS were shown to result in null alleles. RNA blot hybridization, immunostaining, and metabolic labeling experiments demonstrated that SVAS cells and WBS cells have reduced elastin mRNA levels and that they consequently deposit low amounts of insoluble elastin. Although SVAS cells laid down approximately 50% of the elastin made by normal cells, WBS cells deposited only 15% of the elastin made by normal cells. The observed difference in elastin-gene expression was not caused by a difference in the stability of elastin mRNA in SVAS cells compared with WBS cells, but it did indicate that gene-interaction effects may contribute to the complex phenotype observed in patients with WBS. Abnormally low levels of elastin deposition in SVAS cells and in WBS cells were found to coincide with an increase in proliferation rate, which could be reversed by addition of exogenous insoluble elastin. We conclude that insoluble elastin is an important regulator of cellular proliferation. Thus, the reduced net deposition of insoluble elastin in arterial walls of patients with either SVAS or WBS leads to the increased proliferation of arterial SMCs. This results in the formation of multilayer thickening of the tunica media of large arteries and, consequently, in the development of hyperplastic intimal lesions leading to segmental arterial occlusion.     

Diagnosis of supravalvular stenosis of the aorta

The authors analyzed the potentialities of combined radiodiagnosis of supravalvular stenosis (SS) and concomitant diseases of the heart and major vessels (Williams-Beuren syndrome) in 7 patients aged 7 mos. to 24 yrs. Polypositional chest and heart x-ray procedure, catheterization of the cardiac cavities, pulmonary artery and aorta, left ventriculography (6), right ventriculography (4) and angiopulmonography were used. The diagnostic potentialities of each method were defined. It was proved that SS practically in all patients was accompanied by major vascular changes in the pulmonary, vertebral, coronary, carotid, subclavicular, renal and other arteries, heart failure (atrial septal defect, and mitral incompetence).     

A calcified sinutubular junction: the discovery of a supravalvular aortic stenosis in an elderly woman

We report a case of a 64 year old woman with a calcified ring at the level of the sinotubular junction. Echocardiography and Computed Tomography showed a supravalvular aortic stenosis, without known associated lesions, except for the existence of an aberrant right subclavian artery. These combination of abnormalities makes it an unique case. Differential diagnosis of sinutubular calcification is added. From the literature a short review of supravalvular aortic stenosis is presented with indications for surgical intervention. Lifelong and regular follow up is necessary.     

Study on coacervation of the repeat pentapeptide model of tropoelastin: effect of cations

The effects of various cations on coacervation of the polypentapeptide (Val-Pro-Gly-Val-Gly)n, a sequential model of tropoelastin, were studied by following the turbidity at 300 nm and the fluorescence intensity due to the interaction between the polypentapeptide and a hydrophobic probe (4-benzoylamido-4'-aminostilbene-2,2'-disulfonic acid). In the absence of cations, as the polypentapeptide concentration was increased, the turbidity curves shifted to lower temperatures and became much steeper, while the fluorescence intensity increased markedly. This suggests that a conformational change is induced by the association of the polypentapeptide molecules and indicates that coacervation occurs predominantly by intermolecular hydrophobic association. In the presence of Mg2+, the temperature profile of the coacervation curve of the polypentapeptide obtained by turbidity measurement moved to higher temperature and the fluorescence intensity decreased significantly. This suggests that Mg2+ inhibits the hydrophobic interaction of the polypentapeptide molecules on coacervation but does not induce conformational change on association of the molecules. In the presence of Ca2+, Na+, and K+, the temperature profile of the coacervation curve of the polypentapeptide was not affected appreciably, but the fluorescence intensity was decreased by these cations in the order of Na+, K+, and Ca2+. Circular dichroism spectra of the polypentapeptide in 80% trifluoroethanol in the presence of cations showed a less ordered state of the polypentapeptide. This less ordered state implies inhibition by the cations of the hydrophobic interaction between the molecules.     

Differential expression of two tropoelastin genes in zebrafish.

