Molecular-level characterization of elastin-like constructs and human aortic elastin

Matrix Biology 38 (2014) 12–21

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Molecular-level characterization of elastin-like constructs and human aortic elastin Andrea Heinz a,⁎, Christoph U. Schräder a, Stéphanie Baud b,c, Fred W. Keeley d, Suzanne M. Mithieux e, Anthony S. Weiss e,f,g, Reinhard H.H. Neubert a, Christian E.H. Schmelzer a a

Institute of Pharmacy, Faculty of Natural Sciences I, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany Laboratoire SiRMa, FRE CNRS/URCA 3481, Université de Reims Champagne-Ardenne, Reims, France Plateforme de Modélisation Moléculaire Multi-échelle, UFR Sciences Exactes et Naturelles, Université de Reims Champagne-Ardenne, Reims, France d Molecular Structure and Function, Hospital for Sick Children, Toronto, Canada e School of Molecular Bioscience, University of Sydney, Sydney, Australia f Bosch Institute, University of Sydney, Sydney, Australia g Charles Perkins Centre, University of Sydney, Sydney, Australia b c

a r t i c l e

i n f o

Article history: Received 1 July 2014 Received in revised form 17 July 2014 Accepted 18 July 2014 Available online 25 July 2014 Keywords: Desmosine Allysine aldol Mass spectrometry Cross-links

a b s t r a c t This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS2 data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin. © 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction Abbreviations: AA, allysine aldol; ACN, acetonitrile; ANAO, Aspergillus nidulans amine oxidase; ANAO-TE, tropoelastin cross-linked in vitro after addition of ANAO; CTR, chymotrypsin; DES, desmosine; DSSP, define secondary structure of proteins; EP, elastin polypeptide; ESI, electrospray ionization; FA, formic acid; GROMACS, Groningen machine for chemical simulations; HPLC, high performance liquid chromatography; IDES, isodesmosine; k, allysine residue; LC, liquid chromatography; LOX, lysyl oxidase; MALDI, matrix-assisted laser desorption/ionization; MD, molecular dynamics; MS, mass spectrometry; MS2, tandem mass spectrometry; MS3D, studies of three-dimensional protein structure using mass spectrometry; NMR, nuclear magnetic resonance; OPLSAA, optimized potentials for liquid simulations all atoms; PE, pancreatic elastase; PQQ, pyrroloquinoline quinone; SEM, scanning electron microscopy; TE, tropoelastin; TFA, trifluoroacetic acid; TOF, time of flight; TR, trypsin; Tris, 2-amino-2-hydroxymethylpropane-1,3-diol. ⁎ Corresponding author at: Institute of Pharmacy, Martin Luther University HalleWittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany. Tel.: +49 345 5525220; fax: +49 345 5527292. E-mail address: [email protected] (A. Heinz).

Elastin is an essential extracellular matrix protein of vertebrates and possesses unique properties including elasticity and extreme durability, which make it critical for the long-term function of tissues and organs such as aorta, skin and cartilage. Elastin is formed through K-mediated cross-linking of its monomeric precursor tropoelastin, which results in the formation of a variety of cross-links such as allysine aldol (AA), desmosine (DES) and its isomer isodesmosine (IDES) (Eyre et al., 1984; Reiser et al., 1992; Akagawa and Suyama, 2000; Mithieux and Weiss, 2005). However, even though the types of cross-links in elastin have been identified decades ago, almost nothing is known about the cross-linking pattern of elastin in different tissues. Elastin not only provides mechanical integrity to various tissues, but also plays an important role in the regulation of the cell behavior via bioactive elastin peptides (elastokines) that result from enzymatic degradation of

http://dx.doi.org/10.1016/j.matbio.2014.07.006 0945-053X/© 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

