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T. Oya-Ito, S. Padival, A.K. Padival, D.G. Smith, J.W. Crabb, R.H. Nagaraj; Argpyrimidine Modification of Heat Shock Protein 27 in the Human Lens . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2348.
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Purpose: Argpyrimidine is a blue fluorescent product generated by the reaction of methylglyoxal with arginine residues in proteins and accumulates in relatively large amounts in brunescent cataractous lenses (Padayatti et al, IOVS, 42:1299-1304, 2001). Here we investigate argpyrimidine formation in a human lens epithelial cell line and in whole lenses. Methods: Cytosolic proteins of the human lens epithelial cells (HLE B-3 from Dr. Usha Andley, St. Louis, MO) were subjected to 2-D Western analysis using a monoclonal antibody to argpyrimidine. Water-soluble human lens proteins from a young (22 years) and an aged lens (66 years) were also probed by 2-D Western analyses with antibodies to heat shock protein 27 (Hsp27) or to phospho-Hsp27 (ser 82), or to argpyrimidine. Immunohistochemical studies were carried out with the same antibodies. Immunoreactive gel components were excised, digested in situ with trypsin and identified by LC MS/MS. Results: Human lens epithelial cells exhibited three strongly immunoreactive 2-D gel spots with the anti-argpyrimidine antibody. The major component was identified as Hsp27. Minor, more acidic spots may be phospho-Hsp27s. 2-D Western analysis of human lens proteins showed that most of the argpyrimidine immunoreaction was with proteins that also reacted with antibodies to Hsp27 or to phospho-Hsp27. Immunoprecipitation of water-soluble human lens proteins with anti-argpyrimidine antibody followed by Western analyses with anti-Hsp27 antibody confirmed that argpyrimidine formation occurs on Hsp27 in vivo. Immunohistochemical studies in the human lens revealed that the immunoreactivity for argpyrimidine and Hsp27 occur mostly in the outer cortical layers and epithelial cells. Immunofluorescence studies in the human lens epithelial cells showed Hsp27 throughout the cytoplasm but argpyrimidine mostly in the nucleus and in the perinuclear region. Conclusions: Hsp27 appears to be preferentially modified by methylglyoxal in epithelial cells and in fiber cells of the human lens. The functional consequences of this argpyrimidine modification is under investigation.
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