Characterization of the Molecular Interaction between Tropoelastin and DANCE/Fibulin-5

Abstract

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca(2+), Ba(2+) and Mg(2+) significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2-15 and exons 16-36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

“…3), we examined whether oxidative modifications of TE alter the process of coacervation. Similar to previous studies (47) unoxidized TE coacervated at ϳ42°C (Fig. 4A).…”

“…Hydrophobic amino acid residues (Tyr, Phe, Ile, or Leu) next to the Lys residues play a critical role in maintaining the optimal microenvironment for proper cross-linking (8,(47)(48)(49)(50)(51)(52) (Fig. 7, step 3).…”

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

“…Therefore, there were no other proteins in this assay system that could reasonably bridge between tropoelastin and integrin ␣ V ␤ 3 . Interestingly the whole of the tropoelastin molecule has been shown to be necessary for fibulin-5-tropoelastin association (40), and so fibulin-5 should not associate with our cell adhesive peptide 36 and GRKRK. Therefore, it is unlikely that fibulin-5 will bind to this small section of tropoelastin, making it unlikely that fibulin-5 is acting as a bridging molecule for cell adhesion.…”

Alternative Splicing and Tissue-specific Elastin Misassembly Act as Biological Modifiers of Human Elastin Gene Frameshift Mutations Associated with Dominant Cutis Laxa

“…In vitro studies and studies with transgenic mice showed that tropoelastin with altered or deleted sequences in this region is still capable of interacting with microfibrils to form an elastic fiber, although with abnormal cross-linking (7,42,43). How this region of tropoelastin facilitates fiber assembly is still unknown, although recent studies have identified binding sites for fibulin-4 and fibulin-5-, two proteins that facilitate elastin organization (44,45). Inactivation of these two fibulins in mice results in abnormal elastic fiber formation, and particularly relevant to our study, mutations in the human genes for fibulin-4 and fibulin-5 have been linked to an autosomal recessive form of cutis laxa (ARCL Type I) (46 -50).…”