A. Heinz et al. / Matrix Biology 38 (2014) 12–21

mature elastin (Maquart et al., 2005). In this context, it is noteworthy that the destruction of elastic fibers and the release of elastokines have been shown to be associated with pathological conditions including emphysema, atherosclerosis and actinic elastosis (photoaging) (Vrhovski and Weiss, 1998; Saarialho-Kere et al., 1999; Mithieux and Weiss, 2005). Therefore, it is important to investigate the molecularlevel structure of human elastin to better understand the biomechanical properties of elastin as well as elastic-tissue diseases and their functional consequences. The diverse functions of elastin and its degradation products as well as its unique properties such as extensibility and recoil, chemical stability and capacity to self-assemble have made the protein of interest for biomedical applications including tissue engineering. In particular recombinant human tropoelastin, elastin polypeptides and elastin-like polypeptides have been increasingly used as versatile, soluble components for the preparation of a variety of biomaterials in recent years (Keeley et al., 2002; Wise and Weiss, 2009; Almine et al., 2010; MacEwan and Chilkoti, 2010). Among other methods, elastin-based biomaterials have been produced using chemical cross-linkers such as bis(sulfosuccinimidyl) suberate (Mithieux et al., 2004) or pyrroloquinoline quinone (PQQ) (Bellingham et al., 2003; Vieth et al., 2007). These materials have been mainly characterized through their rheological and mechanical properties, e.g. swelling behavior and shear modulus, and their structure using a variety of microscopic techniques such as atomic force microscopy, confocal laser scanning microscopy and electron microscopy (Bellingham et al., 2003; Mithieux et al., 2004; Li et al., 2005; Vieth et al., 2007; Lim et al., 2008). However, no study has provided significant insight into the cross-linking pattern of such materials. Structural analysis of elastin-based biomaterials could contribute to understanding not only the structure of the materials but also that of mature elastin. Due to its low accessibility for structure analysis, investigations on elastin have mainly been carried out after total hydrolysis of the protein in a strongly acidic or basic environment or by treatment with elastases and subsequent analysis of the resulting hydrolytic products, in particular the cross-linked amino acids DES and IDES (Partridge et al., 1955; Foster et al., 1974; Gerber and Anwar, 1975; Baig et al., 1980; Spacek et al., 1998). Analytical methods to characterize the structures of elastin and elastin-derived peptides include circular dichroism, vibrational spectroscopy, NMR spectroscopy and different microscopic techniques (Keeley et al., 2002; Bochicchio et al., 2004; Kumashiro et al., 2006; Vieth et al., 2007; Dyksterhuis et al., 2009). In recent years, liquid chromatography–mass spectrometry (LC–MS) has become an important tool in protein analytics. Since available software only allows for the identification of chemically introduced and bifunctional cross-links from MS2 data, in the scope of an earlier project the software PolyLinX (Heinz et al., 2013) was developed to facilitate the sequence elucidation of peptides containing polyfunctional elastin cross-links including DES/ IDES based on LC/MALDI MS/MS data. The present study deals with the structural characterization of two elastin-derived materials of different structural complexity and native human aortic elastin to establish analytical methods for the structural characterization of elastin, in particular with respect to its crosslinking pattern. Another aim was to investigate how similar the materials are as compared to native elastin with respect to their crosslinking degree and ultrastructure. One of the constructs was composed of cross-linked elastin-like polypeptide (EP) 20-24-24 (Bellingham et al., 2001), a 200 amino acid residue long recombinant elastin peptide and the other one was cross-linked tropoelastin, isoform 2 (McGrath et al., 2011), the natural precursor of mature elastin. First, the morphology of these samples was investigated using scanning electron microscopy. Then, the susceptibility of the three materials to enzymatic degradation was studied and the degree of cross-linking was analyzed using mass spectrometric and bioinformatics methods recently developed for the identification of cross-linked species (Heinz et al., 2013). Moreover, molecular dynamics (MD) simulations were used to better

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understand the formation of cross-links, i.e. typical conformations that occur in linear elastin peptides prior to cross-linking as well as conformations present in already cross-linked elastin peptides. 2. Materials and methods 2.1. Materials Tropoelastin lacking domains encoded by exons 22, 24A and 26A (isoform 2, UniProt accession number P15502-2) and the elastin-like polypeptide EP20-24-24 were recombinantly produced as described by Martin et al. (1995) and Bellingham et al. (2001), respectively. Human aortic punch biopsies (5 mm in diameter) were taken from patients of both genders (aged between 66 and 72) during coronary artery bypass grafting. Pure elastin was isolated from these biopsies using a newly developed, gentle method, which removes all other extracellular matrix components and prevents damage to elastin (Schmelzer et al., 2012). The work on human samples was approved by the ethics committee of the Medical Faculty, Martin Luther University HalleWittenberg (Germany), and performed in compliance with the Helsinki Declaration. Porcine pancreatic elastase (PE), proteomics grade trypsin (TR) and sequencing grade chymotrypsin (CTR) were purchased from Elastin Products Company (Owensville, MO, USA), Sigma-Aldrich (Steinheim, Germany) and Roche Diagnostics (Mannheim, Germany), respectively. Aspergillus nidulans amine oxidase (ANAO) was purified from A. niger culture (McGrath et al., 2011). Pyrroloquinoline quinone (PQQ) and α-cyano-4-hydroxycinnamic acid were obtained from Sigma-Aldrich. HPLC-grade acetonitrile (ACN) (VWR Prolabo, Leuven, Belgium) was used. Analytical grade 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), formic acid (FA) and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany). 2.2. In vitro cross-linking experiments Tropoelastin was coacervated and subsequently cross-linked in vitro through addition of ANAO (McGrath et al., 2011). The resulting insoluble material was lyophilized prior to analysis and is referred to as ANAO cross-linked tropoelastin (ANAO-TE). The elastin-like polypeptide EP20-24-24 was dissolved at a concentration of 5 mg mL− 1 in 50 mM Tris buffer, pH 7.5, containing 1.5 M NaCl and cross-linked upon addition of PQQ as described earlier (Heinz et al., 2013). The cross-linked material was washed three times with a solution of water and ethanol (7:3, V/V), dried at room temperature and stored at −26 °C prior to further analysis. 2.3. Scanning electron microscopy Lyophilized cross-linked EP20-24-24, lyophilized ANAO-TE and human aortic elastin, dried at room temperature after isolation from human aortic tissue biopsies, were analyzed by scanning electron microscopy (SEM) using an environmental scanning electron microscope ESEM XL 30 FEG (Philips, Amsterdam, Netherlands) as described previously (Heinz et al., 2013). 2.4. Proteolysis of tropoelastin, human aortic elastin and the cross-linked materials Cross-linked EP20-24-24, ANAO-TE and human aortic elastin were dispersed in 50 mM Tris buffer, pH 7.5, at a concentration of 1 mg mL−1, respectively. All samples were digested with PE, TR and CTR for 24 h at 37 °C using enzyme-to-substrate ratios of 1:100 (w/w), respectively. All digestions were stopped by adding TFA to a final concentration of 0.5% (V/V), and the samples were stored at −26 °C prior to further analysis.

