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

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. 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 2The abbreviations used are: ECMextracellular matrixGAGglycosaminoglycanHSheparan sulfateCSchondroitin sulfateDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminFBCfetal bovine chondrocytesCHOChinese hamster ovary. assembly to directly or indirectly assemble monomers of ECM proteins into their functional polymeric form. extracellular matrix glycosaminoglycan heparan sulfate chondroitin sulfate Dulbecco's modified Eagle's medium bovine serum albumin fetal bovine chondrocytes Chinese hamster ovary. Elastin is the major extracellular matrix protein capable of elastic recoil in tissues repeatedly subjected to cycles of reversible extension (1Mecham R.P. Heuser J.E. Cell Biology of Extracellular Matrix. Plenum Press, Inc., New York1991: 79-109Crossref Google Scholar, 2Partridge S.M. Adv. Protein Chem. 1962; 17: 227-302Crossref Scopus (126) Google Scholar, 3Rosenbloom J. Abrams W.R. Mecham R.P. FASEB J. 1993; 7: 1208-1218Crossref PubMed Scopus (506) Google Scholar). It functions as an insoluble polymer made up of cross-linked tropoelastin molecules enmeshed in a network of filamentous microfibrils (4Albert E.N. Am. J. Pathol. 1972; 69: 89-102PubMed Google Scholar, 5Cleary E.G. Gibson M.A. Int. Rev. Connect. Tissue Res. 1983; 10: 97-209Crossref PubMed Google Scholar, 6Ross R. J. Histochem. Cytochem. 1973; 21: 199-208Crossref PubMed Scopus (200) Google Scholar). 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 ϵ-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 (7Blood C.H. Sasse J. Brodt P. Zetter B.R. J. 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Chem. 1989; 264: 10614-10620Abstract Full Text PDF PubMed Google Scholar). Karnik et al. (13Karnik S.K. Brooke B.S. Bayes-Genis A. Sorensen L. Wythe J.D. Schwartz R.S. Keating M.T. Li D.Y. Development. 2003; 130: 411-423Crossref PubMed Scopus (370) Google Scholar) found that an elastin-derived peptide activates a pertussis toxin-sensitive G-protein-coupled pathway that stimulates Gαi, inhibits adenylate cyclase, reduces cAMP levels, and stimulates Rho-induced actin polymerization. Other studies have found that elastin peptides signal through a Ras-independent mechanism requiring both p110g/Raf-1 and protein kinase A/B-Raf (23Duca L. Lambert E. Debret R. Rothhut B. Blanchevoye C. Delacoux F. Hornebeck W. Martiny L. Debelle L. Mol. Pharmacol. 2005; 67: 1315-1324Crossref PubMed Scopus (45) Google Scholar). The sequence in elastin assumed responsible for these activities is the hexapeptide VGVAPG and similar sequences (24Grosso L.E. Scott M. Arch. Biochem. 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Other documented elastin-binding proteins include a 120-kDa protein (elastonectin) up-regulated in the presence of fragments of insoluble elastin (9Hornebeck W. Tixier J.M. Robert L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5517-5520Crossref PubMed Scopus (78) Google Scholar), and a 59-kDa VGVAPG-binding protein identified on tumor cells (7Blood C.H. Sasse J. Brodt P. Zetter B.R. J. Cell Biol. 1988; 107: 1987-1993Crossref PubMed Scopus (89) Google Scholar). Elastin does not contain an RGD sequence and does not interact with most RGD-binding integrins, although αvβ3 integrin has been shown to bind to tropoelastin in solid phase binding assays (29Rodgers U.R. Weiss A.S. Biochimie (Paris). 2004; 86: 173-178Crossref PubMed Scopus (94) Google Scholar). Whereas numerous biological activities have been demonstrated for VGVAPG and similar hydrophobic peptides from insoluble elastin, one thing these hydrophobic sequences not well is cell adhesion. in cell and that with a that is different from that the VGVAPG This a specific cell interaction activity to a highly at the terminus of with of integrin receptors and of elastin-binding protein as binding to this Through the use of and mutant cell lines defective in glycosaminoglycan the of the COOH-terminal binding as heparan sulfate and chondroitin sulfate cell-surface with a for chondrocytes R.P. 1987; PubMed Scopus Google Scholar) from a fetal were in serum and The cells were used and were the use in adhesion Chinese hamster cells and their in cellular J.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and surface were in fetal serum and the use in adhesion with were with bovine serum albumin then at for with of use in adhesion and of and and tropoelastin fragments were as proteins the in the of proteins were chromatography and the to the tropoelastin or tropoelastin fragments were and were phase high chromatography on a a were by in then that the fragments were both and tropoelastin from the bovine C into the a in of the as P.W. L.E. Rosenbloom J. Abrams W.R. Mecham R.