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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Matrix Biol. Author manuscript; available in PMC Sep 1, 2009.
Published in final edited form as:
PMCID: PMC2586719
NIHMSID: NIHMS79121
Modification and Functional Inactivation of the Tropoelastin Carboxy-terminal Domain in Cross-linked Elastin
Thomas J. Broekelmann, Christopher H. Ciliberto, Adrian Shifren, and Robert P. Mecham
Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO
Corresponding author: Dr. Robert P. Mecham Washington University School of Medicine Department of Cell Biology and Physiology 660 South Euclid Ave, Campus Box 8228 St. Louis, MO 63110 Email: bmecham/at/wustl.edu
The carboxyl terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is ~0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysine-derived cross-link in this region, which would alter the extractability and antigenicity of the carboxy-terminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.
Keywords: elastin, elastic fiber assembly, tropoelastin, exon-36, elastin diseases
Elastin is the major extracellular matrix protein capable of elastic recoil in tissues subjected to repeating cycles of extension and contraction; such as the aorta, skin, and lung. The secreted form of elastin (tropoelastin) is a protein comprised of alternating hydrophobic and lysine rich sequences (Bressan et al., 1987; Mithieux and Weiss, 2005). All but ~10% of the 35-40 (depending on species) lysine residues are modified to form unique bifunctional or tetrafunctional cross-links that covalently link monomeric tropoelastin molecules into the mature, functional polymer (Franzblau et al., 1969; Gerber and Anwar, 1974). The mature protein is insoluble, hydrophobic in character, resistant to most proteases, and has a very low turnover rate (Partridge, 1962; Shapiro et al., 1991).
How tropoelastin monomers assemble prior to crosslinking is still not completely understood. Like collagen, tropoelastin monomers are capable of self-association but proper elastic fiber assembly requires the participation of both cells and ancillary proteins. Functional mapping studies identified domains in the C-terminal half of tropoelastin that facilitate fiber assembly. Tropoelastin lacking the sequence in domain1 30, for example, does not interact with microfibrils in the extracellular matrix and, hence, does not assemble into fibers (Kozel et al., 2003). Mutations in the elastin gene that delete this region of the protein are associated with supravalvular aortic stenosis, an autosomal dominant disease arising from elastin haploinsufficiency (Li et al., 1997; Urban et al., 2000). A second important assembly site is located at the C-terminus of the molecule and is encoded by exon 36, the last exon in the gene. Antibodies directed to this domain prevent elastin assembly (Brown-Augsburger et al., 1996) and deletion of this sequence leads to a dramatic reduction in levels of cross-linked elastin in in vitro culture systems (Hsiao et al., 1999). This region of the protein is highly conserved (Chung et al., 2006) and interacts with microfibrillar proteins (Brown-Augsburger et al., 1994), integrins (Rodgers and Weiss, 2004) and sulfated proteoglycans (Broekelmann et al., 2005). An autosomal dominant form of cutis laxa has been linked to mutations that alter the sequence of this region of the molecule (Szabo et al., 2006; Zhang et al., 1999).
The sequence encoded by domain-36 is unique; it is not a typical crosslinking or hydrophobic domain, but it does contain a hydrophobic cluster at the N-terminal end of the sequence as well as a cluster of positively charged amino acids (usually RKRK) at the C-terminus. This sequence also contains the protein's only two cysteine residues, which are disulfide bonded to form an intrachain loop structure (Brown et al., 1992; Floquet et al., 2005). How this region of the protein facilitates elastin assembly is still not known. Based on pulse-chase studies of tropoelastin production/degradation by rat aortic smooth muscle cells, it was proposed that the C-terminal region is important in directing fiber assembly similar to the role played by extension peptides of the fibrillar collagens (Chipman et al., 1985; Franzblau et al., 1989). Pulse-chase data suggested that after facilitating incorporation of the tropoelastin molecule into the growing elastic fiber, the C-terminal “pro” peptide is removed by a specific tryptic-like protease and is not incorporated into the mature elastic structure. Other investigators, however, have identified C-terminal sequences in solubilized peptides from insoluble elastin using specific antibodies (Rosenbloom et al., 1986) or by mass spectrometry (Getie et al., 2005; Schmelzer et al., 2005), implying that the C-terminus may not be processed off during fiber assembly.