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A. Heinz et al. / Matrix Biology 38 (2014) 12–21

2.5. NanoHPLC/MALDI-TOF/TOF MS analysis Analysis of the enzymatic digests of EP20-24-24, ANAO-TE and human aortic elastin was carried out offline by using an UltiMate 3000 RSLCnano system (Thermo Fisher, Idstein, Germany), a Probot microfraction collector (Thermo Fisher) and a 4800 MALDI-TOF/TOF Analyzer (AB Sciex) as described previously (Heinz et al., 2013). 2.6. NanoHPLC–nanoESI–QqTOF MS analysis Digests of ANAO-TE with PE, TR and CTR were analyzed online using an UltiMate 3000 nanoHPLC system (Thermo Fisher) coupled to a QqTOF mass spectrometer Q-TOF-2 (Waters/Micromass, Manchester, UK). The mass spectrometer was equipped with a nanoESI Z-spray source and a tip adapter for PicoTips (New Objective, Woburn, MA, USA), which was used with SilicaTip emitters (10 μm I.D.) from New Objective. Chromatographic separation of the peptides was performed by loading 1 μL of the sample onto a trapping column (Acclaim PepMap 100 C18, 5 μm, 100 Å, 300 μm I.D. × 5 mm; Thermo Fisher) and washing for 10 min with a solvent consisting of 98% water and 2% ACN containing 0.1% FA at a flow rate of 6 μL min−1. The trapped peptides were then eluted onto the separation column (Acclaim PepMap C18, 3 μm, 100 Å, 75 μm I.D. × 150 mm; Thermo Fisher), which had been equilibrated with 88% solvent A (0.1% FA) and 12% solvent B (80% ACN and 20% water containing 0.1% FA). The peptides were separated using a solvent system of solvent A and solvent B: linear gradient of 12% to 40% solvent B in 51 min, linear gradient of 40% to 90% solvent B in 10 min, maintenance at 90% solvent B for 5 min, linear gradient of 90% to 12% solvent B in 15 min and equilibration for 5 min prior to the next run. The column was kept at 40 °C, and the flow rate was 300 nL min−1. The typical operating conditions for the mass spectrometer were used as described earlier (Heinz et al., 2010) except for the sample cone voltage which was lowered to 20 V to minimize in-source decay. 2.7. Peptide sequencing The tandem mass spectra obtained from nanoESI–QqTOF and MALDI-TOF/TOF experiments were processed into de-isotoped peak lists with the MassLynx add-on MaxEnt3 (version 4.1; Waters) and Mascot Distiller (Matrix Science, London, UK), respectively. Peptides were identified by database-driven sequencing using a local Mascot server (version 2.2.1; Matrix Science, London, UK) (Perkins et al., 1999) and by automated de novo sequencing followed by database matching with the software Peaks Studio (version 6; Bioinformatics Solutions, Waterloo, Canada) (Zhang et al., 2012). The searches were taxonomically restricted to Homo sapiens. The enzyme was set to ‘none’ for PE and CTR digests, respectively, and to ‘trypsin’ for TR digests. The formation of allysine residues and hydroxylated proline residues were considered as variable modifications. For the MALDI TOF/TOF MS/MS data, mass error tolerances for precursor and fragment ions were set to 50 ppm and 0.3 Da, respectively, and for the QqTOF MS/MS data, mass tolerances for precursor and fragment ions were set to 40 ppm and 0.1 Da, respectively. For searching purposes, the sequence of EP20-2424 was implemented into a Swiss-Prot database. Sequencing of crosslinked species was performed using the in-house developed software PolyLinX (Heinz et al., 2013).

