Biophysical Characterisation of Fibulin-5 Proteins Associated with Disease Ralf Schneider 1 , Sacha A. Jensen 1 , Pat Whiteman 1 , James S. O. McCullagh 2 , Christina Redfield 3 and Penny A. Handford 1 ⁎ 1 Laboratory of Genes and Development, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK 2 Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK 3 Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK Received 26 April 2010; received in revised form 18 June 2010; accepted 19 June 2010 Available online 25 June 2010 FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycopro- tein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degener- ation (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. © 2010 Elsevier Ltd. All rights reserved. Edited by M. F. Summers Keywords: fibulin-5; protein misfolding; age-related macular degeneration; cutis laxa; cbEGF Introduction Fibulin-5 (also known as “DANCE” or “EVEC”) is an extracellular matrix (ECM) glycoprotein that plays an essential role in the formation and repair of elastic fibres. It is expressed in elastic-fibre-rich tissues such as blood vessels, heart, ovary, colon, lung, uterus and kidney during embryogenesis, and its production is down-regulated in adult tissues. 1,2 Elastic fibres contain a fibrillin-rich microfibril scaffold onto which elastin is deposited. The soluble form of elastin, tropoelastin, is secreted by cells into the ECM and polymerised by lysyl oxidase (LOX) and LOX-like enzymes. Fibulin-5 is thought to be a key component in elastogenesis by interacting with fibrillin, 3,4 tropoelastin 4,5 and LOX-like 6,7 proteins, thus bringing these proteins into close proximity. It also contains an RGD motif in its N-terminus that binds to cell surface integrins, 8,9 which may facilitate the anchoring of elastic fibres to the cell surface. Fibulin-5 knockout mice display a loose skin phenotype with severe lung and vascular abnormalities such as emphyse- ma and extended and tortuous arteries, emphasis- ing the crucial role of fibulin-5 in these elastic-fibre- rich tissues. 5,8 *Corresponding author. E-mail address: penny.handford@bioch.ox.ac.uk. Present address: R. Schneider, Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Straße 15, 44227 Dortmund, Germany. Abbreviations used: AMD, age-related macular degeneration; CL, cutis laxa; ECM, extracellular matrix; LOX, lysyl oxidase; cbEGF, calcium-binding epidermal growth factor like; ESI-MS, electrospray ionisation mass spectrometry; RT, reverse transcription; EGTA, ethylene glycol bis(b-aminoethyl ether) N,N′-tetraacetic acid. doi:10.1016/j.jmb.2010.06.039 J. Mol. Biol. (2010) 401, 605–617 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.