The status of the carboxyl terminus in mature elastin is important. Its presence or absence in the mature protein will provide important clues as to its role in fiber assembly. Furthermore, as others and we have shown, the C-terminal sequence can interact with adhesive receptors on cells and, in this capacity, could provide a substrate for cellular adhesion to elastic fibers. Alternatively, the sequence could serve a signaling function, particularly if liberated from the insoluble polymer by proteolytic events associated with inflammation or tissue remodeling. Indeed, elastin peptides have been shown to recruit inflammatory cells to regions of damage in the lung (Houghton et al., 2006) and are responsible for progression of vascular injury in animal models of aneurysmal disease (Hance et al., 2002).
In this report, we use a domain-36-specific antibody to evaluate the presence or absence of the C-terminal sequence in mature elastin. We show that the sequence recognized by the antibody is present but underrepresented in mature elastin, and modified most likely due to crosslinking of one or more of the lysine residues.
2.1 Antibody Specificity for C-terminal Sequence
A key reagent in our identification and quantification of the C-terminal domain in elastin is an antibody generated to a synthetic peptide containing a sequence encoded by exon 36 of bovine tropoelastin (anti-CTP) (Brown-Augsburger et al., 1996). This antibody blocks the association of tropoelastin with microfibrils and decreases the amount of cross-linked elastin when added to cells in vitro (Brown-Augsburger et al., 1996). Hence, the antibody recognizes an epitope important for elastic fiber assembly. To show that the antibody is specific for elastin and only for the C-terminal sequence to which it was generated, we undertook an extensive characterization of specificity. Figure 1 is an immunoblot showing that the CTP antibody reacts with a single band of appropriate size for tropoelastin (~65 kDa) in a cell extract of FBAF cells (Panel A lane 1). Reactivity of the antibody to this band is completely blocked by pre-incubation with the synthetic peptide to which the antibody was produced (Panel A lane 2).
Figure 1
Figure 1
The CTP antibody specifically reacts with the C-terminus of tropoelastin in western blots
Western blot analysis using bacterially produced 6-His tagged and purified fragments of tropoelastin shows reactivity only with molecules that contain domain-36 at the C-terminus. The first panel in Figure 1B is a Coomassie-blue stained gel confirming that approximately equivalent amounts of purified protein were loaded for each fragment. The second panel is a western blot using the CTP antibody and shows reactivity to full-length tropoelastin (domains 1-36), domains 15-36 and domains 29-36, but no reactivity to fragments lacking the C-terminus (domains 1-15 and 15-29). The third panel is the negative control for the immunoblot using the CTP antibody blocked by pre-incubation with the immunizing peptide.
Figure 2A shows a series of ELISA dilution curves for the CTP antibody on plates coated with different bacterially expressed tropoelastin fragments. The half maximal reactivity for this antibody is approximately 160 μg/ml for substrates coated with full-length tropoelastin and 80 μg/ml for substrates coated with either the domain 15-36 or the domain 29-36 fragments. The antibody does not react with substrates coated with domain 1-15 or domain 15-29 fragments. Similar results are obtained when the ELISA plates are coated with synthetic peptides containing sequences at the C-terminus of tropoelastin (Figure 2B). Half maximal reactivity occurs at approximately 80 μg/ml for a substrate coated with the CT-25 peptide and at 100 μg/ml for a substrate coated with the CT-14 peptide. There is slight antibody reactivity towards the e35 peptide and minimal reactivity towards a scrambled e35-36s peptide. Together, the western blot and ELISA assays show that the CTP antibody is specific for the C-terminal sequence.