mass spectrometer Finnigan LCQ (Thermo Fisher, San Jose, CA, USA) via an electrospray interface. Isocratic chromatographic separation was performed over 8 min at a flow rate of 0.2 mL min− 1 on a Reprosil-Pur 120 C18 AQ 3 μm column (150 × 2 mm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). 0.1% FA/methanol (97.5:2.5, V/V) was used as mobile phase and the column temperature was kept at 40 °C. Under these chromatographic conditions DES and IDES co-elute. For mass spectrometric analyses, the following parameters were used: positive ion mode, electrospray voltage: 4.5 kV, and heated capillary temperature: 220 °C. 2.9. Molecular dynamics simulations The structures of two intramolecularly cross-linked peptides containing allysine aldol cross-links, AAkYGVGTPAAAAAkAAAK and SAAkVAAkAQLR (allysine residues are denoted by k; cross-linked residues are underlined), identified in enzymatic digests of ANAO-TE were investigated using MD simulations (Table 1). The corresponding linear peptides AAKYGVGTPAAAAAKAAAK, AAkYGVGTPAAAAAkAAAK, SAAKVAAKAQLR and SAAkVAAkAQLR were also modeled to determine differences in the conformations of K- and k-containing peptides, in particular with respect to the formation of cross-links in k-containing peptides. Moreover, modified sequences of the linear peptides AAKYGVGTAAAAAAKAAAK and AAkYGVGTAAAAAAkAAAK, where the central P residues had been replaced by A residues, respectively, were modeled to investigate the role of P during formation of cross-links. Simulations were carried out using the GROMACS simulation package (Van der Spoel et al., 2005). OPLSAA force field was chosen as the set of parameters for atoms of standard amino acids and their interactions (Jorgensen and Tiradorives, 1988). Parameters for the non-standard amino acid k and AA were implemented. Isolated peptides were placed in cubic boxes with a side length of 60 Å or 65 Å depending on the peptide. These values were chosen so that a given peptide would not interact with its images when the periodic boundary conditions are applied. Water (SPC/E model) and counter ions were added prior to the simulation. To relax the structures 500 steps of energy minimization were performed using the steepest descent algorithm. The subsequent equilibration of the system was conducted in two phases. At first, a 100 ps isothermal-isochoric ensemble was executed to stabilize the temperature at 310 K, which was followed by a 100 ps isothermalisobaric ensemble. After equilibration, MD simulations were carried out for 100 ns, maintaining a pressure of 1 bar and a temperature of 310 K. The Verlet algorithm was used to integrate the equation from classical mechanics in parallel with an integration step of 2 fs since the length of the bonds implicating hydrogen atoms was frozen. For the non-bonded terms, we used the Particle Mesh Ewald (PME) algorithm with a cut-off at 1.49 Å for the Coulombic interactions and a potentialshifting function for van der Waals interactions applied at 1.3 Å and a cut-off at 1.4 Å. Table 1 Linear and cross-linked peptides that were investigated using MD simulations. Small letter k denotes allysine residues and underlined small letter k denotes allysine residues which are involved in cross-linking. Peptide sequence

Peptide Mr

Residues (based on tropoelastin IF 2)

2.8. Quantification of desmosine/isodesmosine

AAkYGVGTPAAAAAkAAAK P1

1666.87 449–467

Human aortic elastin, ANAO-TE and cross-linked EP20-24-24 were hydrolyzed at a concentration of 1 mg mL−1 in 6 M HCl at 105 °C for 24 h, respectively, to allow the liberation of DES and IDES. After incubation, the samples were evaporated to dryness at 60 °C and the residues were taken up in ACN/water (1:1, V/V) prior to LC–MS analysis. LC–MS analysis of DES and IDES was carried out using an Agilent 1100 LC system (Agilent, Waldbronn, Germany) coupled to a quadrupole ion trap

SAAkVAAkAQLR AAkYGVGTPAAAAAkAAAK AAKYGVGTPAAAAAKAAAK AAkYGVGTAAAAAAkAAAK AAKYGVGTAAAAAAKAAAK SAAkVAAkAQLR SAAKVAAKAQLR

1192.66 1684.88 1686.94 1658.86 1660.93 1210.67 1212.73

P2 P3 P4 P5 P6 P7 P8

530–541 449–467 449–467 – – 530–541 530–541

Identified in sample ANAO-TE, cross-linked EP20-24-24 ANAO-TE – – – – – –

A. Heinz et al. / Matrix Biology 38 (2014) 12–21

3. Results and discussion 3.1. Structural characterization of cross-linked EP20-24-24, ANAO-TE and human aortic elastin SEM images revealed the presence of rather porous structures with diameters of about 5 μm to 30 μm in the case of EP20-24-24 (Fig. 1A and B) and ANAO-TE (Fig. 1C and D) with a rough, characteristic sponge-like structure and small fibers of about 1 μm in diameter. In contrast, aortic elastin was composed of a network of fibers of about 1 μm in diameter and fiber bundles of larger diameters (Fig. 1E and F). With respect to enzymatic cleavage, the following results were obtained. Human aortic elastin was cleaved well by PE, but hardly by TR and CTR (Fig. 2). ANAO-TE (Fig. 3) and cross-linked EP20-24-24 (Fig. 4) were readily digested by all three enzymes. The occurrence of a variety of semi-tryptic and semi-chymotryptic peptides in all samples was most likely due to in-source fragmentation during MS measurement which is favored by the presence of proline residues. The higher resistance of human aortic elastin toward enzymatic cleavage as compared to ANAO-TE is due to the extensive cross-linking of native elastin (Vrhovski and Weiss, 1998; Mithieux and Weiss, 2005), which decreases the proteolytic susceptibility of the protein. Since about 88% of the K residues are modified and involved in cross-links (Kozel et al., 2003), in particular cleavage by TR (cleaves C-terminal of K and R) is

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hindered. In contrast, as described in the literature, porcine PE was found to be an aggressive elastase, which cleaves predominantly Cterminal to A, V, L and I but to lesser extent also C-terminal to Y, F, K, P, R and G (Powers et al., 1977; McRae et al., 1980) (Figs. 3 and 4). While cleavages of PE C-terminal to K residues were observed in the case of cross-linked EP20-24-24 and ANAO-TE, respectively, no such cleavages were identified in human aortic elastin due to the previously mentioned cross-linking. However, interestingly, four unmodified K residues were identified in the elastin digests with PE and two unmodified K residues in digests with CTR, which suggests that these residues remain at least partially non-cross-linked in the five human aortic samples that were analyzed. The residues originate from the KP motifs of domains 4, 8 and 12 (K residues 61, 64,137, 140, 209 and 212 based on numbering of tropoelastin isoform 9; see Fig. 2), which have been hypothesized to be less likely to form tetrafunctional cross-links (Chung et al., 2006). However, the overall lack of identified peptides from the cross-linked KA and KP domains in human aortic elastin (Fig. 2) can be attributed to the fact that these parts of the tropoelastin molecules are cross-linked and such peptides still cannot be sequenced with available bioinformatics tools. Even with the in-house developed software PolyLinX it is still a substantial challenge to identify such peptides in mature elastin due to sequence repetitions and similarities within the protein which lead to a high number of possible candidate peptides of the same mass.

