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L. Ervin, R.K. Crouch, K.L. Schey; Identification of novel lens protein modifications in membrane protein MP20 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3990.
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Purpose: Membrane protein 20 (MP20) is lens specific and is the second most abundant membrane protein in the lens. The function of MP20 is unknown; however, changes in the expression and localization of MP20 with fiber cell age indicating a possible role in lens development.1 Furthermore, mutations of MP20 have been linked to cataract.2 Recently, galectin–3, a protein that binds to beta–galactosides, has been found to bind to MP20 suggesting that the protein is glycosylated3; however, little is known about the specific types and sites of post–translational modifications present in MP20. The purpose of this work is to identify the post–translation modifications of MP20. Methods: Bovine lenses were homogenized in 10 mM NaF/5 mM EDTA/10 mM bicarbonate buffer (pH 8). Membrane proteins were isolated by centrifugation and washed with the buffers in succession as follows: Tris buffer (10mM Tris/1mM EDTA/1 mM CaCl2 at pH 9), 4 M urea (in Tris buffer), 7 M urea (in Tris buffer), 50 mM NaOH (in Tris buffer), and then deionized water. MP20 was further purified by SDS–PAGE or anion exchange chromatography. Glycosylation analysis of the fractions was done by dot blotting on nitrocellulose membrane and staining with Periodic Acid–Schiff (PAS). Tryptic digestion was done in solution or in–gel for 20–36 hours. Masses of intact protein and sequences of tryptic peptides were analyzed by MALDI–TOF, MALDI–TOF/TOF, and by LC–ESI–MS/MS. Results: Analysis of the MP20 tryptic peptides shows single phosphorylation site at Ser–170 and double phosphorylation with the second, heretofore unreported, site at Thr–171. The intact protein was found to be glycosylated by the PAS method and specific sites and structures have been identified by MALDI–TOF/TOF. A recently discovered modification, C–mannosylation4, of tryptophan residues was discovered at W–43 and W–61 of MP20. Conclusions: A new lens protein modification was discovered in addition to sites of phosphorylation on MP20. Studying the post–translational modifications of MP20 may provide information about the normal function of this protein in the lens and the involvement in cataract formation. 1Grey AC, Jacobs MD, Gonen T, Kistler J, Donaldson PJ. Exp. Eye Res. 2003, 77:567–574. 2Chen T, Li X, Yang Y, Church RL. Mol Vis. 2002, 8: 372–388. 3Gonen T, Grey AC, Jacobs MD, Donaldson PJ, Kistler J. BMC Cell Biol. 2001, 2:17–28. 4Furmanek A, Hofsteenge J. Acta Biochim. Pol. 2000, 47:781–789.
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