Oxidative Modifications of the C-terminal Domain of Tropoelastin Prevent Cell Binding

Abstract

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin ␣ v ␤ 3 and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO ؊ ) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO ؊ treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO ؊ treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO ؊ prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.Elastic fibers are key extracellular matrix structures that provide the stretch and recoil properties of tissues such as arteries, lungs, and skin. Elastic fibers consist of two major components, elastin and microfibrils. Elastin is the predominant component of elastic fibers, comprising Ͼ90% of the total mass. Tropoelastin (TE), 2 the soluble precursor of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, endothelial cells, and smooth muscle cells (SMCs). Assembly of monomeric TE into elastic fibers is a multistep process. Upon secretion from cells, TE monomers organize into aggregates on the cell surface. These aggregates are then deposited onto pre-existing microfibrils. Microfibrillar components align the TE aggregates, which undergo cross-linking to form mature elastic fibers. Cell surface molecules, such as the 67-kDa elastin-binding protein (EBP), ␣ v ␤ 3 integrin, and glycosaminoglycans (GAGs), have been proposed to be involved in elastic fiber assembly either by promoting aggregation of TE monomers or by keeping the microfibrils close to the cell membrane through interactions with microfibrillar components (1-5).TE is mainly synthesized during late fetal and early postnatal stages of development. Synthesis of TE in normal adult tissues is negligible; however, in several cardiovascular and pulmonary diseases, such as atherosclerosis and emphysema/chronic obstr...

A Novel Cell Adhesion Region in Tropoelastin Mediates Attachment to Integrin αVβ5

“…GAG-mediated cell binding has been observed between HDFs and the C-terminal 25 amino acids of bovine tropoelastin. This is in contrast with our current results, as HS does not inhibit HDF attachment to human tropoelastin (12,21). The C termini of human and bovine tropoelastin share a high degree of homology in the domain encoded by exon 36; however, human tropoelastin does not contain domains 34 and 35, which are required in bovine tropoelastin for HS-dependent cell binding.…”

“…The overlapping construct containing domains 17-27 (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) confirmed that the major cell-binding site in N18 is in domains 17 and 18 of tropoelastin (Fig. 4A).…”

“…Their mapped shared sequence is domains 17-18, which are distinct from the EBPbinding region (domain 24 (14)), the GAG-binding site (C terminus (11)), or the RKRK/integrin ␣ V ␤ 3 -binding site (domain 36 (12)). (21). Furthermore, HS blocks GAG-mediated cell binding (11).…”

“…This GAG-mediated cell-binding activity is located in the C-terminal 25 amino acid residues of bovine tropoelastin. Heparan sulfate (HS)-mediated interactions have also been noted for binding of human fibroblasts and smooth muscle cells to bovine tropoelastin (21). Although GAG-tropoelastin interactions are well established for bovine tropoelastin, this same interaction has not been shown for human tropoelastin.…”

Colocalization in vivo and association in vitro of perlecan and elastin

“…The Voigt model provided an estimate on the thickness of the adsorbed material on the crystal surface (Voinova et al 1999). Use of a truncated form of tropoelastin devoid of its C-terminal region demonstrated the important role of the C-terminus in binding to HS-perlecan, and identified perlecan as the likely donor of HS chains (Akhtar et al 2011). The protein core of perlecan also mediated interactions with tropoelastin, so it is likely that this region also contributed to the assembly process between these protein moieties.…”