Tropoelastin Interacts with Cell-surface Glycosaminoglycans via Its COOH-terminal Domain

Abstract

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.The ability to interact with receptors or binding proteins on the cell surface is a property of all proteins of the extracellular matrix. This interaction provides important temporal and spatial signals to cells and influences such cellular processes as adhesion, migration, gene expression, proliferation, and apoptosis. In addition, several receptors function in ECM 2 assembly to directly or indirectly assemble monomers of ECM proteins into their functional polymeric form.Elastin is the major extracellular matrix protein capable of elastic recoil in tissues repeatedly subjected to cycles of reversible extension (1-3). It functions as an insoluble polymer made up of cross-linked tropoelastin molecules enmeshed in a network of filamentous microfibrils (4 -6). The secreted form of elastin, tropoelastin, is a highly cationic protein because of an enrichment of lysine residues that serve as precursors to covalent cross-links that form between and within tropoelastin monomers. Lysine side chains are modified through oxidative deamination of ⑀-amino groups that then condense with unmodified lysines or with other modified side chains to form a covalent linkage. It is important to note that almost all of the lysine residues in tropoelastin are modified to form cross-links in the mature protein. This changes the physical character of the protein from cationic to slightly anionic and hydrophobic in nature. In this respect, mature elastin has very different chemical and physical properties than does the precursor molecule tropoelastin.Like other ECM proteins, elastin has been shown to specifically interact with binding proteins on the cell surface (7-10). Binding occurs with high affinity and to multiple cell types, including bacteria (11). In responsive cells, elastin has been shown to alter gene expression, influence mobilization of intracellular ions, modulate cell adhesion and movement, effect cell proliferation, and to induce actin polymerization (7,(12)(13)(14)(15)(16)(17)(18). There is evidence from multiple studies that implicate changes in cal...

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3

“…In contrast to other data, regions encoded by the N-terminal exons (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), the central exons (18 -27), and the C-terminal exons (18 -36) all supported human umbilical vein endothelial cell attachment.…”

“…Other studies have proposed a second mechanism involving the necessity of cell surface heparan and chondroitin sulfatecontaining glycosaminoglycans for bovine chondrocyte interaction with bovine tropoelastin (14). Peptide binding analysis implicated the last 17 amino acids at the C terminus of bovine tropoelastin in this cell adhesive activity, with higher binding requiring the C-terminal 25 amino acids.…”

“…This region is of interest, as in humans a mutation of Gly-773 to Asp in exon 33 results in blocked elastin network assembly and modulates cell binding to a peptide corresponding to exons 33 and 36 of human tropoelastin (28). Indeed Broekelmann et al (14) have shown that synthetic peptides containing the C-terminal 29 amino acids of bovine tropoelastin possess cell adhesive activity; however, when the G773D mutation was incorporated into the peptide, it prevented cell adhesion to that peptide.…”

“…Elastin is the major protein in large elastic blood vessels such as the aorta, where it is likely to inhibit the proliferation of vascular smooth muscle cells and so preventing vessel occlusion (11), which is a major cause of death in developed countries. Previous studies have shown that human and bovine tropoelastin can bind directly to a variety of cell types directly through a number of cell surface receptors (12)(13)(14) and also bind indirectly to cells through ECM proteins such as fibulin-5 (15,16).…”

Elastin Based Constructs

“…Glycosaminoglycans on the SMC and chondrocyte cell surface dominate binding to the C-terminus of bovine tropoelastin (Broekelmann, Kozel et al 2005;Akhtar, Broekelmann et al 2011). Cell interactions with human tropoelastin C-terminus specifically occur through the integrin v 3 (Rodgers and Weiss 2004;Bax, Rodgers et al 2009).…”