title: Biophysical characterisation of fibulin-5 proteins associated with disease.
abstract: FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments. Limited proteolysis, NMR spectroscopy and chromophoric calcium chelation experiments showed that the G267S and S227P substitutions cause long-range structural effects consistent with protein misfolding. Cellular studies using fibroblast cells further demonstrated that these recombinant forms of mutant fibulin-5 were not present in the extracellular medium, consistent with retention. In contrast, no significant effects of I169T and G202R substitutions on protein fold and secretion were identified. These data establish protein misfolding as a causative basis for the effects of G267S and S227P substitutions in AMD and CL, respectively, and raise the possibility that the I169T and G202R substitutions may be polymorphisms or may increase susceptibility to disease.
authors.affiliation: Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Ralf
authors.initials: R
authors.lastname: Schneider
authors.forename: Sacha A
authors.initials: SA
authors.lastname: Jensen
authors.forename: Pat
authors.initials: P
authors.lastname: Whiteman
authors.forename: James S O
authors.initials: JS
authors.lastname: McCullagh
authors.forename: Christina
authors.initials: C
authors.lastname: Redfield
authors.forename: Penny A
authors.initials: PA
authors.lastname: Handford
mesh_terms: D003483:Cutis Laxa / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D020125:Mutation, Missense; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D016326:Extracellular Matrix Proteins; C492373:FBLN5 protein, human