Key points are not available for this paper at this time.
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 ∼3 μm 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. 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 ∼3 μm 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. Human tropoelastin is the soluble monomer of elastin, which is largely responsible for the elastic properties of human lungs, large blood vessels and skin. Interactions of human tropoelastin with proteins such as microfibril-associated glycoprotein (MAGP-1) and 67-kDa elastin-binding protein (EBP) 1The abbreviations used are: EBP, elastin-binding protein; GAG, glycosaminoglycan; SHEL and SHELΔ26A, related tropoelastin isoforms corresponding to natural splice variants; GST, glutathione S-transferase; PBS, phosphate-buffered saline. have been described (1Brown-Augsburger P. Broekelmann T. Mecham L. Mercer R. Gibson M.A. Cleary E.G. Abrams W.R. Rosenbloom J. Mecham R.P. J. Biol. Chem. 1994; 269: 28443-28449Abstract Full Text PDF PubMed Google Scholar, 2Brown-Augsburger P. Broekelmann T. Rosenbloom J. Mecham R.P. Biochemical J. 1996; 318: 149-155Crossref PubMed Scopus (111) Google Scholar, 3Hinek A. Rabinovitch M. J. Cell Biol. 1994; 126: 563-574Crossref PubMed Scopus (150) Google Scholar, 4Hinek A. CIBA Found. Symp. 1995; 192: 185-196PubMed Google Scholar). The assembly of the elastin fiber is also affected by glycosaminoglycans (GAGs) which may participate in the regulation of elastin synthesis and deposition during periods of new elastogenesis (5Fornieri C. Baccarani-Contri M. Quaglino Jr., D. Pasquali-Ronchetti I. J. Cell Biol. 1987; 105: 1463-1469Crossref PubMed Scopus (73) Google Scholar, 6McGowan S.E. Liu R. Harvey C.S. Arch. Biochem. Biophys. 1993; 302: 322-331Crossref PubMed Scopus (18) Google Scholar). These GAGs are the substantially major components of the relevant proteoglycans. In this context, appreciable amounts of GAGs have been found in close association with the elastic fiber, prompting the view that these molecules could serve to modulate elastic fiber function (7Baccarani-Contri M. Vincenzi D. Cicchetti F. Mori G. Pasquali-Ronchetti I. Eur. J. Cell Biol. 1990; 53: 305-312PubMed Google Scholar). Using an elastin derivative, Velebny et al. (8Velebny V. Ledvina M. Lankasova V. Connect. Tissue Res. 1981; 9: 63-68Crossref PubMed Scopus (10) Google Scholar) suggested that tropoelastin aggregates upon exposure to chondroitin sulfate. Heparin and heparan sulfate protect elastin from proteolytic attack by human leukocyte elastase (9Walsh R.L. Dillon T.J. Scicchitano R. McLennan G. Clin. Sci. 1991; 81: 341-346Crossref PubMed Scopus (62) Google Scholar) and stimulate elastogenesis (10Grodzinsky A.J. Crit. Rev. Biomed. Eng. 1983; 9: 133-199PubMed Google Scholar). The saccharide units of dermatan and chondroitin sulfates differ from those of heparin-based GAGs because of their galactosamine content, and they display differing negative charge densities. The addition of these GAGs to a culture medium can differentially influence the deposition of elastin in the extracellular matrix (6McGowan S.E. Liu R. Harvey C.S. Arch. Biochem. Biophys. 1993; 302: 322-331Crossref PubMed Scopus (18) Google Scholar). The molecular basis of these interactions is obscure although it is appreciated that GAGs serve to modulate the physical properties of the extracellular matrix by swelling in the presence of water, so bestowing diverse effects including electromechanical and physicomechanical properties, lubrication, impact absorption, and molecular sieving, essentially as proteoglycan components (10Grodzinsky A.J. Crit. Rev. Biomed. Eng. 1983; 9: 133-199PubMed Google Scholar, 11Scott J.E. J. Anat. 1995; 187: 259-269PubMed Google Scholar). Local concentrations of GAGs and their distribution can have profound effects on tissue remodeling and elastin fiber assembly (12Rabinovitch M. Semin. Perinatol. 1996; 20: 531-541Crossref PubMed Scopus (38) Google Scholar). Chondroitin sulfates are closely associated with the developing elastic fiber (13Sherratt M.J. Holmes D.F. Shuttleworth C.A. Kielty C.M. Int. J. Biochem. Cell Biol. 1997; 29: 1063-1070Crossref PubMed Scopus (35) Google Scholar) where these GAGs occur at sites of elastin deposition in the healing of full-thickness wounds (14Lamme E.N. de Vries H.J. van Veen H. Gabbiani G. Westerhof W. Middelkoop E. J. Histochem. Cytochem. 1996; 44: 1311-1322Crossref PubMed Scopus (125) Google Scholar) and may be closely associated with the elastic fiber (15Pasquali-Ronchetti I. Baccarani-Contri M. Microsc. Res. Tech. 1997; 38: 428-435Crossref PubMed Scopus (154) Google Scholar). EBP is believed to accompany tropoelastin to the nascent elastic fiber, at which interactions with endogenous galactoside-containing GAGs mediate release of bound tropoelastin (16Hinek A. Mecham R.P. Keeley F. Rabinovitch M. J. Clin. Invest. 