Figure 2
Figure 2
Reactivity of the CTP antibody for the domain-36 sequence is confirmed by ELISA assay
2.2 Epitope mapping
Fine mapping of the anti-CTP epitope was achieved using a competitive immunoassay comparing competition between tropoelastin and various synthetic peptides (Table I) containing permutations of the C-terminal sequence. As expected, soluble tropoelastin blocks anti-CTP binding to a tropoelastin-coated substrate (Figure 3A). Similarly, the CT-25 peptide in solution fully competes, at the same molar concentration as tropoelastin, for anti-CTP binding to the tropoelastin substrate. Using a substrate coated with the CT-25 peptide, soluble CT-25 fully blocked anti-CTP binding whereas the CT-14 peptide blocked binding by ~75% (Figure 3B). When the substrate was coated with the CT-14 peptide, both CT-25 and CT-14 peptides fully blocked anti-CTP binding (data not shown). This confirms that the terminal 25 residues contain all the major epitopes for the anti-CTP antibody. The major epitopes are in the terminal 14 residues (encoded by exon 36), but there is a minor contribution by the 11 residues at the N-terminus of CT-25 that are contributed by exon 35 (exon 33 in human). The control scrambled e36-36s peptide failed to inhibit anti-CTP binding to a CT-25 coated plate.
Table 1
Table 1
Synthetic peptides used for epitope mapping of the CTP antibody. Sequences of domains 35 and 36 are provided for reference.
Figure 3
Figure 3
Fine mapping of anti-CTP epitope by competition assay
2.3 Protease sensitivity of anti-CTP epitope
The susceptibility of the CTP epitope in tropoelastin to elastase treatment was determined by comparing the antigenic reactivity of elastase-digested tropoelastin with tropoelastin not exposed to protease. Elastase digestion did not decrease the reactivity of tropoelastin to the CTP antibody even through the protein was digested to small fragments (Figure 4A).
Figure 4
Figure 4
The epitope for the anti-CTP antibody in tropoelastin and in the CT-25 peptide is insensitive to elastase treatment
The stability of the CTP epitope was further characterized by assessing the effects of pancreatic elastase, pepsin, and trypsin on CT-25 peptide (Figure 4B). Anti-CTP immunoreactivity was eliminated from the peptide by a 15-minute exposure to trypsin and reduced by half after prolonged pepsin treatment. Treatment with elastase, however, had no effect on the immunological properties of the CT-25 peptide, even though it was cleaved by elastase at multiple sites (Figure 5). Characterization of the elastase-derived fragments by HPLC and mass spectrometry identified cleavage sites on either side of the alanine at position 7, N-terminal to the glycine at position 9, and C-terminal to the alanine at position 14. These findings show that elastase can be used to solubilize insoluble elastin without compromising the reactivity of the C-terminal epitope for the CTP antibody.
Figure 5
Figure 5
The CT-25 peptide is cleaved at multiple sites by pancreatic elastase
2.4 CTP in insoluble elastin
The stability of the anti-CTP epitope makes it possible to quantify the amount of CTP in elastin following solubilization by elastase. Figure 6A shows the amount of elastin solubilized from 50 mg neutral salt purified elastin (~770 nmoles using 65,000Da for the molecular mass of tropoelastin) by different concentrations of pancreatic elastase. Soluble protein concentrations were determined by amino acid analysis of the reaction supernatant. A 500μg dose of elastase visibly solubilized all of the elastin in this assay, and increasing the dose to 1mg elastase failed to increase the amount of solubilized elastin as determined by amino acid analysis or competitive ELISA. This confirms that elastin was completely solubilized at elastase levels of 500 μg and higher. Complete solubilization was also confirmed by recovery of desmosine in the reaction supernatant (120 ng desmosine/mg protein, which is equivalent to desmosine levels measured in insoluble elastin prior to enzymatic digestion).
