Molecular Basis of Elastic Fiber Formation

Abstract

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.

Nanostructure of fibrillin-1 reveals compact conformation of EGF arrays and mechanism for extensibility

“…6 and 7, which are published as supporting information on the PNAS web site) to confirm the fragments were the right size and monodisperse. The protein fragments also were found to have native conformations in binding studies with microfibril-associated proteins (15,16), integrins (13), and heparan sulfate (14). Immediately before data collection, the protein fragments were chromatographed to ensure the sample was monomeric.…”

“…Recombinant human fibrillin-1 fragments encoded by the following exons were expressed and purified by using 293-EBNA cells as described (13)(14)(15)(16) (Fig. 1): PF2, exons 9-17; PF5, exons 18-25; PF7, exons 24-30; PF8, exons 30-38; PF9, exons 37-43; PF10, exons 41-52; PF11, exons 37-52; PF12, exons 50-58; and PF13, exons 57-64.…”

Marfan Syndrome-causing Mutations in Fibrillin-1 Result in Gross Morphological Alterations and Highlight the Structural Importance of the Second Hybrid Domain

“…Protein Purification and MALLS-The expression and purification of the recombinant PF5 fibrillin-1 fragment has been described (31). The same procedures were used to express and purify the PF5 mutant fragments.…”

“…Recombinant Human Fibrillin-1 Protein Fragments-The cloning of recombinant fragment PF5 (residues 723-1069) into the mammalian expression vector pCEP-pu/AC7, modified by incorporation of an N-terminal His 6 tag, has been described (31). Four point mutations were engineered into the wild-type PF5 fragment (Fig.…”

“…A central region of fibrillin-1 (exons 24 -32), often referred to as the "neonatal region", is a hotspot for mutations which give rise to an early lethal form of MFS (30), but it also contains mutations that result in classical MFS. The neonatal region interacts with elastin (31), and localizes to the microfibril interbead region (1,19).…”