1991; 88: 2083-2094Crossref PubMed Scopus (120) Google Scholar). At this point, tropoelastin is likely to encounter these molecules in the extracellular matrix. This warrants a study of the binding of GAGs to human tropoelastin. We show here that GAGs can provide a significant role in elastogenesis by promoting the association of tropoelastin molecules by coacervation. Tropoelastin containing 26A (SHEL) was obtained as described previously (17Martin S.L. Vrhovski B. Weiss A.S. Gene (Amst.). 1995; 154: 159-166Crossref PubMed Scopus (168) Google Scholar). The SHELΔ26A gene was constructed from pSHELF by site-directed mutagenesis (Transformer Mutagenesis Kit,CLONTECH) using the oligonucleotide 5′-CGG GTT TCG GTG CTG TTC CGG GCG CGC TGG-3′ that flanked either side of exon 26A by 15 base pairs, resulting in its precise deletion. This was followed by subcloning to provide the final construct in pET3d, which was confirmed by DNA sequencing. For SHEL and SHELΔ26A, full-length protein was obtained (17Martin S.L. Vrhovski B. Weiss A.S. Gene (Amst.). 1995; 154: 159-166Crossref PubMed Scopus (168) Google Scholar) and purified further by C18 reverse phase high performance liquid chromatography using a linear gradient of 20–80% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid. The masses of SHEL and SHELΔ26A were estimated, respectively, by electrospray mass spectrometry as 63,495 and 60,026, which compare well with the calculated masses of 63,489 and 60,017. Tropoelastin for Figs. 1 and 2was obtained using protein from shaker flasks, and subsequent experiments used protein from cells grown in a BioFlo III fermentor where final purification was on a Waters PrepLC C18 reverse phase high performance liquid chromatography coupled with a Millennium-driven Waters 600 controller and pump.Figure 2Comparison of the concentration dependence of SHEL and SHELΔ26A on coacervation temperature. Temperatures at 50% coacervation (Ref. 18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar; Fig. 1A) are shown on the same plot to permit comparison of the midpoint transition temperature behavior of the two isoforms. ●, SHEL; ○, SHELΔ26A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An ultrafiltration assay was developed to examine interactions between heparin and two forms of tropoelastin, SHEL and SHELΔ26A. N-sulfonate-35Sheparin (Amersham Pharmacia Biotech; 1 μl) was incubated with 200 μl of 50 nm to 10 μm protein in phosphate-buffered saline (PBS) overnight at 4 °C to model buffer and salt conditions in the extracellular matrix (18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar). The final concentration of heparin was ∼1.6 pm. In each case after mixing, a 150-μl aliquot was transferred to a centrifugal filter (Millipore Ultrafree-MC; 30,000 nominal molecular weight limit) and spun for 10 min at 4 °C. This was washed with 150 μl of PBS. 5 μl of filtrand samples from before and after washing were transferred to 1 ml of ACSII liquid scintillation fluid (Amersham Pharmacia Biotech) for counting using a Wallac (Finland) 1409 System 1400 liquid scintillation counter. Coacervation was assessed using the method of Vrhovski et al. (18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar). Varying concentrations of either heparin (H-3393, Sigma) or chondroitin sulfate B (dermatan sulfate; Sigma) were added to a solution of SHEL or SHELΔ26A at either 1 or 2 mg/ml in PBS prior to each coacervation assay. Blockage of lysine residues in tropoelastin was achieved using sulfosuccinimidyl acetate (Pierce) in a 25-fold molar excess in 100 mmNaHCO3, pH 8.5, for 1 h at room temperature. Blocked protein was dialyzed extensively against PBS at 4 °C. The extent of blockage (>95%) was assayed using picryl sulfonic acid (Sigma) according to standard Pierce protocol Technical Tip 7, where unblocked tropoelastin was used to produce a standard curve. SHELΔ26A was compared with SHEL for its ability to reversibly associate by coacervation (18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar). This feature served to compare protein association behavior of the two forms of the protein and provided the basis for subsequent studies. SHEL (17Martin S.L. Vrhovski B. Weiss A.S. Gene (Amst.). 1995; 154: 159-166Crossref PubMed Scopus (168) Google Scholar) and SHELΔ26A were expressed inEscherichia coli to provide polypeptide that was identical to that inferred from cDNA sequences that had been isolated from human fetal aorta (19Indik Z. Yeh H. Ornstein-Goldstein N. Sheppard P. Anderson N. Rosenbloom J.C. Peltonen L. Rosenbloom J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5680-5684Crossref PubMed Scopus (266) Google Scholar, 20Urry D.W. Starcher B. Partridge S.M. Nature. 