Purchase this article with an account.
K.M. Hardy, E.A. Hoffman, B.S. McKay, W.D. Stamer; Biochemical Properties of Extracellular Myocilin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3664.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Myocilin (MYOC) is the protein product of GLC1A, the first gene linking to primary open–angle glaucoma. In most cells types, MYOC has an intracellular distribution. However, in human trabecular meshwork (HTM) cells MYOC localizes to both the intracellular and extracellular compartments. Thus, HTM cells appear to traffic MYOC differently than other cell types. To investigate this difference, we compared the cellular and extracellular distribution of MYOC to a conventionally secreted protein, tissue plasminogen activator (tPA). Methods: Cell homogenates and conditioned medium were collected from confluent HTM cell cultures and processed separately. Cell homogenates were subjected to differential centrifugation to isolate cytosol and membrane fractions. Intracellular membranes were segregated by sucrose gradient sedimentation. Fractions were analyzed by SDS–PAGE/western blot using antibodies against MYOC, tPA, and organelle specific markers. Conditioned medium was precleared of dead cells/debris, then extracellular membranes were isolated by centrifugation at 100,000xg. The density of these membrane precipitates was verified by their upward mobility in sucrose gradient analyzes. Fractions from these gradients were analyzed by SDS–PAGE/western blotting as described above. Results: Intracellularly, MYOC was abundant in both cytosol and membranes fractions while tPA exclusively associated with membranes. Interestingly, MYOC localized to a membrane fraction distinct from, and less dense than, traditional secretory vesicles, where tPA was distributed. Extracellularly, the majority of MYOC associated with dead cells/debris or was soluble in the medium. A proportion of MYOC partitioned with an extracellular membrane fraction having unique properties consistent with that of exosomes. In contrast, tPA was completely excluded from the extracellular membrane fraction. Conclusions: Disparity between the subcellular localization of MYOC and tPA suggests that these proteins are processed differently. The association of MYOC with extracellular membranes, free of tPA but with a density typical of exosomes, indicates that the appearance of MYOC in the extracellular space differs from a conventionally secreted protein.
This PDF is available to Subscribers Only