Abstract
Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.
Highlights
Elastic fibers are composed of an amorphous core, consisting mainly of elastin, surrounded by 10 –12 nm microfibrils composed of fibrillins, MAGPs1 and several other components
microfibril-associated glycoproteins (MAGPs)-1 is known to bind tropoelastin, and recent immunoprecipitation studies indicated the possibility of binding to fibrillin (18)
The type VI collagen binding site was localized to a region at the N terminus of MAGP-1 that lacks cysteine residues
Summary
Elastic fibers are composed of an amorphous core, consisting mainly of elastin, surrounded by 10 –12 nm microfibrils composed of fibrillins, MAGPs1 and several other components. The C-terminal domain, which contains the only two cysteine residues of tropoelastin has been proposed as the site of interaction with the microfibrillar components through the formation of a basic, intramolecular disulfide-bonded loop (3) Antibodies directed against this region disrupt fiber formation in vitro (4), while in lamb ductus arteriosus, loss of the C terminus in a 52-kDa proteolytically derived tropoelastin product prevents incorporation into the fiber (5). The interactive repertoire of microfibrillar and elastic fiber components determines the development and integrity of these functional entities in various tissues Toward this end it has been documented that MAGP-1 can bind to the Cterminal portion of tropoelastin (4, 12). Refolding of the purified protein produced a soluble form of MAGP-1 that was used to define binding epitopes on tropoelastin and fibrillin-1 These results suggest a model for the interaction of elastin with the microfibrillar scaffold of the elastic fiber.
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