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Yogita Kanan, Robert A. Hamilton, Muayyad R. Al-Ubaidi; Identification Of Tyrosine-O-sulfated Proteins In The Retina And RPE. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3346.
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To investigate the role of protein tyrosine-O-sulfation (sulfation) in the function of the retina and the RPE.
An affinity column generated with the PSG2 anti-sulfotyrosine antibody was used to isolate sulfated proteins from bovine retinal and RPE extracts. The identity of the sulfated protein was determined following SDS-PAGE, in gel digestion and MS/MS mass spectrometric analysis. Sulfation of identified proteins was confirmed by either non-radioactive method 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.
Two major sulfated proteins identified were vitronectin and lumican. These proteins are known to be expressed in the retina and RPE. Immunoprecipitation and western analysis with PSG2 showed that these proteins are sulfated in-vivo in bovine retina and RPE. Their sulfated status was further confirmed by successfully transfecting the two genes into HEK cells and radiolabeling with sodium 35Sulfate
We have identified lumican and vitronectin as two proteins that are sulfated in the RPE and retina. The role of sulfation of these proteins in the overall function of the retina and RPE will be studied by mutating the tyrosines that are normally sulfated and the generation of knockin mice that express unsulfated forms of both proteins
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