Fibulin-5 mutations: mechanisms of impaired elastic fiber formation in recessive cutis laxa

To elucidate the molecular mechanisms of impaired elastic fiber formation in recessive cutis laxa, we have investigated two disease-causing missense substitutions in fibulin-5, C217R and S227P. Pulse-chase immunoprecipitation experiments indicated that S227P mutant fibulin-5 was synthesized and secreted by skin fibroblasts at a reduced rate when compared with the wild-type protein. Both mutants failed to be incorporated into elastic fibers by transfected rat lung fibroblasts. Purified recombinant fibulin-5 with either mutation showed reduced affinity for tropoelastin in solid-phase binding assays. Furthermore, S227P mutant fibulin-5 also showed impaired association with fibrillin-1 microfibrils. The same mutation triggered an endoplasmic reticulum (ER) stress response, as indicated by the strong co-localization of this mutant protein with folding chaperones in the ER, including calreticulin, immunoglobulin-binding protein and protein disulfide isomerase, and by increased rates of apoptosis in patient fibroblasts. Histological analysis of skin sections from a cutis laxa patient with a homozygous S227P mutation showed a lack of fibulin-5 in the extracellular matrix and a concomitant disorganization of dermal elastic fibers. By electron microscopy, elastic fibers in the skin of this patient showed a failure of elastin globules to fuse into a continuous elastic fiber core. We conclude that recessive cutis laxa mutations in fibulin-5 result in misfolding, decreased secretion and a reduced interaction with elastin and fibrillin-1 leading to impaired elastic fiber development. These findings support the hypothesis that fibulin-5 is necessary for elastic fiber formation by facilitating the deposition of elastin onto a microfibrillar scaffold via direct molecular interactions.