Figure 6
Figure 6
Elastase solubilization of the C-terminal fragment from mature elastin
Figure 6B shows immunoreactive CTP in the same sample as determined by competitive ELISA. Liberation of the CTP epitope by elastase followed the same kinetics observed for solubilization of elastin based on total protein (Figure 6A). However, the total amount of CTP measured by competitive ELISA is less than what would be expected based on the total amount of elastin. Our recovery of ~1.5 nmoles of CTP epitope is well below the expected value of 770 nmoles that is assumed if each tropoelastin monomer incorporated into the insoluble polymer has an intact C-terminus. Expressed as a percentage, the amount of immunologically reactive C-terminus is ~0.2% of the expected value, which corresponds to one CTP epitope per 455 molecules.
2.5 Relative content of the C-terminus in elastin isolated using different procedures
To determine if the relatively harsh procedures typically used to purify insoluble elastin contribute to the low CTP levels, we assessed the amount of anti-CTP reactivity in elastase digests of several common elastin preparations as well as in crude ligamentum nuchae. Figure 7 shows that the amount of CTP expressed as a percent of total elastin protein is low (0.1-0.3%) for all samples. This includes crude ligamentum nuchae, which has not undergone extraction. Interestingly, no detectable CTP epitope was evident in elastase digests of elastin purified by hot base hydrolysis (Lowry et al., 1941). This result is not unexpected since hot NaOH is known to introduce extensive peptide bond cleavage and protein degradation into the purified polymer (Daamen et al., 2001; Soskel and Sandburg, 1983; Starcher and Galione, 1976).
Figure 7
Figure 7
Relative content of immunoreactive C-terminus in elastin purified using different protocols
2.6 Remaining CTP epitope in insoluble elastin is trypsin insensitive
The Starcher method for elastin isolation begins with elastin purified by neutral salt extraction and then uses trypsin and CNBr digestion followed by extraction with urea and dithiothreitol (Starcher and Galione, 1976). The results in Figure 7 indicate that Starcher elastin contains the same amount of CTP epitope as neutral salt elastin, even though our studies with CT-25 show that trypsin digestion is capable of destroying the CTP epitope. To verify that the CTP antibody-reactive peptides released from insoluble elastin by elastase are resistant to trypsin treatment, neutral salt elastin was solubilized by elastase digestion and fractionated by reverse phase HPLC. Fractions with reactivity toward CTP antibody were pooled, treated with trypsin, and assayed for immunoreactivity by ELISA. Figure 8 confirms that the small amounts of CTP epitope obtained from mature elastin is not destroyed by trypsin digestion.
Figure 8
Figure 8
The epitope for the anti-CTP antibody in elastase-derived fragments from mature elastin is insensitive to trypsin digestion
Assessing the presence or absence of the C-terminal sequence in insoluble elastin has been problematic. It was originally proposed that this region served a pro-peptide function, similar to the pro-peptides of collagen, and was removed concurrent with assembly (Chipman et al., 1985; Franzblau et al., 1989). Other evidence, however, suggested that it is retained in the crosslinked polymer. Several biochemical studies have tried to use the cysteine residues unique to this region as markers during peptide purification (Price et al., 1993), but contaminants from cysteine-rich microfibrils make this approach difficult. Another complicating factor is suggested by our finding that purification of elastin using hot NaOH modifies or removes the C-terminal sequence. Thus, the nature of the starting material one chooses to study is perhaps more important than previously appreciated. Our findings described in this report suggest that the domain-36 sequence is, in one form or another, present in mature elastin that has not be prepared using NaOH. However, the low levels that we detect using a quantitative immunological approach suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. It is also important to note that low levels of CTP were detected in unprocessed ligamentum nuchae, a tissue highly enriched in elastin. This suggests that CTP modification occurs coincident with the incorporation of tropoelastin into the mature polymer. As mentioned above, domain-36 of tropoelastin has properties that are important for the biological activity of the molecule. The results shown here indicate that there is little or no intact, unmodified domain-36 in mature elastin. Thus, the cell and matrix interaction properties found in this region of tropoelastin are lost as elastin matures.