Fig. 1. Scanning electron micrographs of cross-linked insoluble materials formed after incubation of EP20-24-24 with PQQ (A, B), after incubation of recombinant human tropoelastin with ANAO (C, D), and of isolated human aortic elastin (E, F). The white bars represent either 100 μm (A, C, E) or 30 μm (B, D, F).

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Fig. 2. Cleavage sites identified after digestion of human aortic elastin with PE, TR and CTR, shown based on the sequence of tropoelastin isoform 9 (Swiss-Prot accession number P155029). Cleavage sites are marked with triangles (PE: red, TR: green, CTR: blue) and sequenced regions are labeled with solid lines. Identified hydroxylated prolines are shown as capital letter P in orange.

With respect to the splicing of tropoelastin, it was found that exons 24A and 26A are spliced out from human aortic elastin, which is in agreement with previous studies on human skin elastin (Schmelzer et al., 2005; Taddese et al., 2008; Heinz et al., 2012) (Fig. 2). Moreover, peptides comprising residues from both domains 23 and 24 indicate that exon 24A is spliced out (Fig. 2). The data furthermore points to the absence of exon 22 as no linear peptides from this domain were identified in any of the three enzymatic digests even though it is a hydrophobic domain which does not contain cross-linking motifs. It is worth mentioning that the possible absence of domain 22 is consistent with earlier results of our group on skin elastin samples (Taddese et al., 2008; Heinz et al., 2012) and with work by Fazio et al. (1988a). Peptides of domain 32, which has been reported to be often spliced out of human tropoelastin mRNA (Holzenberger et al., 1993), prove the presence of this domain in the analyzed aortic elastin samples (Fig. 2). Similar results were obtained in previous studies on human skin elastin (Schmelzer et al., 2005; Taddese et al., 2008; Heinz et al., 2011). With respect to P hydroxylation, 19 partially hydroxylated sites were detected in human aortic elastin of which 13 have already been identified in human skin elastin (Schmelzer et al., 2005) and 6 (P147,

P337, P405, P433, P595, and P726 based on the numbering of isoform 9 of tropoelastin) are first time reports (Fig. 2). The role of hydroxyproline (HyP) in elastin has not been determined yet. Several reports suggest that HyP plays a minimal role during elastic fiber formation (Rosenbloom and Cywinski, 1976; Narayanan et al., 1978), however, on the other hand, a recent study on hydroxylated elastin peptides showed that the presence of HyP increases the coacervation temperature and alters the self-assembly process, which in turn may have consequences on the ability of tropoelastin to cross-link and form mature elastin (Bochicchio et al., 2013). As mentioned previously, cross-linked EP20-24-24 and ANAO-TE were cleaved extensively by all three proteases indicating that the two constructs differ structurally from human aortic elastin, i.e. show less complex cross-linking patterns. This hypothesis is supported by the fact that upon MS analysis of ANAO-TE 29 of the 35 K residues were found to be unmodified, i.e. non-oxidized and non-cross-linked. Thirteen of these 29 K residues were also detected in the form of allysine residues (k residues) in linear peptides, which suggests that both oxidation by ANAO and subsequent cross-linking were incomplete (Fig. 3). As for cross-linked EP20-24-24, in addition to unmodified K residues, free

A. Heinz et al. / Matrix Biology 38 (2014) 12–21

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Fig. 3. Cleavage sites identified after digestion of ANAO-TE with PE, TR and CTR, shown based on the sequence of tropoelastin isoform 2 (Swiss-Prot accession number P15502-2). Cleavage sites are marked with triangles (PE: red, TR: green, CTR: blue) and sequenced regions are labeled with solid lines. Identified allysine residues are shown as small letter k in orange. Two cross-linked peptides that were identified in digests of ANAO-TE (P1 and P2 in Table 1) are highlighted in light orange.

k residues were also detected for all four K residues present in EP20-2424 (Fig. 4). Overall, it can be concluded that in the case of ANAO-TE and cross-linked EP20-24-24 partial oxidation of K residues during the in vitro cross-linking experiments most likely resulted in the formation of incompletely cross-linked products. The two in vitro cross-linked materials were also analyzed for the presence of cross-links typical of mature elastin including tetrafunctional DES and its isomer IDES. Using PolyLinX (Heinz et al., 2013), bifunctional cross-links containing intramolecular AA were

identified in two peptides, of which one was found in TR digests of ANAO-TE (cross-linked P2) and the other one (cross-linked P1) was present in TR digests of both cross-linked EP20-24-24 and ANAO-TE (Table 1). A tandem mass spectrum of cross-linked P2 with annotated b, y and immonium ions is shown in Fig. 5. The lack of the ions b4 to b7 and y5 to y8 and in particular the mass shift of 20.07 Da for the precursor and the product ions b8 to b11 and y9 to y11 as compared to the nonoxidized, linear peptide prove the existence and position of the intramolecular AA cross-link indicated above the sequence. However, it

Fig. 4. Cleavage sites identified after digestion of cross-linked EP20-24-24 with PE, TR and CTR, shown based on the sequence of EP20-24-24 using the numbering of tropoelastin isoform 2. The repeat of domains encoded by exons 21, 23 and 24, respectively, is indicated using orange brackets. Identified allysine residues are shown as small letter k in orange.

