Structural characterization of human elastin derived peptides containing the GXXP sequence

The degradation of elastin, the insoluble biopolymer of tropoelastin, can lead to the production of small peptides. These elastin-derived peptides (EDPs) are playing a key role in cellular behavior within the extracellular matrix, showing a great variety of biological effects such as chemotaxis, stimulation of cell proliferation, ion flux modifications, vasorelaxation, and inflammatory enzymes secretion. It has also been demonstrated recently that EDPs containing the GXXPG motif could induce pro-MMP1 and pro-MMP3 upregulation. Elastolysis could then cause collagen degradation and play an important role in the aging process. Many experimental studies have been devoted to EDPs, but their structure/activity relationships are not well elucidated yet. However, the assumption that their active conformation is a type VIII beta-turn on GXXP was highly suggested on the basis of predictive statistical calculations. Investigation of the EDPs three-dimensional (3D) structure would provide useful information for drug-design strategies to propose specific inhibitors. The work presented here reports theoretical results obtained from molecular dynamics simulations performed over 128 human EDPs containing the GXXP motif. We show that all the peptides, for which the central residues are not glycines, adopt a canonical (or very close to) type VIII beta-turn structure on the GXXP sequence. Amino acids surrounding this motif are also important for the structural behavior. Any residue located before the GXXP motif (XGXXP) increases the beta-turn stabilization, whereas the residue located after GXXP (GXXPX) has no significant structural effect. Moreover, we show their biological activity can be correlated with their ability to exhibit a type VIII beta-turn conformation.     

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.     

Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides

Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized.     

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24

Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S₄ to S₄′) are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP.     

Molecular and supramolecular structural studies on human tropoelastin sequences

One of the unusual properties of elastin is its ability to coacervate, which has been proposed to play an important role in the alignment of monomeric elastin for cross-linking into the polymeric elastin matrix. The temperature at which this transition takes place depends on several factors including protein concentration, ionic strength, and pH. Previously, polypeptide sequences encoded by different exons of the human tropoelastin gene have been analyzed for their ability to coacervate and to self-assemble. Few of them were indeed able to coacervate and only one, that encoded by exon 30 (EX30), gave amyloid fibers. In this article, we report on two chemically synthesized peptides-a decapeptide and an octadecapeptide-whose sequences are contained in the longer EX30 peptide and on a polypeptide (EX1-7) of 125 amino-acid residues corresponding to the sequence coded by the exons 1-7 and on a polypeptide (EX2-7) of 99 amino-acid residues encoded by exons 2-7 of human tropoelastin obtained by recombinant DNA techniques. Molecular and supramolecular structural characterization of these peptides showed that a minimum sequence of approximately 20 amino acids is needed to form amyloid fibers in the exon 30-derived peptides. The N-terminal region of mature tropoelastin (EX2-7) gives rise to a coacervate and forms elastinlike fibers, whereas the polypeptide sequence containing the signal peptide (EX1-7) forms mainly amyloid fibers. Circular dichroism spectra show that beta-structure is ubiquitous in all the sequences studied, suggesting that the presence of a beta-structure is a necessary, although not sufficient, requirement for the appearance of amyloid fibers.     

Examination of the secondary structure of the kringle 4 domain of human plasminogen

The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation.     

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.     

Mimicry of beta II'-turns of proteins in cyclic pentapeptides with one and without D-amino acids.

The solution structure of eight cyclic pentapeptides has been determined by two-dimensional 1H-NMR spectroscopy combined with spectra simulations and restrained molecular dynamic simulations. Six of the cyclic pentapeptides were derived from the C-terminal cholecystokinin fragment CCK-4 enlarged with Asp1 resulting in the sequence (Asp-Trp-Met-Asp-Phe), one L-amino acid after the other was substituted by its D-analog. In addition, two peptides, including an all-L-amino-acid-containing cyclic pentapeptide, cyclo(Asp-Phe-Lys-Ala-Thr) and cyclo(Asp-Phe-Lys-Ala-D-Thr) were investigated. All D-amino-acid-containing peptides show beta II'-turn conformations with the D-amino acid in the i + 1 position, excepting the D-aspartic-acid-containing peptides. These two peptides are characterized by the lack of beta-turns at pH values less than 4, suggesting that D-aspartic acid in the full-protonized state avoids the formation of beta-turns in these compounds. At pH values greater than 5, a conformational change into the beta II'-turn conformation was also observed for these peptides. Conformations without beta-turns are expected for cyclic all-L pentapeptides, but both cyclo(Asp-Phe-Lys-Ala-Thr) and the D-Thr analog cyclo(Asp-Phe-Lys-Ala-D-Thr) exhibit beta II'-turn conformations around Thr-Asp and D-Thr-Asp. Thus cyclic all-L pentapeptides and those with one D-amino acid are able to form similar structures preferably with a beta II'-turn. The beta-turn formation in cyclic pentapeptides containing a D-aspartic acid is dependent on the ionization state. The relevance of the work to the design of beta'-turn mimetics is discussed.     

