Analysis of TIGR/myocilin Secretion, a Protein Implicated in Glaucoma

Abstract

Abstract: : 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 MYOC^delta2-32, 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 MYOC^M1I, MYOC^M15I and the WT form may be explained by SP cleavage during ER translocation. Our results suggest an extracellular function for TIGR/myocilin protein.

Keywords: proteins encoded by disease genes • protein structure/function • protein modifications-post translational