1969; 222: 795-796Crossref PubMed Scopus (86) Google Scholar). These structural proteins are capable of thermodynamically reversible association by coacervation (e.g. 18, 20). SHELΔ26A was found to display similar coacervation characteristics to SHEL at comparable concentration, NaCl concentration, and pH. Thus, an increase in the concentrations of SHELΔ26A and NaCl substantially decreased the temperature of coacervation. An increase in pH more subtly also decreased the temperature of coacervation (Fig. 1; Ref.18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar). At similar protein concentrations, repeated studies revealed that the temperatures of coacervation were slightly lower for SHELΔ26A than for SHEL and tended toward concentration independence at just below 37 °C (Fig. 2). The overall characteristics of both proteins showed the same trends. When all the variables are superimposed, coacervation of both tropoelastin forms (>20 mg/ml) at pH 7–8 and 150 mm NaCl is essentially complete at 37 °C. These in vitro conditions are similar to those found in vivo in the extracellular matrix (18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar). Circular dichroism of SHELΔ26A and SHEL revealed similar profiles indicative of comparable folding (results not shown; Ref. 18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar). These assays demonstrated that the two splice forms of tropoelastin could be compared in heparin binding studies. To test for the ability of GAGs to bind tropoelastin, radiolabeled heparin was mixed with protein then separated by ultrafiltration. Binding of heparin to SHEL and SHELΔ26A revealed indistinguishable responses (Fig. 3A), with half-maximal binding in the low micromolar range. Binding by these proteins accounted for ∼50% radiolabeled heparin; this is explained by heterogeneity commonly observed in commercial sources of heparin (21Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar). Accordingly, binding of heparin to SHEL and to SHELΔ26A were each quantified using Scatchard analyses according to the method of Lee and Lander (21Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar), where it was assumed that the concentration of GAG was much less than the Kd, the GAGs were not multivalent, and multiple high affinity sites in each tropoelastin form bound with similarKd. Considering these assumptions, theKd values for the interaction between heparin and SHEL and with SHELΔ26A were estimated to be 2.5 and 4.5 μm, respectively (Fig. 3B). These comparable values provided an average Kd ∼3 μm. When isolated domain 26A was assayed as a fusion with glutathioneS-transferase (GST26A), it showed a propensity to bind radiolabeled heparin. However, this association was weaker than the interaction of tropoelastin with heparin (results not shown). This implicated a dominant participation by the most abundant positively charged amino acids (there are 35 lysines per molecule; Refs. 18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholarand 19Indik Z. Yeh H. Ornstein-Goldstein N. Sheppard P. Anderson N. Rosenbloom J.C. Peltonen L. Rosenbloom J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5680-5684Crossref PubMed Scopus (266) Google Scholar) in tropoelastin. Chondroitin sulfate B (1 μg/ml to 1 mg/ml) added to SHEL or SHELΔ26A significantly lowered the coacervation temperature. A similar effect was seen for both isoforms. Using tropoelastin (2 mg/ml), the addition of 1 mg/ml chondroitin sulfate B dramatically reduced the temperature required for coacervation, whereas as little as 5 μg/ml noticeably lowered the temperature for this process (Fig. 4,A and B). Thus the presence of this GAG can lower the coacervation of tropoelastin to below physiological temperatures and at higher concentrations to below room temperature. The effect of heparin, while effective in the same concentration ranges as those for chondroitin sulfate B, clearly displayed a more substantial effect on coacervation at lower temperatures (Fig. 4,C and D). This indicated an aggregation of tropoelastin molecules prior to reaching the transition temperature because of multiple intermolecular interactions. Interaction with negatively charged GAGs was probably a consequence of the abundance of positively charged residues in tropoelastin, which contribute to a high, calculated isoelectric point of 11.2. The net positive charge status of tropoelastin drops in vivo upon cross-linking because of substantial lysine modification and participation in the production of oxidized and condensed lysine derivatives that are essential to the formation of elastin. In the presence of chondroitin sulfate B, the coacervation curves for both forms of tropoelastin were displaced to lower temperatures by about 20–25 °C (Fig. 4, A and B). Heparin similarly displaced coacervation to lower temperatures, but in doing so it also flattened the curves, probably as a consequence of its different charge density and distribution compared with chondroitin sulfate B (Fig. 4, C and D). These effects were concentration-dependent to as little as 5 μg/ml GAG, whereas protein concentrations were considerably higher at 2–10 mg/ml. The GAG effects assumed that charge interactions played an important role in assisting aggregation of tropoelastin molecules. To examine the involvement of lysine side-chains in GAG interactions, the two forms of tropoelastin were treated with sulfosuccinimidyl acetate, which predominantly acetylates the amino and it to charge W. H. C. M.A. Arch. Biochem. Biophys. 