title: 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 α(v)β(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.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Kamal
authors.initials: K
authors.lastname: Akhtar
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Broekelmann
authors.forename: Haowei
authors.initials: H
authors.lastname: Song
authors.forename: John
authors.initials: J
authors.lastname: Turk
authors.forename: Tom J
authors.initials: TJ
authors.lastname: Brett
authors.forename: Robert P
authors.initials: RP
authors.lastname: Mecham
authors.forename: Tracy L
authors.initials: TL
authors.lastname: Adair-Kirk
mesh_terms: D000818:Animals; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D019902:Chondrocytes / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014333:Tropoelastin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D039302:Integrin alphaVbeta3; D010455:Peptides; D014333:Tropoelastin; D030421:Peroxynitrous Acid