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J. Escribano, D. Aroca–Aguilar, F. Sánchez–Sánchez, S. Ghosh, M. Coca–Prados; Myocilin is Processed in the Secretory Pathway by an Endoproteolytic Cleavage at Arg226 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):36.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Myocilin is a secreted glycoprotein of unknown function that is ubiquitously expressed in many human organs, including the eye. Mutations in this protein cause glaucoma, a leading cause of blindness worldwide. To investigate the elusive biological function of this protein we have analyzed myocilin expression and secretion in the non–ocular cell lines COS1 and 293T and in the human ocular cell lines 26HCMsv and 59HIsv. Methods: Cells were transiently transfected with a myocilin cDNA construct containing both c–myc and His tags in the 3'–end. The presence of myocilin in the culture medium and in the intracellular medium was tested by Western immunoblot using anti–myc and anti–myocilin antibodies. Recombinant myocilin was isolated by Ni–chelating chromatography and analyzed by amino acid microsequencing and MALDI–TOF. The subcellular location of myocilin was studied in 293T cells transiently transfected with a DNA construct encoding C–terminal GFP–tagged myocilin. Results: All the transfected cell lines secreted different ratios of two major forms of myocilin with apparent molecular masses around 55 and 35 kDa. The 55 kDa form corresponds to the full–length protein. N–terminal sequencing and mass spectrometry analysis of the 35 kDa form, isolated by Ni–chelating chromatography, demonstrated that it corresponds to a myocilin C–terminal fragment containing the olfactomedin–like domain, which arises from an endoproteolytic cleavage at the C–terminus of Arg226. Treatment of transfected 293T cells with the protein secretion inhibitor brefeldin A showed that this cleavage occurs intracellularly in the endoplasmic reticulum. Accordingly, the protein was detected in the endoplasmic reticulum and Golgi apparatus by fluorescence microscopy examination of 293T cells transfected with a myocilin–GFP construct. Conclusions: Myocilin is endoproteolytically processed intracellularly in the ER. The cleavage takes place at the C–terminal end of the amino acid residue Arg226. The procesed olfactomedin–like domain is secreted to the extracellular medium. We hypothesize that the activity of myocilin could be regulated by means of a specific proteolytic cleavage at Arg226.
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