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S. Yang, F. Liu, H. Wang, H. Yanagisawa, R. Doliana, A. Colombatti, J. Bennett; Distribution of Fibulin 5 in Bruch’S Membrane and Defects Due to Missense Variations: Pathological Implications for Age–Related Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2315.
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The specific molecular mechanisms underlying age–related macular degeneration (AMD) are largely unknown. Fibulin 5 is an extracellular matrix protein abundantly expressed in tissues with enriched elastic fibers. This protein plays a critical role in the assembly and crosslinking of tropoelastin. Missense variations of fibulin 5 have been reported to occur exclusively in 1.7 percent of AMD patients (EM Stone et al, 2004, NEJM351:346–53). We characterized the distribution of fibulin 5 in healthy and diseased retina and, in vitro, investigated functional alterations of fibulin 5 due to missense variations found in AMD.
The expression of fibulin 5 in mouse retina and human macula was assessed by RT–PCR, immunohistochemistry and Western blotting. Expression constructs containing the seven variants (EM Stone et al, 2004, NEJM351:346–53) were generated by site–directed mutagenesis from the human fibulin–5 cDNA (generously provided by B. Schiemann). Secretion rates/levels of the different fibulin 5 proteins were evaluated by quantitative Western blotting and pulse–chase assay. The binding capacity of fibulin 5 (and its variants) with Elastin or EMILIN–1 was assessed by co–immunoprecipitation and quantitative Western blotting.
Fibulin 5 transcript and protein were detected in mouse retina by RT–PCR and western blotting. Immunofluorescence revealed presence of fibulin 5 in cornea, neural retina and Bruch’s membrane. A similar distribution pattern of fibulin 5 was observed in retinal sections from a patient with AMD. In vitro assays showed reductions in secretion rates/levels in four fibulin 5 variants compared to wildtype. The most dramatic reductions were identified in the Ile169Thr variant. The effect was similar to that observed in cells expressing wildtype fibulin 5 protein after being placed in calcium–free medium. There was no difference between wildtype and variant fibulin 5 proteins in interaction with Elastin Microfibril Interface Located Protein–1 (EMILIN–1).
The results suggest that secretion defects exist in four of seven AMD–associated fibulin 5 variants. The EGF–Calcium binding domain in fibulin 5 appears to play a key role in this process.
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