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were from the The a and amino residues The at the and to the of the molecule and amino residues and the and amino residues The by and amino residues The of residues is on the bovine with the J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). The bovine C has and peptides were on an on peptides were in with and with phase high chromatography on a a were by in then were in and were to form use Mecham L. C. Mecham R.P. Biochem. Biophys. Res. PubMed Scopus Google Scholar). that the peptides were chromatography with a by the Protein and at the of peptide and the presence of The of the peptide residues is on the bovine with the J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). The COOH-terminal peptide the sequence in and to the of the molecule The other and residues and The peptides are on the peptide and are as the the tropoelastin the the last in elastin Protein and peptide were by amino a high amino Cell adhesion to protein and peptide as J. Cell Sci. 2002; PubMed Google Scholar). protein fragments were at in The were with in the and cells in were and the at for The were and cells by at for cells were as a U. J. 1984; 67: PubMed Scopus Google Scholar) at and to peptides with the peptides in then with the cells to the adhesion and the at for cells were and cells as adhesion used to adhesion between the different cell and and adhesion to serum as and adhesion to as peptides with adhesive activity were to the of cells or to the with the of integrin activity not alter cell adhesion to adhesion of fetal bovine chondrocytes to peptide or serum in the presence of of the integrin peptide or the adhesion of fetal bovine chondrocytes to or in the presence or of adhesion of fetal bovine chondrocytes to or serum in the presence of of the of cell on The of fetal bovine chondrocytes to in the or presence of the integrin The the In cell adhesion assays were as a and results are as at for the elastin not cell adhesion to of fetal bovine chondrocytes to in the presence of of elastin peptides or and sugars Cell adhesion as a and results are as at adhesion to the terminus of tropoelastin. adhesion of to with in the presence of of chondroitin chondroitin and sulfate adhesion is by the presence of of and cells to tropoelastin and to to tropoelastin or serum by cells, cells heparan sulfate chains or cells in all adhesion used to adhesion between the different cell and adhesion to serum as and adhesion to as Cell adhesion to serum as a and results are as at of treatment on adhesion to adhesion of and and cells to were at for with of adhesion adhesion to serum as and adhesion to as of fetal bovine chondrocytes to with tropoelastin, or serum as the cells were by with cells were with in and with with by The actin and were directly with and cells were and a with an Cell were from a of the function in the Heparin affinity chromatography a Heparin on a were the in and with a in were used to the of to the sulfate and chondroitin sulfate C were from sulfate with to sulfate U. M. Res. Full Text PDF PubMed Scopus Google Scholar), a from P. of sulfate B. A. S. Weiss A.S. Biol. 2005; PubMed Scopus Google Scholar) a from of Cell at the of tropoelastin and a of tropoelastin fragments were as proteins and to The protein found to adhesion of multiple cell lines and including from and the bovine cells from fetal bovine cells, and fetal bovine chondrocytes from elastic not cells for studies because of their in and their well that cells to with tropoelastin and fragments the of the protein and the cells to to the of the molecule or to the at These results the binding activity to within the peptides for peptide were then used to the adhesive activity to a sequence in the last of the molecule. adhesion of cells to a sequence that the in and all of of bovine elastin, and and of of the bovine sequence or peptides the or of and not adhesion and adhesion the of by the at an for at cellular binding in the the of by to effect and sequences for tropoelastin fragments and peptides used in this in a amino acids of are to to an elastin sequences from different were Our that amino acids for the biological activity of the peptide multiple an of the of from from to The most an or amino acids from the COOH-terminal of the This by a hydrophobic amino or in all that an from the bovine sequence the amino and to the amino terminus of adhesive of tropoelastin and the in from different in a in with or the COOH-terminal Elastin and cells in the presence of to the as cells with serum the the cells and the that were for cells on similar for cells on with tropoelastin that the adhesion properties were similar for both on and the major of adhesion receptors for extracellular matrix The RGD inhibits binding to of the integrin receptors R. S. S. E. 1987; PubMed Scopus Google Scholar). the effect of the peptide or a peptide on adhesion to serum or and specifically adhesion effect on adhesion by the of to integrin function through the of on adhesion to adhesion of the a specific of αvβ3 integrin