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Yogita Kanan, Robert A. Hamilton, Muayyad R. Al-Ubaidi; Investigating the Role of Protein-Tyrosine Sulfation in the Function of Fibulin 2. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1590.
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Fibulin 2, an extracellular protein present in elastic and basement membranes, has been shown to exhibit tumor suppressive and anti-angiogenic properties. The purpose of this study is to investigate the role of tyrosine-O-sulfation (sulfation) in the function of fibulin 2 and its relevance to vision.
An affinity column generated with an anti-sulfotyrosine antibody was used to isolate sulfated proteins from bovine retinal and RPE extracts. The identity of the sulfated protein was determined by MS/MS mass spectrometric analysis. Sulfation of identified proteins was confirmed by either non-radioactive methods involving the immunoprecipitation of the protein of interest, SDS-PAGE and immunoblotting with the anti-sulfotyrosine antibody or by radioactive method. The latter process included transfecting HEK cells with the gene of interest, metabolic labeling with radioactive sodium sulfate followed by barium hydroxide hydrolysis and thin layer electrophoresis. Identity of the sulfotyrosine residues was done by site-directed mutagenesis followed by transfection of the wild-type fibulin 2 and mutant clones followed by immunoprecipitation and immunoblotting with the anti-sulfotyrosine antibody.
Fibulin 2 was a major sulfated protein identified from the retina and RPE by the immunoaffinity column. Immunoprecipitation, western analysis and radioactive sodium sulfate incorporation showed that the protein was sulfated in-vitro and in-vivo. Their tyrosine sulfated status was further confirmed by barium hydroxide hydrolysis and thin layer electrophoresis. Site directed mutagenesis identified tyrosines 192, 196 and 198 as sulfotyrosines.
We have identified fibulin 2 as a sulfated protein in the RPE and retina. The role of sulfation in fibulin 2 in the retina and RPE will be studied by anti-angiogenic assays and the generation of knockin mice that express unsulfated forms of the protein.
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