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B. Govindarajan, S. J. Spurr-Michaud, S. Heimer, M. Gilmore, P. Argueso, I. K. Gipson; Purification of a Protein Secreted by Non-Encapsulated Streptococcus pneumoniae That Induces MUC16 Shedding in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3886.
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Membrane-tethered mucins on the ocular surface epithelium act as barriers to microbes present in the environment. Infections at the ocular surface occur by opportunistic pathogens that bypass the mucin barrier through epithelial wounds, or by non-opportunistic pathogens that cross the mucin barrier by unknown mechanisms. Our laboratory has shown that an infectious, nontypeable strain of Streptococcus pneumoniae that causes epidemic conjunctivitis secretes a protein that induces shedding of the membrane-tethered mucin MUC16 from corneal epithelial cells. The purpose of this study was to isolate and determine the identity of this protein.
Human corneal epithelial cells (HCLE) cultured for optimal mucin production were treated with exoproducts of non-encapsulated Streptococcus pneumoniae (SPN) strain 168 for 1 hour. The SPN exoproducts sheddase was purified using a 50 kDa cut off filter followed by sequential anionic exchange (DEAE) and size exclusion chromatography (CL-4B). Fractions were collected and tested for MUC16 ectodomain shedding activity. Active fractions were assayed for protein content and subjected to gel electrophoresis followed by Coomassie blue staining.
99.9 % of extraneous proteins from the SPN culture supernatants were removed by the 50 kDa cut off membrane, whereas the mucin shedding activity was retained. Active fractions of the retentate were recovered in low salt concentrations (150-300 mM) upon anionic exchange, providing additional 10 fold enrichment. Size exclusion chromatography yielded a single active peak with a 10 fold purification. SDS-PAGE of the active fractions, followed by Coomassie blue staining revealed a protein band of apparent 170-180 kDa molecular weight.
We have purified a protein from the culture media of pathogenic non-encapsulated Streptococcus pneumoniae strain 168 that induces shedding of MUC16 from HCLE cells. The molecular mass of the candidate protein is between 170 and 180 kDa and its identity is currently under study using mass spectroscopy.
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