April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Identification Of Tyrosine-O-sulfated Proteins In The Retina And RPE
Author Affiliations & Notes
  • Yogita Kanan
    Dept Cell Biology, OUHSC, Oklahoma City, Oklahoma
  • Robert A. Hamilton
    Dept Cell Biology, OUHSC, Oklahoma City, Oklahoma
  • Muayyad R. Al-Ubaidi
    Dept Cell Biology, OUHSC, Oklahoma City, Oklahoma
  • Footnotes
    Commercial Relationships  Yogita Kanan, None; Robert A. Hamilton, None; Muayyad R. Al-Ubaidi, None
  • Footnotes
    Support  OCAST HR10-069, R01 EY014052, R01 EY018137, FFB, NRCC P20RR017703 and NEI P30EY012190
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3346. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To investigate the role of protein tyrosine-O-sulfation (sulfation) in the function of the retina and the RPE.

Methods: : 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.

Results: : 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

Conclusions: : 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

Keywords: retina • retinal pigment epithelium • protein modifications-post translational 

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