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Fig. 5. Annotated MALDI-TOF/TOF fragment spectrum of the peptide SAAkVAAkAQLR containing an intramolecular AA identified in a trypsin digest of ANAO-TE. The peptide sequence on top of the spectrum visualizes the detected backbone fragments (b and y ions) that occur upon fragmentation of the cross-linked peptide during MS/MS measurements. In addition to y ions (shown in red) and b ions (highlighted in blue) that are labeled in the spectrum, immonium ions of the single amino acids A, S, V, G, Q and R are shown in green.

was impossible to clarify whether the cross-links have formed before or after enzymatic digestion. Given that size- and temperature-dependent condensation is required for the formation of AA crosslinks, it is more likely that they were present in the material prior to enzymatic digestion rather than having occurred after the digestion. Since the crosslinked K residues are only three residues apart in the case of crosslinked P2 derived from domain 25, the cross-link may easily have formed prior to enzymatic degradation. With respect to native elastin, however, it is possible that this specific AA is not stable, but further condenses with K or k residues to form tri- or tetrafunctional cross-links. Cross-linked P1 contains an AA between two residues (K451 and K463; Table 1) of domains 21 and 23 that are relatively far apart (Figs. 3 and 4). The cross-link can, therefore, only form when K residues are brought into proximity. Earlier, the presence of a double β-turn between the residues GVGTPA of domain 23 has been demonstrated by CD and NMR spectroscopies (Tamburro et al., 2006). This conformation has been hypothesized to bring K451 and K463 closer together, which may facilitate the formation of the AA cross-link identified in this work. Moreover, data from further CD spectroscopic measurements (Miao et al., 2005), predictions of secondary structures (Miao et al., 2005), NMR spectroscopic measurements (Kumashiro et al., 2006) and MD simulations (Djajamuliadi et al., 2009) on peptides composed of domains 21 and 23 revealed that the center of these two cross-linking domains, i.e. the residues GVGTP, represents a flexible hinge region which is speculated to be essential for elastic fiber formation. All of these experimental results indicate that the cross-linked peptide P1 (Table 1) found in this study may have formed prior to enzymatic degradation of the two in vitro cross-linked materials. This hypothesis is also supported by a bis(sulfosuccinimidyl) suberate cross-link between K451 and K463, which was found in a polypeptide consisting of domains 17–27 of human tropoelastin lacking domain 22 (Dyksterhuis et al., 2007). Moreover, SAXS and SANS structures of the entire tropoelastin molecule (Baldock et al., 2011) suggest that the formation of a crosslink between K451 and K463 may be facilitated due to the tertiary structure of the tropoelastin molecule, which allows the residues to come close together. This cross-link is likely to have biological relevance whenever exon 22 is spliced out. Indeed, exon 22 is omitted from

most human elastin isoforms as indicated by cDNA sequencing (Fazio et al., 1988b), mass spectrometry (Heinz et al., 2011, 2012) and the present study. The in vivo formation of the cross-link between K451 and K463 may occur with the formation of an intramolecular crosslink between domains 19 and 25, which has been described previously (Brown-Augsburger et al., 1995), and forms the basis for the recently proposed head-to-tail model of arrangement of tropoelastin molecules (Baldock et al., 2011). Detection of DES/IDES after total hydrolysis of the three substrates revealed that all of them contained the tetrafunctional cross-link. Cross-linked EP20-24-24 showed the lowest amount of tetrafunctional cross-links (1.1 μg ± 0.3 μg per mg substrate), while human aortic elastin exhibited the highest amount of DES/IDES (24.8 μg ± 5.3 μg per mg substrate). With 3.8 μg ± 0.2 μg per mg substrate, the amount of DES/ IDES in ANAO-TE is 6.5 fold lower as compared to human aortic elastin. With respect to the two constructs, the results are consistent with the findings of the SEM analysis, which showed that ANAO-TE and crosslinked EP20-24-24 were more porous and of a lower density than human aortic elastin. More importantly, as mentioned previously, MS analysis revealed that cross-linking had only partially occurred in the case of the two constructs because many free K and k residues were identified in both substrates. 3.2. Studying the formation of cross-links using MD simulations In addition to the two peptides containing the bifunctional cross-link AA identified from TR digests of ANAO-TE and cross-linked EP20-24-24 (Table 1, P1 and P2), analogous linear peptides containing either K or k residues (Table 1, P3–P8) were modeled to gain insight into a possible way of formation of bifunctional cross-links. The peptides were analyzed on secondary structure elements over a 100 ns trajectory. Moreover, the distance between the Cε and Cδ atoms of either two k residues or two K residues within a peptide molecule was investigated. Regarding the evolution of Cε–Cδ distances in the linear peptides (P3– P8), it was found that it was strongly modified when K residues were replaced by k residues (Fig. 6). In the case of the k-containing elastin peptides P3 and P7, the distributions were shifted to the left (to smaller

