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S. Gobeil, L. Burke, M.A. Walter, V. Raymond; Tandem mass spectrometry analysis of secreted myocilin protein complexes . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4375.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations in the myocilin (MYOC) gene, also known as the trabecular induced glucocorticoid responsive (TIGR) gene, are associated with 3–4 % of open–angle glaucomas. The protein forms dimers, migrating at 115–125 kDa, as well as multiple higher molecular weight complexes at above 180 kDa. It has been suggested that the myocilin polypeptide was interacting with several proteins, for example fibronectin. We assessed the nature of MYOC complexes secreted in the extracellular media of transfected cell lines using tandem mass spectrometry. Methods: A pRcCMV expression vector encoding wild–type (WT) myocilin tagged at its C–terminal with a c–myc epitope (MYOCWT–myc) was transiently transfected in immortalized human trabecular meshwork (iHTM) and COS–7 cell lines. Forty–eight hours after transfection, extracellular media from both cell lines were immunoprecipitated (IPP) using an anti–c–myc antibody. IPP products were migrated under non–reducing conditions on TRIS–glycine 7% protein gels and stained using Coomassie blue. Specific protein bands were excised, in gel–digested with trypsin and analyzed on a Q–Tof 2 tandem mass spectrometer. Micromasses were thereafter analysed with Mascot software using the MSDB and NCBInr databases. Results: No IPP products were detected in cell lines transfected with an empty expression vector. The migration patterns of immunoprecipitated MYOC proteins were identical between the two tested cell lines. Five protein complexes were detected migrating at about 116, 180, 200 and at above 200 kDa yielding a migration pattern similar to myocilin immunoreactive migration patterns detected by Western analysis using an anti–MYOC antibody. Tandem mass spectrometry analysis performed on the protein bands (5 from each cell lines from 116 to above 200 kDa) revealed that they were solely composed of myocilin polypeptides. Conclusions: Our data demonstrated that myocilin complexes observed in extracellular media of transfected cell lines resulted from self–interaction generating homomultimers, rather than by association with other protein(s).
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