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J. Biol. Chem., Vol. 280, Issue 22, 21043-21051, June 3, 2005

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Myocilin Mutations Causing Glaucoma Inhibit the Intracellular Endoproteolytic Cleavage of Myocilin between Amino Acids Arg²²⁶ and Ile^227*

J. Daniel Aroca-Aguilar¶, Francisco Sánchez-Sánchez, Sikha Ghosh||, Miguel Coca-Prados||, and Julio Escribano**

From the Área de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Avda. de Almansa sn, 02006 Albacete, Spain and the ^||Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510

Myocilin is a secreted glycoprotein of unknown function that is ubiquitously expressed in many human organs, including the eye. Mutations in this protein produce glaucoma, a leading cause of blindness worldwide. To explore the biological role of myocilin and the pathogenesis of glaucoma, we have analyzed the expression of recombinant wild type and four representative pathogenic myocilin mutations (E323K, Q368X, P370L, and D380A) in transiently transfected cell lines derived from ocular and nonocular tissues. We found that wild type myocilin undergoes an intracellular endoproteolytic processing at the C terminus of Arg²²⁶. This cleavage predicts the production of two fragments, one of 35 kDa containing the C-terminal olfactomedin-like domain, and another of 20 kDa containing the N-terminal leucine zipper-like domain. Here we have analyzed the 35-kDa processed fragment, and we have found that it is co-secreted with the nonprocessed protein. Western immunoblot analyses showed that human aqueous humor and some ocular tissues also contain the processed 35-kDa myocilin, indicating that the endoproteolytic cleavage occurs in vivo. Mutant myocilins accumulated in the endoplasmic reticulum of transfected cells as insoluble aggregates. Interestingly, the four pathogenic myocilins inhibited the endoproteolytic processing with varying efficiency. Furthermore, the mutation P370L, which produces the most severe glaucoma phenotype, also elicited the most potent endoproteolytic cleavage inhibition. We propose that the endoproteolytic processing might regulate the activity of myocilin and that the inhibition of the processing by pathogenic mutations impairs the normal role of myocilin.

Received for publication, February 4, 2005 , and in revised form, March 28, 2005.

^* This work was supported in part by "Ministerio de Ciencia y Tecnología," "Consejería de Sanidad," and "Consejería de Ciencia y Tecnología de la Junta de Comunidades de Castilla-La Mancha" Grants SAF2002-03086, 02021-00, and PAI-02-049, respectively (to J. E.), National Institutes of Health Grant EY04873, and a Research to Prevent Blindness Lew Waserman Merit Award (to M. C.-P.). 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 an additional figure.

These authors contributed equally to this work.

^¶ Recipient of a fellowship from the "Consejería de Sanidad de la Junta de Comunidades de Castilla-La Mancha."

^** To whom correspondence should be addressed. Tel.: 34-967-599200 (ext. 2928); Fax: 34-902-204130; E-mail: julio.escribano{at}uclm.es.

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