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L. Letartre, S. Gobeil, M. Rodrigue, J. Morissette, V. Raymond; Functional Analysis of the Glaucoma-associated TIGR/myocilin Protein: A Major Role for the Coiled-coil Motif at Residues 78-105 in Oligomerization . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1171.
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Purpose: Mutations in TIGR/myocilin 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 (MW) complexes above 180 kDa. Understanding the mechanisms generating these complexes may help to decipher the still unknown function of the polypeptide. We investigated the role of a putative coiled-coil motif at amino acids (AA) 78 to 105; the leucine zipper at AA 114 to 183; and 5 cysteine residues, in complex formation. Methods: Expression vectors encoding myc-tagged TIGR/myocilin mutants were created by site-directed mutagenesis. Mutated cDNAs were tested by transient transfection in COS-7 and human trabecular meshwork cells in culture. Intra- and extra-cellular complexes were separated by non-reducing SDS-PAGE and analyzed by Western blotting using anti-myc antibodies. Results: Deletion of the first coiled-coil motif (AA 78-105) severely impaired TIGR/myocilin oligomerization above 180 kDa without affecting its dimerization. In contrast, deletion of the leucine zipper (AA 114-183) did not impair oligomerization nor dimerization. Single substitution of critical AAs in the first coiled-coil motif (78-105) by alpha-helix breaker glycine residues significantly abrogated the ability of the protein to form stable oligomers. Triple substitutions (between AA 84-99) abolished high MW complex formation without affecting dimerization. The single cysteine to alanine substitution at position 185 and double cysteine to alanine substitution at positions 47 and 61 abolished covalent TIGR/myocilin oligomerization. Simultaneous substitution of cysteines 47, 61 and 185 resulted only in the expression of monomeric species. Mutation of the 245 and/or 433 cysteines blocked TIGR/myocilin secretion without altering neither its oligomerization or dimerization. Conclusions: Our experiments demonstrate that the putative coiled-coil motif between AA 78 to 105 was essential for efficient TIGR/myocilin oligomerization whereas deletion of the leucine zipper motif had no effect on complex formation. These data led us to suggest that the coiled-coil motif at residues 78 to 105 may act as a nucleation site (a region involved in specific recognition of oligomerization partners), the oligomers being subsequently stabilized through disulfide bonds involving cysteines 47, 61 and 185.
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