The role of calcium in the organization of fibrillin microfibrils

AbstractThe microfibrillar glycoprotein fibrillin has a multidomain structure which contains forty-three epidermal growth factor-like motifs with calciumbinding consensus sequences. We have utilized intact microfibrils isolated from human dermal fibroblast cultures to investigate the putative influence of bound calcium on microfibrillar organization and integrity. Incubation with EDTA or EGTA rapidly resulted in gross disruption of microfibril morphology. The treatment induced disorganization of the interbead domains although the regular beaded arrangement was always apparent. These changes were readily reversible on replacing calcium, indicating that the treatment had not compromised microfibrillar integrity. The data localize calcium binding EGF-like repeats to the interbead domains and indicate that lateral packing of fibrillin monomers is calcium-dependent. This arrangement suggests how mutations in epidermal growth factor-like domains of fibrillin might cause the disruption in microfibril organization and interactions which underlies the clinical symptoms of some Marian syndrome patients

Fibrillin: Evidence that chondroitin sulphate proteoglycans are components of microfibrils and associate with newly synthesised monomers

AbstractWe have investigated the potential association of proteoglycans with intact fibrillin-containing microfibrils from foetal bovine elastic tissues and with newly synthesised fibrillin in human and bovine cell cultures. Microfbril integrity was disrupted by chondroitinase ABC lyase and chondroitinase AC lyase, but not by keratanase or hyaluronidase. Following chondroitinase treatment, beads were disrupted but the underlying fibrillar scaffold appeared intact. Cuprolinic blue was prominently associated with beaded domains at a critical electrolyte concentration. Electron-dense rods were often associated with cuprolinic blue-treated microfbrils isolated from fixed tissues. Positive staining revealed charged foci at the beads. Newly synthesised fibrillin could be labelled with 35S TransLabel, [3H]glucosamine or 35SO4 but its electrophoretic mobility was not influenced by treatment with chondroitinase ABC or AC lyase. A diffuse 35SO4-labelled chondroitinase-sensitive component with a resistant band (Mr 35000) co-immunoprecipitated with fibrillin. These experiments indicate that chondroitin sulphate proteoglycans associate with fibrillin and contribute to microfibril assembly. This association has major implications for microfibril function in health and disease