We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dosedependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these Nand C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with Nethylmaleimide reduced homotypic binding. Microfibrilassociated glycoprotein-1 inhibited N-to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly.
Fibrillin microfibrils are widely distributed extracellular matrix assemblies that endow elastic and nonelastic connective tissues with long-range elasticity. They direct tropoelastin deposition during elastic fibrillogenesis and form an outer mantle for mature elastic fibers. Microfibril arrays are also abundant in dynamic tissues that do not express elastin, such as the ciliary zonules of the eye. Mutations in fibrillin-1-the principal structural component of microfibrils-cause Marfan syndrome, a heritable disease with severe aortic, ocular, and skeletal defects. Isolated fibrillin-rich microfibrils have a complex 56 nm "beads-on-a-string" appearance; the molecular basis of their assembly and elastic properties, and their role in higher-order elastic fiber formation, remain incompletely understood.
We have investigated the molecular basis of elastic fiber formation on fibrillin microfibrils. Binding assays revealed high affinity calcium-independent binding of two overlapping fibrillin-1 fragments (encoded by central exons 18 -25 and 24 -30) to tropoelastin, which, in microfibrils, map to an exposed "arms" feature adjacent to the beads. A further binding site within an adjacent fragment (encoded by exons 9 -17) was within an eightcysteine motif designated TB2 (encoded by exons 16 and 17). Binding to TB2 was ablated by the presence of Nterminal domains (encoded by exons 1-8) and reduced after deleting the proline-rich region. A novel transglutaminase cross-link between tropoelastin and fibrillin-1 fragment (encoded by exons 9 -17) was localized by mass spectrometry to a sequence encoded by exon 17. The high affinity binding and cross-linking of tropoelastin to a central fibrillin-1 sequence confirm that this association is fundamental to elastic fiber formation. Microfibril-associated glycoprotein-1 showed calcium-dependent binding of moderate affinity to fibrillin-1 N-terminal fragment (encoded by exons 1-8), which localize to the beads. Microfibril-associated glycoprotein-1 thus contributes to microfibril organization but may also form secondary interactions with adjacent microfibril-bound tropoelastin.
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