1997; PubMed Scopus Google Scholar). In the of of lysine slightly the coacervation curves from those of protein (Fig. This that positively lysine residues are not major of the temperature of transition of tropoelastin, probably because they are the D.W. N. Biochem. Biophys. Res. 1995; PubMed Scopus Google Scholar). In modification of the amino on lysine side showed a substantial effect on the influence of GAGs on coacervation. When the positive on lysine side were the effects of chondroitin sulfate B and heparin on coacervation were essentially Tropoelastin assembly by coacervation is dramatically in the presence of the GAGs These effects are in the relevant range. For heparin to with high affinity and including the with 2 Thus binding probably on an domain each and at multiple sites S.M. C.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). human matrix elastin in the where its heparin in the micromolar C.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The two human tropoelastin isoforms SHEL and SHELΔ26A, differ by the presence or of 26A which the most hydrophilic in human tropoelastin. However, the two isoforms show similar coacervation properties at comparable protein concentrations, in the presence or of glycosaminoglycans. the presence of this 26A not substantially the coacervation ability of SHEL the of this of the The coacervation (18Vrhovski B. Jensen S. Weiss A.S. Eur. J. Biochem. 1997; 250: 92-98Crossref PubMed Scopus (193) Google Scholar) with SHEL to a of a further splice et al. D. P. Abrams W. Rosenbloom J. H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) found that the isoform 26A is a for lysyl oxidase and is not as The shown here that this is not by a in the ability of the isoforms to coacervate under these conditions. The similar coacervation behavior of these two splice forms that this feature is likely to be a of tropoelastin splice where the transition temperature is to occur under extracellular physiological conditions. The heparin binding studies were also under these conditions. is little for the role of 26A to as Ref. 19Indik Z. Yeh H. Ornstein-Goldstein N. Sheppard P. Anderson N. Rosenbloom J.C. Peltonen L. Rosenbloom J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5680-5684Crossref PubMed Scopus (266) Google Scholar) in tropoelastin and for its physiological This lysyl oxidase on tropoelastin containing the D. P. Abrams W. Rosenbloom J. H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), and isolated 26A for F. M.A. S. A. A. A. PubMed Scopus Google Scholar). 26A is in human have to be in When to and the shows propensity to multiple and In of have to solution studies on the isolated 26A W. and A. and 26A dichroism that 26A is essentially or in from a with a acid in high concentrations of this is with the of et al. F. M.A. S. A. A. A. PubMed Scopus Google Scholar). to be heparin binding by than GST, a by 26A to GAG association not that be relevant in the elastic fiber after Heparin binding by either tropoelastin isoform shows dissociation in the low micromolar range. This with the binding displayed by heparin to in S.M. C.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). modification of lysine residues in tropoelastin the to this It is that the effects on coacervation are a consequence of the participation of an of The of low concentrations of GAGs that interactions are effective with a of for coacervation that the effects of heparin and chondroitin sulfate B may be because of in lysine binding and because of differing negative charge in these The effect on coacervation be less lysyl oxidase most of the lysines in tropoelastin to the prior to significant exposure to EBP (16Hinek A. Mecham R.P. Keeley F. Rabinovitch M. J. Clin. Invest. 1991; 88: 2083-2094Crossref PubMed Scopus (120) Google Scholar) in of tropoelastin to the nascent elastic fiber while association with GAGs prior to to form elastin. GAG interactions occur in the extracellular matrix, which to tropoelastin aggregation in its the We that tropoelastin is encouraged to participate in elastogenesis by association with a consequence of this of these GAGs may in the nascent elastic fiber, as observed by et al. (5Fornieri C. Baccarani-Contri M. Quaglino Jr., D. Pasquali-Ronchetti I. J. Cell Biol. 1987; 105: 1463-1469Crossref PubMed Scopus (73) Google Scholar). This an role for assisting in the release of tropoelastin from its partner EBP (16Hinek A. Mecham R.P. Keeley F. Rabinovitch M. J. Clin. Invest. 1991; 88: 2083-2094Crossref PubMed Scopus (120) Google Scholar), then promoting coacervate formation and elastin In this GAGs serve to mediate tropoelastin interactions and We for in the production and of in E. coli for and during and for and in studies of 26A.
Wu et al. (Thu,) studied this question.