The quantitative recovery of CTP activity when tropoelastin or CT-25 is treated with elastase (Figure 4) confirms that the estimate of CTP levels in insoluble elastin are not artifactually low due to inactivation of the antigenic epitope by elastase digestion. The question then becomes whether something other than removal of the CTP during assembly can explain the low levels. The presence of the CTP epitope in Starcher-purified elastin is particularly informative in this regard. Starcher elastin, which utilizes trypsin treatment in the purification protocol, contains the same amount of CTP epitope as neutral salt purified elastin even though trypsin digestion is capable of destroying the CTP epitope. Resistance to trypsin digestion suggests that one or more of the lysine residues normally recognized by trypsin is modified in the domain-36 sequence once tropoelastin is incorporate into the insoluble polymer. The most likely modification is crosslinking to another lysine residue, which is supported by studies of Mithieux et al (2005) who demonstrated that lysines in the RKRK (domain-36) sequence of purified tropoelastin are particularly sensitive to lysyl oxidase modification and are capable of forming a bifunctional cross-link with lysine elsewhere in the molecule. A high crosslinking frequency associated with lysines in domain-36 was also demonstrated using bifunctional crosslinkers (Wise et al., 2005). Lysine alteration through cross-link formation would likely alter the antigenicity of the CTP epitope and covalently lock the CTP into the mature polymer. Hence, epitope modification and low extractability would explain the low levels of CTP reactivity found in the elastase digests. It is also possible that the C-terminal region is removed from some molecules during assembly, while that which remains participates in a crosslink.
Our studies are similar to those of Rosenbloom et al. (1986) who, using an antibody generated to a similar C-terminal peptide, found reactivity in neutrophil elastase digests of insoluble elastin. Other than demonstrating the presence of the C-terminal epitope amongst the solubilized peptides, the amount of recovered peptide was not quantified. It is interesting to note that these authors identified CTP reactivity in digests of NaOH-treated elastin whereas we did not. Nevertheless, both studies support the presence of C-terminal sequences in insoluble elastin. Also consistent with retention of the CTP in mature elastin are mass spectrometry studies that have identified fragments of the CTP in proteolytic digests. Getie et al. (2005), for example, identified a peptide in an elastase digest of skin elastin that contains the residues N-terminal to the first cysteine in domain-36, which is consistent with our finding of an elastase cleavage site at this sequence (Figure 5). An analysis of a thermolysin digest of the same elastin identified sequence immediately upstream of RKRK, including the two cysteine residues (Schmelzer et al., 2005). In neither study, however, were the lysine residues in domain-36 identified, which is consistent with their modification to form cross-links.
The presence of a cross-link in domain-36 suggests how this domain may play a critical role in elastin assembly. Expression of tropoelastin deletion constructs in vitro and as transgenes in mice have identified sequences encoded by exon 30 as being critical for the self-association of tropoelastin molecules (Kozel et al., 2003). Tropoelastin constructs lacking domain-36, however, were able to associate with existing elastic fibers, but crosslinking was greatly attenuated (Hsiao et al., 1999; Kozel et al., 2003; Sato et al., 2007). These studies support a multistep process for elastin assembly that involves tropoelastin self-association (mediated by domain 30) followed by crosslinking and maturation mediated by domain-36 (Czirok et al., 2006; Kozel et al., 2004; Kozel et al., 2006; Sato et al., 2007). The data from the current study are consistent with this model and suggest that domain-36 may function to facilitate fiber maturation by forming an initial cross-link that serves to help register the multiple tropoelastin crosslinking sites for efficient oxidation by lysyl oxidase. A crosslinking function would explain why mutations that alter the domain-36 sequence, such as frame shift mutations associated with dominant cutis laxa (Szabo et al., 2006; Zhang et al., 1999), have detrimental effects on elastic fiber assembly.
