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J. Biol. Chem., Vol. 280, Issue 32, 28917-28926, August 12, 2005

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Extracellular Trafficking of Myocilin in Human Trabecular Meshwork Cells^*

Katharine M. Hardy, Emely A. Hoffman, Pedro Gonzalez¶, Brian S. McKay, and W. Daniel Stamer||**

From the Departments of Cell Biology, Ophthalmology, and ^||Pharmacology, the University of Arizona, Tucson, Arizona 85724 and the ^¶Department of Ophthalmology, Duke University, Durham, North Carolina 27705

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.

Received for publication, May 2, 2005 , and in revised form, June 7, 2005.

^* This work was supported by National Institutes of Health Grant EY12797, the Research to Prevent Blindness Foundation, and the Lions Clubs of Arizona. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table S1, additional Refs., and Fig. S1.

^** To whom correspondence should be addressed: University of Arizona, 655 North Alvernon Way, Suite 108, Tucson, AZ 85711. Tel.: 520-626-7767; Fax: 520-626-1757; E-mail: dstamer{at}eyes.arizona.edu.

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