Proc. Natl. Acad. Sci. U. S. A. 1987; 84: PubMed Scopus Google Scholar), adhesion effect on adhesion by There also effect of and on the ability of cells to on these that adhesion to is not by integrins. Elastin on and with the elastin (EBP67) through the sequence The sequences and have also been shown to peptides these sequences effect on adhesion to at up to adhesion not by the or or by the There also effect of the VGVAPG peptide on cell on not COOH-terminal of residues at the terminus of tropoelastin is of in binding is a property of the COOH-terminal the fragments and peptides were to a affinity and with an the of to of the fragments and peptides from the tropoelastin and bind and to from the The of the molecule also is at The of the COOH-terminal to the middle binding with tropoelastin. the COOH-terminal the for and the major in tropoelastin. to from the is with the for tropoelastin. The residues of to the affinity of the peptide for as the and for the sequence at the terminus a in binding as of the for peptide by to The of the peptide altered with the of an of the sequence with of The binding of to is as the peptide and the peptide to for to and the binding on cells is a side of a and cell lines, and were for their ability to to cells are in the in the of a on their cell surface is greatly cells are in and all that cells were to to tropoelastin or in the cell that are for cell to tropoelastin. with the cells, that is not to all cell lines in the presence of serum that is a binding cellular side chains were by treatment with that treatment adhesion of cells to the peptide effect on cell These results that is capable of adhesion to that adhesion by a as of adhesion cells with of or were to with the at by at and in to adhesion. at the used on adhesion The effect on adhesion The tropoelastin molecule has a by hydrophobic in with The lysine residues in that tropoelastin monomers into the functional The hydrophobic in are thought to to the elastic properties of the protein. The of the protein by the last is different from the of the molecule. It the residues of the protein as well as a of amino acids evidence that this of the protein is important in elastic although is molecules that this sequence not assemble into and the sequence by interactions between tropoelastin and proteins T. Rosenbloom J. Mecham R.P. Biochem. J. 1996; PubMed Scopus Google Scholar, B.A. Mecham R.P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (120) Google Scholar). we use cell adhesion assays to show that the tropoelastin COOH-terminal also a cell interaction fragments of tropoelastin in cell adhesion we identified adhesive activity for and cells with a tropoelastin by adhesion of cell with the of the molecule this the VGVAPG sequence to with the elastin-binding protein. adhesion to the of the molecule for both and cells, at peptide than for the not studies peptides sequences within the COOH-terminal the cell adhesive activity to the last 17 amino acids of the protein. These peptides also several that cell adhesion to this The is a for of the lysine and residues at the terminus of the Cell adhesive activity the residues were the sequence is a of hydrophobic amino acids the with the last amino acids of the to the In elastin these amino acids are by and are from the that the amino sequence of to in other This is also shown in the peptide with in the adhesion Cell to the COOH-terminal sequence is not through the elastin-binding protein (EBP67) because neither VGVAPG-like sequences nor galactoside sugars altered adhesion to the we not cell adhesion to fragments of tropoelastin that contain the VGVAPG fragments from insoluble elastin that contain VGVAPG are also adhesive have chemotactic and This a functional for these peptides that is from the binding protein that adhesion to the tropoelastin Our results also show that adhesion to the COOH-terminal peptide is not promoted by integrins. an of integrin binding a effect on adhesion to The of the peptide also not the adhesion as both and greatly cell binding to the αvβ3 effect on cell adhesion to adhesion of these cells to serum the presence of αvβ3 on cells on tropoelastin or on the peptide were to on the not actin and induce as cells on studies with cells also a for in adhesion to the terminus of cells in and other important for with ECM proteins, not to tropoelastin, that for in the COOH-terminal cells the from αvβ3 integrins, their ability to bind the elastin COOH-terminal sequence are provides evidence that αvβ3 is not for binding to this et al. 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Our of a major cell interaction site to the last of elastin, the most highly that the ability to interact with cell-surface is a and property of the tropoelastin molecule. is to tropoelastin binding to of assembly on the or to important intracellular signals through and for and tropoelastin and tropoelastin

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