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Yogita Kanan, Dan Brobst, Muayyad Al-Ubaidi; Understanding The Role Of Fibulin 2, A Tyrosine-O-Sulfated Protein, In Vision. Invest. Ophthalmol. Vis. Sci. 2013;54(15):345.
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Fibulins are a 7-member family of proteins associated with Bruch’s membrane (BrM) and elastic tissues. Mutations in multiple members of this family result in Macular Degeneration. Our goal is to understand the role of fibulin 2 in the eye and its relevance to vision.
An immuno-affinity column was generated with PSG2, an anti-sulfotyrosine antibody to isolate sulfated proteins from bovine RPE extracts. Mass spectrometry was used to identify fibulin 2. Immunohistochemistry was performed to determine the cellular localization of fibulin 2. Sulfation of fibulin 2 was confirmed by transfecting HEK cells with fibulin 2, metabolic labeling with radioactive sodium sulfate followed by barium hydroxide hydrolysis and thin layer electrophoresis. Site directed mutagenesis was used to identify the sulfotyrosines in the protein. Permanent transfectants of sulfated and unsulfated mutants of fibulin 2 were generated to study the role of sulfation. Retinal detachments and adhesion assays were performed to study the role of fibulin 2 in-vivo and in-vitro.
Fibulin 2 was localized to the RPE, Bruchs membrane and choriocapillaries. It was determined to be sulfated in-vivo and in-vitro at tyrosines 192, 196 and 198. Sulfated fibulin 2 permanent transfectants grew slower than the unsulfated fibulin 2 transfectants. However, sulfated fibulin 2 was incorporated in the extracellular matrix (ECM) more efficiently than the unsulfated counterpart. Upon retinal detachments, fibulin 2 was up-regulated 24 hours post detachment and remained elevated for 7 days after that. In-vitro adhesion assays showed that ARPE19 cells adhered better in presence of fibulin 2.
Fibulin 2’s up-regulation following retinal detachment suggest a role for it in the resolution of the detachment and that its sulfation enhances this process by playing a role in fibulin 2’s incorporation in the ECM.
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