Abstract
Following cellular secretion into the extracellular matrix, tropoelastin is transported, deposited, and cross-linked to make elastin. Assembly by coacervation was examined for an isoform of tropoelastin that lacks the hydrophilic domain encoded by exon 26A. It is equivalent to a naturally secreted form of tropoelastin and shows similar coacervation performance to its partner containing 26A, thereby generalizing the concept that splice form variants are able to coacervate under comparable conditions. This is optimal under physiological conditions of temperature, salt concentration, and pH. The proteins were examined for their ability to interact with extracellular matrix glycosaminoglycans. These negatively charged molecules interacted with positively charged lysine residues and promoted coacervation of tropoelastin in a temperature- and concentration-dependent manner. A testable model for elastin-glycosaminoglycan interactions is proposed, where tropoelastin deposition during elastogenesis is encouraged by local exposure to matrix glycosaminoglycans. Unmodified proteins are retained at approximately 3 microM dissociation constant. Following lysyl oxidase modification of tropoelastin lysine residues, they are released from glycosaminoglycan interactions, thereby permitting those residues to contribute to elastin cross-links.
Highlights
Human tropoelastin is the soluble monomer of elastin, which is largely responsible for the elastic properties of human lungs, large blood vessels and skin
Tropoelastin Lacking Domain 26A (SHEL⌬26A) Shows Coacervation Performance Similar to That of Tropoelastin (SHEL)—SHEL⌬26A was compared with SHEL for its ability to reversibly associate by coacervation [18]
Circular dichroism of SHEL⌬26A and SHEL revealed similar profiles indicative of comparable folding. These assays demonstrated that the two splice forms of tropoelastin could be compared in heparin binding studies
Summary
Production and Expression of Tropoelastin Lacking 26A (SHEL⌬26A)—Tropoelastin containing 26A (SHEL) was obtained as described previously [17]. The SHEL⌬26A gene was constructed from pSHELF by site-directed mutagenesis (Transformer Mutagenesis Kit, CLONTECH) using the oligonucleotide 5Ј-CGG GTT TCG GTG CTG TTC CGG GCG CGC TGG-3Ј that flanked either side of exon 26A by 15 base pairs, resulting in its precise deletion. This was followed by subcloning to provide the final construct in pET3d, which was confirmed by DNA sequencing. For SHEL and SHEL⌬26A, full-length protein was obtained [17] and purified further by C18 reverse phase high performance liquid chromatography using a linear gradient of 20 – 80% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid. Standard Binding Assay—An ultrafiltration assay was developed to examine interactions between heparin and two forms of tropoelastin, This paper is available on line at http://www.jbc.org
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