A. Heinz et al. / Matrix Biology 38 (2014) 12–21

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Table 2 Secondary structure contents (%) of different linear elastin peptides determined applying the DSSP algorithm on the corresponding MD trajectories. Peptide

P1 P2 P3 P4 P5 P6 P7 P8

Fig. 6. Distributions of the distance between the Cε and Cδ of the lysine residues (K) or allysine residues (k). From top to bottom, pairs of linear peptides with and without allysine residues are considered. For each peptide, the mean distance between Cε and Cδ ± SD in Ǻ is shown in brackets.

distances), which means that the atoms were generally closer than in the K-containing peptides P4 and P8. This presumably facilitates the reaction of k residues during in vivo formation of cross-links. The smallest minimal distances of 3.2 Å and 3.3 Å were obtained for the k-containing peptides P3 and P7, respectively, which are the precursor peptides of the actually cross-linked peptides P1 and P2 that were identified in digests of the elastin-like constructs. Moreover, the lowest mean distance between the Cε and Cδ atoms was observed for P7 (7.7 Å ± 2.3 Å) and P8 (10.3 Å ± 2.4 Å), which is due to the fact that the two k/K residues are only three residues apart in the peptide chain, while the k/K residues in P3–P6 are eleven residues apart. Interestingly, for P5 and P6, where the P residue present in naturally occurring elastin is replaced by an A residue, a shift of the distribution to lower distances occurs for K-containing P6 (Fig. 6). Moreover, a narrowing of the width of the distribution is observed for k-containing P5 as compared to P6. Overall, this leads to a lower mean distance in the case of P5 (14.5 Å ± 2.1 Å), however, a smaller minimal distance in the case of P6 (4.7 Å). Visualization of the trajectories as well as the detailed analysis of local secondary structures were performed in parallel using the DSSP (define secondary structure of proteins) algorithm (Kabsch and Sander, 1983) as implemented in the GROMACS package (Table 2). It was found for both P7 and P8 that α-helical and β-turn structures formed within the 100 ns, which allowed the Cε and Cδ atoms of the k/K residues to be on the same side of the peptide backbone and come close together (Fig. 7A). The overall conformation of their cross-linked version P2 was found to be stable over 100 ns, showing α-helical structures and structures close to α-helices (Fig. 7B). Regarding the four longer peptides P3–P6, the results of DSSP analysis (Table 2) revealed that coiled-coil conformations predominated in all peptides (Fig. 7C). Striking differences concerned α-helix and 3-helix structures which

Conformation (%) Coil

β-Sheet

β-Bridge

Bend

Turn

α-Helix

3-Helix

52.7 52.8 39.5 6.5 5.1 3.9 47.9 49.5

1.3 5.1 0.1 0.2 1.5 0.2 0 0.4

1.7 1.6 0.2 0.9 2.3 1.7 2.2 4.5

29.9 12.1 22.9 24.5 40.8 35.4 14.7 22.9

8.7 17.6 17.1 9.4 4.1 23.2 34.4 17.7

2.1 2.8 14.1 0.1 0 0 0 0

3.5 8.0 6.1 0.3 0 0 0.8 4.9

were present only in P1 and P3 (the small amount detected in P4 is b1% and, therefore, not significant). Another important structure element that was observed in the cross-linked peptide P1 was a double β-turn comprising residues GVGTPA (Fig. 7D) (Tamburro et al., 2006). In peptides P3 to P6, turns were also detected, but not necessarily in the GVGTPA region. Turns were, however, found in adjacent regions covering residues located before the G or after the A residues. Replacing the P residue by a A residue (P5 and P6) has consequences on the average and local secondary structure that is adopted by the peptides. Overall, when studying the trajectories, these peptides are less flexible as compared to the P-containing peptides P3 and P4. As for the K-containing peptides, when the P residue (P4) is replaced by an A residue (P6), an increase of the amount of turns can be observed which reflects the change of secondary structure of the GTAAAAA region in comparison to the GTPAAAA region. This modification of the local secondary structure causes different locations of the K side chains with respect to the peptide backbone and, therefore, has an impact on the Cε–Cδ distances. When K residues (P6) are replaced by k residues (P5), the amount of turns decreases whereas the number of coil structures increases. This may be due to a competition between the stabilization through the backbone hydrogen bonds (turns) and the favorable interactions between the k side chains. The shift to lower Cε–Cδ distances (Fig. 6) as well as the narrowing of the distribution are associated with the predominance of k side chain interaction. Overall, these findings underline the importance of the GVGTPA hinge region for the formation of elastin cross-links. The results of this study are consistent with an MD simulation study which revealed that a peptide composed of domains 21 and 23 is able to adopt a closed hairpin conformation, which may even stabilized by salt bridge formation and hydrogen bonding (Djajamuliadi et al., 2009). As described in Section 3.1 above, conformations like this would bring the K residues 451 and 463 closer together prior to cross-linking. Moreover, it has been shown previously that the exchange of the GVGTP hinge residues by five A residues is accompanied by an increase in α-helical content (Miao et al., 2005), which means that the structure of this part of domain 23 is significantly altered and, therefore, cross-linking between domains 21 and 23 would be dramatically affected. The structural results obtained in this study suggest the following scenario concerning the formation of aldol cross-links: in regular elastin-like peptides, the side chains of k residues interact attractively and drive the backbone of the peptides to adopt helices and turns that place the Cε and Cδ of the side chain k residues on the same side of the backbone and at a distance short enough to create the internal crosslink. In peptides where the P residue is substituted by an A residue, no amount of helices has been found during the simulation of P5 (or P6). Since the minimal distance between Cε and Cδ atoms in P5 is more than twice as big as the one observed for P3, it seems unlikely that P5 is able to form internal cross-links. 4. Conclusion In this study, native aortic elastin and two cross-linked constructs based on either tropoelastin or shorter elastin domains were analyzed

