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M. Narayan, S. P. Mirza, S. S. Twining; Identification of Phosphorylation Sites on Maspin Secreted by Corneal Epithelial Cells using Tandem Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4603.
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Maspin, a 42 kDa non-classical serpin (serine protease inhibitor), secreted by corneal epithelial cells may regulate migration and adhesion of fibroblasts and prevent angiogenesis during corneal wound healing by unknown mechanisms. One approach to understand the mechanism of these maspin-mediated effects is to characterize post-translational modifications (PTMs) such as phosphorylation on maspin synthesized by the corneal epithelial cells.
To study phosphorylation of maspin in the corneal epithelium, immortalized human corneal epithelial cells (HCEC) and primary epithelial cels were lysed directly in iso-electric focusing (IEF) buffer. The proteins in the cell lysates were separated by IEF and then by SDS- electrophoresis in the second dimension (2D-PAGE). Maspin was detected by western blotting. Maspin was also immunoprecipitated (IP) from lysates and conditioned medium of primary and immortalized corneal epithelial cells in culture, and analyzed by tandem mass spectrometry for phosphorylation. Maspin was localized by immunofluorescence (IF).
Maspin, synthesized by human corneal epithelial cells exists in multiple forms displaying differences in size and charge. IEF-2D-PAGE analysis shows that, in normal corneal epithelium, maspin exists at the predicted molecular weight of ~42 kDa; however, differences in charge are evidenced by isoelectric points (pIs) lower and higher than the predicted value of 5.6. Tandem mass spectrometry analysis indicates secreted maspin is phosphorylated at multiple serine and threonine residues (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310 and Ser316). No phosphorylation was detected on intracellular maspin. IF analysis localized maspin to the cytosol and nucleus of corneal epithelial cells.
Corneal epithelial cells phosphorylate maspin targeted for secretion. This modification may be important for inducing conformational changes in maspin, and regulating maspin’s interactions with other proteins.
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