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M. Narayan, D. J. Warejcka, M. A. Horswill, S. S. Twining; Post-Translational Modifications Facilitate Maspin Diversity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2722.
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Post-translational modifications (PTMs) diversify the repertoire of protein functions. 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 PTMs on maspin synthesized by the corneal epithelial cells in the normal and wounded cornea.
To study PTMs of maspin in normal corneal epithelium, human donor corneal epithelial cells were scraped off and lysed directly in iso-electric focusing (IEF) buffer. Alternatively epithelial cells were isolated by dispase treatment and trypsinized, and grown in culture, simulating wounding conditions. At confluence, cells were lysed for IEF. 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 of cultured epithelial cells and analyzed by western blotting for phosphorylation and ISG15ylation. Maspin in the medium from cultured epithelial cells was compared to maspin from IPs by western blotting. Maspin was localized by immunofluorescence (IF).
Maspin, synthesized by human corneal epithelial cells under normal and wounded conditions, 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. In contrast, in cultured epithelial cells, maspin displays differences both in charge and molecular weight; sizes include ~42, 57, 85 kDa. IP of maspin, from cultured epithelial cells, also yielded the same species. Maspin from epithelial cell culture medium is glycosylated and is larger than ~42kDa. Western blot analysis of maspin from IPs indicated that this molecule is modified with ISG15, an ubiquitin-like molecule, and phosphate groups. IF analysis localized maspin to the cytosol and nucleus of corneal epithelial cells.
In cultured corneal cells, which are similar to those present in the wounded cornea, maspin is phosphorylated, glycosylated and ISG15ylated. These modifications may be important for stabilization of maspin and regulating maspin’s interactions with other proteins. In addition, glycosylation of maspin probably facilitates its secretion.
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