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J.D. Aroca–Aguilar, Y. Li, S. Ghosh, F. Sánchez–Sánchez, J. Escribano, M. Coca–Prados; Interaction of Myocilin With the C–Terminal Region of Hevin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):199.
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© ARVO (1962-2015); The Authors (2016-present)
To identify and characterize new proteins that interact with myocilin, a matricellular protein that is mutated in glaucoma.
The yeast two–hybrid system was used to identify myocilin interacting proteins. In order to confirm the interaction, candidate cDNA was cloned and co–expressed with myocilin in 293–T cells. The co–localization of proteins was detected by Western immunobloting and immunofluorescence of transfected cells. In addition the co–localization of the candidate protein and myocilin was analyzed by immunohistochemistry of bovine ciliary body.
Here we report the identification and characterization of a putative interacting protein with myocilin. One of the positive clones obtained by the yeast two–hybrid exhibited 100% identity with the carboxyl–terminal (C–t) region of hevin, a member of the BM–40/SPARC/osteonectin family of extracellular matrix proteins. Protein interaction was assayed, in doubly transfected 293–T cells, by Western blot and fluorescent microscopy. Western blot analysis of the culture medium and lysates from co–transfected cells indicated that myocilin causes intracellular accumulation of hevin–C–t and impairs its secretion. This effect on hevin–C–t was augmented when co–expressed with the myocilin P370L mutant, known to cause a severe form of glaucoma. By fluorescent microscopy, myocilin localizes with hevin–C–t in the Golgi in co–transfected 293–T cells and with hevin–wt in the ocular ciliary epithelium.
Overall, these results suggest that the C–t of hevin contains important determinants for interaction with myocilin.
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