Flexibility in the solution structure of human tropoelastin

We investigated the flexibility of full-length tropoelastin in solution by using far- and near-ultraviolet circular dichroism (UV CD) and fluorescence spectroscopy to probe for structural flexibility and residue mobility within secondary and tertiary features of the monomer. Fluorescence spectroscopy revealed the presence of exposed hydrophobicity through the binding of the hydrophobic probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ANS), which demonstrates that hydrophobic regions form clusters and are not confined to a molecular core. Near-UV CD indicated substantial mobility of aromatic residues. Structural prediction programs (PONDR, DisEMBL, and Globplot version 2.0) estimated 75 +/- 2% disorder in the tertiary structure of tropoelastin on the basis of primary sequence information. A single-site substitution of Trp for Gln (Q513W) at the tropoelastin domain 25-26 interface facilitated fluorescence spectroscopy for revealing that this region is exposed to solvent. Polarization anisotropy demonstrated substantial flexibility of W513 and little change upon denaturation of the monomer with guanidine hydrochloride. Comparable movement was found for native sequence aromatic residues in the presence of glycosaminoglycans and trifluoroethanol. These data prove the intrinsic flexibility of specific residues and adjacent sequences in any native conformation(s) they may take. This study is the first characterization of the level of mobility in defined regions of the full-length tropoelastin monomer and provides direct evidence for regions of flexible structure in tropoelastin.     

Elastin induces myofibrillogenesis via a specific domain, VGVAPG.

A hallmark of vascular smooth muscle cells (VSMCs) is their dynamic ability to assemble and disassemble contractile proteins into sarcomeric units depending upon their phenotypic state. This phenotypic plasticity plays an important role during vascular development and in obstructive vascular disease. Previously, we showed that the Elastin gene product, tropoelastin, activates myofibrillar organization of VSMCs. Recently, others have suggested that elastin does not have a direct signaling role but rather binds to and alters the interactions of other matrix proteins with their cognate receptors or disrupts the binding of growth factors and cytokines. In contrast, we provide evidence that tropoelastin directly regulates contractile organization of VSMCs. First, we show that a discrete domain within tropoelastin, VGVAPG, induces myofibrillogenesis in a time- and dose-dependent fashion. We confirm specificity using a closely related control peptide that fails to stimulate actin stress fiber formation. Second, the activity of VGVAPG is not affected by the presence or absence of other serum or matrix components. Third, both the elastin hexapeptide and tropoelastin stimulate actin polymerization through a common pertussis toxin-sensitive G protein pathway that activates RhoA-GTPase and results in the conversion of G to F actin. Collectively, these data support a model whereby the elastin gene product, signaling through the VGVAPG domain, directly induces VSMC myofibrillogenesis.     

Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer

Previous studies afforded on peptides and polypeptides containing repetitive sequences of elastin have largely demonstrated that their molecular and supramolecular properties are fully representative of those of tropoelastin, the soluble, linear precursor of elastin itself. In the attempt to synthesize cross-linked elastin-mimetic polypeptides, the repeating sequence VGGVG (V: valine; G: glycine), typical of elastin, was modified to incorporate lysine residues, yielding the polymer poly(KGGVG) (K: lysine). This imparts primary amine functionality susceptible to cross-linking reaction with appropriate bifunctional cross-linking reagents. We report herein the chemical synthesis and cross-linking of poly(KGGVG) with glutaraldehyde (GTA) and with disuccinimidyl glutarate (DSG). In both cases, the characterization of the polymers, both linear and cross-linked, has been carried out by CD spectroscopy and transmission electron microscopy measurements. The obtained results, although not conclusive, demonstrate that poly(KGGVG), both linear and cross-linked, may be considered very similar to tropoelastin and mature elastin, as concerns its molecular and supramolecular properties.     

Circular dichroism studies on repeating polypeptide sequences of abductin

The secondary structure of abductin was investigated by CD and NMR studies of several synthetic peptides. Results obtained with these peptides showed the dominant conformations to be the polyproline II (PPII) structure in aqueous solution and different types of beta-turns in the less polar solvent trifluoroethanol. Accordingly, a preliminary structure-elasticity relationship for abductin, not unlike that currently accepted for elastin, is proposed.     

Investigating by CD the molecular mechanism of elasticity of elastomeric proteins

Elastomeric proteins are widespread in the animal kingdom, and their main function is to confer elasticity and resilience to organs and tissues. Besides common functional properties, elastomeric proteins share a common sequence design. They are usually constituted by repetitive sequences with a high content of glycine residues. From a conformational point of view, all the elastomeric proteins since now analyzed show a dynamic equilibria between folded (mainly beta-turns) and extended (polyproline II and beta-strands) conformations that could be at the origin of the high entropy of the relaxed state. As a matter of fact, elastin, lamprin, abductin, as well as the PEVK domain of titin share the same conformational ensemble, thus pointing to a common molecular mechanism as the origin of elasticity. CD spectroscopy represents the proper spectroscopic technique to be used overall because of its particular sensitivity to the presence of PPII structure. Its use in the molecular studies of elastin, abductin, and lamprin as well as the recently analyzed protein resilin will be presented.     

Amino acid sequences C-terminal to the cross-links in bovine elastin.

All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685--695].