Fibulin 5 Forms a Compact Dimer in Physiological Solutions

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca²⁺ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.Fibulins are a family of seven extracellular matrix glycoproteins, some of which associate with elastic fibers and basement membranes (1, 2). They are involved in the assembly, organization, and stabilization of macromolecular complexes (3). Fibulins contain arrays of cbEGF²-like domains and a fibulin-type C-terminal (Fc) module (4). Fibulins 3–5 have a modified N-terminal cbEGF domain, followed by five cbEGF domains (4).Fibulin 5 (supplemental Fig. S1) is highly expressed in developing arteries with a low expression in adult vessels that is up-regulated following vascular injury and in atherosclerosis (5, 6). Expression has been detected in other elastin-rich tissues, including aorta, skin, uterus, lung, heart, ovary, and colon (5, 6). The extensibility of such tissues is provided by elastic fibers (7), and aging is associated with a loss of elasticity (8). Fibulin 5 is essential for elastinogenesis. The fibulin 5 knock-out mouse exhibits disorganized elastic fibers resulting in severe elastinopathies, with loose skin, vascular abnormalities, and emphysematous lungs. Similar changes are seen in an aged phenotype (9, 10). Mutations in fibulin 5 lead to the elastin disorder cutis laxa (11–13) and have been associated with age-related macular degeneration (14, 15).It has been shown that fibulin 5 binds elastic fibers (16) and interacts with tropoelastin (10), fibrillin 1 (17), lysyl oxidase-like protein 1 (18), -2, and -4 (19), latent transforming growth factor-β-binding protein 2 (19), emilin 1 (20), apolipoprotein (a) (21), and superoxide dismutase (22). Through an RGD motif fibulin 5 interacts with integrins (6, 9, 23).The assembly of elastic fibers is a complex hierarchical process. A model proposes that fibulin 5 associates with microfibrils via interactions with fibrillin 1; tropoelastin molecules bind fibulin 5 and coacervate, and lysyl oxidase-like protein 1 enzymes cross-link tropoelastin to form mature elastin (7, 16). Data that support this model indicate that fibulin 5 potentially increases the coacervation of tropoelastin, enhancing elastic fiber formation (24). However, other data suggest that fibulin 5 slows the maturation of elastin assemblies (25).Rotary-shadowing EM has suggested that fibulin 5 exists as a short rod with a globular domain at one end (26). We used size-exclusion column multiangle laser light scattering (SEC-MALLS), small angle x-ray scattering (SAXS), EM single particle analysis, analytical ultracentrifugation (AUC), CD, and isoelectric focusing to investigate the structures of fibulin 5 in monomeric and dimeric form, and the equilibrium between the two forms.