Structural effects of fibulin 5 missense mutations associated with age-related macular degeneration and cutis laxa.
OBJECTIVE
AMD has a complex etiology with environmental and genetic risk factors. Ten fibulin 5 sequence variants have been associated with AMD and two other fibulin 5 mutations cause autosomal-recessive cutis laxa. 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. Biophysical techniques were used to detect structural changes in the fibulin 5 mutants and to determine whether changes are predictive of pathogenicity.


METHODS
Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the fibulin 5 mutants in the absence and presence of Ca(2+).


RESULTS
CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca(2+) but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca(2+).


CONCLUSIONS
The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study-for example, disruption of heterointeractions.
Jones
Richard P O
RP
Genetic Medicine, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, St. Mary's Hospital, Manchester, United Kingdom. rpojones@yahoo.co.uk
Ridley
Caroline
C
Jowitt
Thomas A
TA
Wang
Ming-Chuan
MC
Howard
Marjorie
M
Bobola
Nicoletta
N
Wang
Tao
T
Bishop
Paul N
PN
Kielty
Cay M
CM
Baldock
Clair
C
Lotery
Andrew J
AJ
Trump
Dorothy
D
D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003483:Cutis Laxa / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017510:Protein Folding
D016326:Extracellular Matrix Proteins; C492373:FBLN5 protein, human; D002118:Calcium