Elastin is the extracellular matrix protein responsible for properties of extensibility and elastic recoil in large blood vessels, lung and skin of most vertebrates. Elastin is synthesized as a monomer, tropoelastin, but is rapidly transformed into its final polymeric form in the extracellular matrix. Until recently information on sequence and developmental expression of tropoelastins was limited to mammalian and avian species. We have recently identified and characterized two expressed tropoelastin genes in zebrafish. This was the first example of a species with multiple tropoelastin genes, raising the possibility of differential expression and function of these tropoelastins in elastic tissues of the zebrafish. Here we have investigated the temporal expression and tissue distribution of the two tropoelastin genes in developing and adult zebrafish. Expression was detected early in skeletal cartilage structures of the head, in the developing outflow tract of the heart, including the bulbus arteriosus and the ventral aorta, and in the wall of the swim bladder. While the temporal pattern of expression was similar for both genes, the upregulation of eln2 was much stronger than that of eln1. In general, both genes were expressed and their gene products deposited in most of the elastic tissues examined, with the notable exception of the bulbus arteriosus in which eln2 expression and its gene product was predominant. This finding may represent a sub-specialization of eln2 to provide the unique architecture of elastin and the specific mechanical properties required by this organ.     

Glycosaminoglycan-mediated coacervation of tropoelastin abolishes the critical concentration, accelerates coacervate formation, and facilitates spherule fusion: implications for tropoelastin microassembly

Elastogenesis and elastin repair depend on the secretion of tropoelastin from the cell, yet cellular production is low in the many biological systems that have been studied. To address the apparent paradox of a paucity of tropoelastin for cell surface microassembly, we examined the effects of the glycosaminoglycans heparin, heparan sulfate, and chondroitin sulfate B, on tropoelastin aggregate formation through coacervation. We found a significant effect, particularly of heparin, on the minimum or critical concentration of tropoelastin, which was required for microassembly, lowering critical concentration to a point that it was no longer detectable. The assemblies resulted in protein droplet formation that was visually indistinguishable from the spherules that typify coacervation. The spherules readily coalesced in the presence of heparin and higher concentrations of tropoelastin, resulting in an almost continuous layer of coacervated tropoelastin. Four stages of droplet behavior were observed: early droplet formation, approximately 6 mum droplet formation, and fusion of droplets followed by the formation of a coalesced layer. We conclude that glycosaminoglycans in the extracellular matrix have the capacity to promote coacervation at low concentrations of tropoelastin.     

Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers.

A number of reports point to the presence of proteoglycans and/or glycosaminoglycans within elastic fibers in normal and in pathological conditions. We present data that heparan sulphate (HS)-containing proteoglycans are associated with normal elastic fibers in human dermis and that isolated HS chains interact in vitro with recombinant tropoelastin and with peptides encoded by distinct exons of the human tropoelastin gene (EDPs). By immunocytochemistry, HS chains were identified as associated with the amorphous elastin component in the human dermis and remained associated with the residual elastin in the partially degenerated fibers of old subjects. HS appeared particularly concentrated in the mineralization front of elastic fibers in the dermis of patients affected by pseudoxanthoma elasticum (PXE). In in vitro experiments, HS induced substantial changes in the coacervation temperature and in the aggregation properties of recombinant tropoelastin and of synthetic peptides (EDPs) corresponding to sequences encoded by exons 18, 20, 24 and 30 of the human tropoelastin gene. In particular, HS modified the coacervation temperature and favoured the aggregation into ordered structures of tropoelastin molecules and of EDPs 18, 20 and 24, but not of EDP30. These data strongly indicate that HS-elastin interactions may play a role in tissue elastin fibrogenesis as well as modulating elastin stability with time and in diseases.     

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.     

Effect of FKBP65, a putative elastin chaperone, on the coacervation of tropoelastin in vitro

FKBP65 is a protein of the endoplasmic reticulum that is relatively abundant in elastin-producing cells and is associated with tropoelastin in the secretory pathway. To test an earlier suggestion by Davis and co-workers that FKBP65 could act as an intracellular chaperone for elastin, we obtained recombinant FKBP65 (rFKBP65) by expressing it in E.?coli and examined its effect on the coacervation characteristics of chicken aorta tropoelastin (TE) using an in vitro turbidimetric assay. Our results reveal that rFKBP65 markedly promotes the initiation of coacervation of TE without significantly affecting the temperature of onset of coacervation. This effect shows saturation at a 1:2 molar ratio of TE to rFKBP65. By contrast, FKBP12, a peptidyl prolyl isomerase, has a negligible effect on TE coacervation. Moreover, the effect of rFKBP65 on TE coacervation is unaffected by the addition of rapamycin, an inhibitor of peptidyl prolyl isomerase (PPIase) activity. These observations rule out the involvement of the PPIase activity of rFKBP65 in modulating the coacervation of TE. Additional experiments using a polypeptide model of TE showed that rFKBP65, while promoting coacervation, may retard the maturation of this model polypeptide into larger aggregates. Based on these results, we suggest that FKBP65 may act as an elastin chaperone in vivo by controlling both the coacervation and the maturation stages of its self-assembly into fibrils.     

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.     

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.