Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

doi:10.1016/j.matbio.2008.06.001

Matrix Biology

Volume 27, Issue 7, September 2008, Pages 631-639

https://doi.org/10.1016/j.matbio.2008.06.001 Get rights and content

Abstract

The carboxy-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.

Introduction

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.

Section snippets

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

Discussion

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

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

Acknowledgement

We thank Jessica Wagenseil, Clarissa Craft, and Hideki Sugitani for helpful discussion and Patricia Brown-Augsburger for the preparation of the C-terminal antibody. We also thank Terese Hall for the administrative assistance. The research reported in this manuscript was supported by National Institutes of Health HL71960, HL74138, and HL53325 to R.P.M.

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Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

Abstract

Introduction

Section snippets

Antibody specificity for C-terminal sequence

Discussion

Insoluble elastin and synthetic peptides

Acknowledgement

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Matrix Biol.

Biomaterials Biomaterials

Matrix Biol.

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J. Biol. Chem.

J. Vasc. Surg.

J. Biol. Chem.

Matrix Biol.

J. Biol. Chem.

Adv. Protein Chem.

J. Struct. Biol.

Matrix

Biochimie

Coll. Relat. Res.

J. Mol. Biol.