4.1 Insoluble elastin and synthetic peptides
Crude defatted bovine ligamentum nuchae and bovine insoluble elastin prepared using the Lansing (Lansing et al., 1951), Starcher (Starcher and Galione, 1976), and neutral salt extraction techniques were obtained from Elastin Products Company. All peptides were synthesized using an ABI-431A synthesizer with FastMoc chemistry on Wang-capped resins. After cleavage, peptides were dissolved in MilliQ water with 0.05% trifluoroacetic acid and purified with reverse phase HPLC2 on a Vydak 218TP1022 C-18 column using a linear 0-50% acetonitrile gradient. Peak fractions were dried by rotary evaporation, dissolved in MilliQ water then lyophilized. Peptides containing cysteine were dissolved in water and disulfide bonds were allowed to form before use (Brown-Augsburger et al., 1996). LC-mass spectrometry performed by the Protein and Nucleic Acid Chemistry Laboratory at Washington University Medical School verified the identity of each purified peptide. Protein and synthetic peptide concentrations were determined by amino acid analysis using a Beckmann 6300 high performance amino acid analyzer. Synthetic peptide sequences are listed in Table I. The CTP peptide was used to generate the anti-CTP antibody and is the C-terminal 19 residues of elastin, CT-25 includes the C-terminal 25 residues, and the CT-14 peptide includes the C-terminal 14 residues of elastin. The e35 peptide includes 11 residues from domain 35, and the e35-36s peptide is a scrambled peptide from domain 35-36 sequences.
4.2 SDS-PAGE, antibodies and western blots
Anti-CTP (Brown-Augsburger et al., 1996) is a rabbit polyclonal raised against a synthetic peptide from the C terminus of elastin (CTP, Table I) and purified by the caprylic acid method of Steinbuch (Steinbuch and Audran, 1969). SDS-PAGE and Western blot analyses were performed as described (Brown-Augsburger et al., 1996) using enhanced chemiluminescent reagents (ECL from GE Healthcare) to detect horseradish peroxidase conjugated secondary antibodies (donkey anti-rabbit horseradish peroxidase from GE Healthcare).
4.3 Expression and purification of tropoelastin and fragments
Tropoelastin and tropoelastin fragments were expressed as 6-His fusion proteins using the pQE expression system (Qiagen, Inc.) in the M-15 strain of E. coli (Broekelmann et al., 2005). Expressed proteins were initially purified using nickel chromatography under denaturing conditions with a batch purification method recommended by the manufacturer. Eluted tropoelastin or tropoelastin fragments were dialyzed against 50mM acetic acid and lyophilized. The proteins were further purified using reverse phase HPLC chromatography on a Vydak 214TP510 C-4 column using a 0-50% linear acetonitrile gradient.
4.4 Cells
Fetal bovine adventitial fibroblasts (FBAF) were obtained from the explanted adventitial layer dissected from the aorta of 170-day gestation fetal calf (Wrenn et al., 1986). Cells were maintained in DMEM + 20% fetal calf serum (Biomeda) and subcultured 1:3 weekly.
4.5 Direct ELISA
EIA plates (96 well, Corning) were coated with various protein fractions at a concentration of 25μg/ml in 0.01M sodium carbonate pH=9.6. The plates were washed and blocked with PBS containing 1% nonfat dry milk, 0.1% fish gelatin (Sigma chemical G-7765), and 0.1% TWEEN 20 (PBS-milk). Dilutions of anti-CTP were added for 1 hour at room temperature, non-bound antibodies were removed and bound antibodies detected with secondary donkey anti-rabbit IgG-horseradish peroxidase conjugates (GE Healthcare). The amount of secondary conjugate bound was determined with the peroxidase substrate 2,2′-azino-di-(3-ethylbenzthiaaoline-6-sulfonate (ABTS) (KPL Labs) at 410nm.
