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S. Gobeil, L. Letartre, J. Morisette, V. Raymond; Analysis of TIGR/myocilin Secretion, a Protein Implicated in Glaucoma . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1172.
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Purpose: Mutations in TIGR/myocilin (MYOC), which encodes a 504 amino acids (AA) glycoprotein, cause primary open-angle glaucoma. The first 32 AA of the polypeptide contain a potential signal peptide (SP) targeting proteins to the endoplasmic reticulum (ER). This N-terminal region also harbors two methionines, at positions 1 and 15, that may initiate the polypeptide. We investigated TIGR/MYOC secretion and the role of its N-terminal region in this process. Methods: Deletion/substitution TIGR/MYOC variants containing a myc-tag at their carboxy-terminals were generated by site-directed mutagenesis in the pRcCMV expression vector. Wild-type (WT) and variants cDNAs were transiently transfected in COS-7 cells. Newly synthesized proteins were separated using Invitrogen NuPAGE gels and analyzed by Western blotting. Pulse-chase experiments were performed with 35S methionine/cysteine using a 2 hr labeling period. Results: Transfection of MYOCdelta2-32, in which AA 2 to 32 have been deleted, revealed that the produced protein remained in intracellular compartements when compared to its WT counterpart which was secreted. Pulse-chase experiments showed that approximately 75 % of the WT polypeptide was released within the medium after a 6 hr chase period. More than 90% of the labeled protein was secreted after a 24 hr period. Half-life of the wt polypeptide was estimated at more than 48 hr in the culture medium. Substitution of methionine 1 or 15 by an isoleucine showed migration and secretion patterns identical to those of the wild-type protein. Conclusion: Our data showed that myocilin N-terminal region targets the protein to extracellular locations. The similarities observed between MYOCM1I, MYOCM15I and the WT form may be explained by SP cleavage during ER translocation. Our results suggest an extracellular function for TIGR/myocilin protein.
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