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A. Heinz et al. / Matrix Biology 38 (2014) 12–21

Fig. 7. Snapshots of MD simulations of the peptides (A) P7, (B) P2, (C) P3 and (D) P1 shown in Table 1. N-termini of the peptides are shown in red and C-termini are shown in blue. K residues and AA cross-links are highlighted in black. Left-handed α-helical backbones are shown in red and turn structures in light blue.

microscopically and at the molecular level. Characterization of human aortic elastin revealed the absence of domains encoded by exons 24A and 26A as well as the possible absence of domain 22. With the aid of PolyLinX software, it was possible to sequence two cross-linked peptides containing intramolecular allysine aldols. One of these is an important cross-link present between lysine residues of domains 21 and 23, of which the latter contains a flexible hinge region comprising the residues GVGTP which facilitates the formation of a cross-link between lysine residues that are otherwise too far apart. Overall, the results of mass spectrometric measurements and MD simulations of this study, which confirm the structural flexibility of this region, underline the importance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Moreover, it was found that the two in vitro cross-linked materials contain the tetrafunctional cross-links DES/IDES characteristic of native elastin. In contrast to mature aortic elastin, however, the constructs show a lower degree of cross-linking and a more porous structure. Further investigations of their mechanical properties will be carried out in the near future to further compare the materials to native elastin and reveal possibilities for potential biomedical applications as biomaterials. Acknowledgments The work was supported by the German Research Foundation (DFG) grants HE 6190/1-1 and HE 6190/1-2 (A.H.), by the European Regional Development Fund of the European Commission (C.U.S.) and by the German Academic Exchange Service (DAAD) in the frame of the PROCOPE program. The authors thank Dr. Frank Heyroth (Interdisciplinary Center for Materials Science, Martin Luther University Halle-Wittenberg, Germany) for assistance with scanning

electron microscopy. Moreover, Dr. Arjen Sein (DSM Biotechnology Center, DSM Food Specialities B.V., Delft, Netherlands) is thanked for providing ANAO and for proof-reading the manuscript. Ruth Fritzsche (Institute of Pharmacy, Martin Luther University HalleWittenberg, Germany) is thanked for help with MS measurements and Prof. Ulrich Stock (University Hospital Frankfurt, Germany) for providing aortic punch biopsies. The authors thank the HPC-Regional Center ROMEO and the Multiscale Molecular Modeling Platform (P3M) from the University of Reims Champagne-Ardenne (France) for providing CPU time and support. A.S.W. acknowledges support from the Australian Research Council. References Akagawa, M., Suyama, K., 2000. Mechanism of formation of elastin crosslinks. Connect. Tissue Res. 41, 131–141. Almine, J.F.,Bax, D.V., Mithieux, S.M.,Nivison-Smith, L.,Rnjak, J., Waterhouse, A., Wise, S.G., Weiss, A.S., 2010. Elastin-based materials. Chem. Soc. Rev. 39, 3371–3379. Baig, K.M., Vlaovic, M., Anwar, R.A., 1980. Amino acid sequences C-terminal to the crosslinks in bovine elastin. Biochem. J. 185, 611–616. Baldock, C., Oberhauser, A.F., Ma, L., Lammie, D., Siegler, V., Mithieux, S.M.,Tu, Y., Chow, J.Y., Suleman, F., Malfois, M., Rogers, S., Guo, L., Irving, T.C., Wess, T.J., Weiss, A.S., 2011. Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity. Proc. Natl. Acad. Sci. U. S. A. 108, 4322–4327. Bellingham, C.M., Woodhouse, K.A., Robson, P., Rothstein, S.J., Keeley, F.W., 2001. Selfaggregation characteristics of recombinantly expressed human elastin polypeptides. Biochim. Biophys. Acta 1550, 6–19. Bellingham, C.M., Lillie, M.A., Gosline, J.M., Wright, G.M., Starcher, B.C., Bailey, A.J., Woodhouse, K.A., Keeley, F.W., 2003. Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties. Biopolymers 70, 445–455. Bochicchio, B., Ait-Ali, A., Tamburro, A.M., Alix, A.J.P., 2004. Spectroscopic evidence revealing polyproline II structure in hydrophobic, putatively elastomeric sequences encoded by specific exons of human tropoelastin. Biopolymers 73, 484–493.

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