4.6 Competitive ELISA
EIA plates were coated with 150nM CT-25 peptide (Table 1) or 25μg/ml tropoelastin in 0.01M sodium carbonate pH=9.6. The plates were washed and blocked with PBS-milk. The soluble competitor (diluted in PBS-milk) and 100μg/ml (final concentration) anti-CTP were added to the plate. After one hour, the amount of bound anti-CTP was quantified as above. Known concentrations of soluble CT-25 were used to generate a standard curve.
4.7 Protease digestions
Bovine pancreatic trypsin (TPCK treated, Fluka) and porcine pancreatic elastase (PPE) (EC134, Elastin Products Company) were added to substrate at 1:100 enzyme to substrate ratio (w:w) unless otherwise indicated in 10mM HEPES, 1mM CaCl2 pH=7.4. The reactions were stopped with the addition of phenylmethylsulfonylfluoride (PMSF) to 1mM. Pepsin (Sigma) digestion was performed in 10mM acetic acid at an enzyme to substrate ratio of 1:100 (w:w). The reaction was stopped by the addition of 1/10 volume of 1M Tris pH=8.8. HPLC purification of CT-25 fragments generated by PPE digestion was performed using reverse phase chromatography with a 4.6 × 25 cm C18 column (Beckman Ultrasphere ODS 235320) using a 0-50% acetonitrile gradient.
To determine the trypsin sensitivity of anti-CTP reactive fragments from insoluble elastin, 0.5 g neutral salt purified elastin was digested with 10mg PPE for 1 hour at 37°C. Soluble fragments were purified with reverse phase HPLC chromatography on a Vydak 218TP1022 C-18 column using a linear 0-50% acetonitrile gradient. Fractions immunoreactive with the CTP antibody were pooled, dried by rotary evaporation, and total protein determined by amino acid analysis. 10 mg of the sample was then digested for 1 hour at 37°C with 0.1mg trypsin. A control sample was treated the same but the trypsin was omitted. The reaction was stopped with the addition of PMSF to 1mM. The trypsin digested elastin or control elastin fragments were initially diluted to 1 mg/ml in 0.01M carbonate buffer then serially diluted and coated onto an ELISA plate. After blocking and washing, 100 μl of anti-CTP at 100 μg/ml was added for 1 hour at room temperature. Non-bound antibodies were removed and bound antibodies were detected with secondary donkey anti-rabbit IgG-horseradish peroxidase conjugates (GE Healthcare). The amount of secondary conjugate bound was determined colorimetrically with the peroxidase substrate ABTS at 410nm. The positive control for trypsin activity was the loss of the anti-CTP epitope in the CT-25 peptide.
4.8 Desmosine and isodesmosine determination
Elastin content in crude ligament or purified elastin was calculated from desmosine and isodesmosine levels determined by amino acid analysis using a Beckman 6300 high performance amino acid analyzer (Brown-Augsburger et al., 1995).
Acknowledgement
We thank Jessica Wagenseil, Clarissa Craft, and Hideki Sugitani for helpful discussion and Patricia Brown-Augsburger for preparation of the C-terminal antibody. We also thank Terese Hall for administrative assistance. The research reported in this manuscript was supported by National Institutes of Health HL71960, HL74138, and HL53325 to R.P.M.
Footnotes
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1Domain numbers refer to sequences encoded by individual exons in the elastin gene.
2Abbreviations: ABTS, 2,2’-azino-di-(3-ethylbenzthiaaoline-6-sulfonate; FBAF, fetal bovine adventitial fibroblasts; CTP, carboxyl terminal peptide; HPLC, high pressure liquid chromatography; AAA, amino acid analysis; ELISA, enzyme linked immunosorbent assay; PPE, porcine pancreatic elastase; PMSF, phenylmethylsulfonylfluoride.
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