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J. Biol. Chem., Vol. 282, Issue 38, 27810-27824, September 21, 2007

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Characterization of the Intracellular Proteolytic Cleavage of Myocilin and Identification of Calpain II as a Myocilin-processing Protease^*

Francisco Sánchez-Sánchez¹, Francisco Martínez-Redondo¹², J. Daniel Aroca-Aguilar³, Miguel Coca-Prados, and Julio Escribano⁴

From the Área de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Avda. de Almansa, no. 14, 02006 Albacete, Spain and the Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.

Received for publication, October 11, 2006 , and in revised form, June 22, 2007.

^* This work was supported in part by "Ministerio de Ciencia y Tecnología," "Consejería de Sanidad," "Consejería de Ciencia y Tecnología" de la Junta de Comunidades de Castilla-La Mancha, and "Fondo de Investigaciones Sanitarias" Grants SAF2002-03086, 02021-00, PAI-02-049, and PI052494 (to J. E.); National Institutes of Health Grants EY04873 and EY00785 (for core facilities); and grants from Research to Prevent Blindness and the Connecticut Lions Foundation (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 supplemental Fig. S1.

¹ Both authors contributed equally to this work.

² Recipient of a fellowship from the "Consejería de Educación y Ciencia."

³ Recipient of a fellowship from the "Consejería de Sanidad de la Junta de Comunidades de Castilla-La Mancha."

⁴ To whom correspondence should be addressed:Área de Genética, Facultad de Medicina, Avda. de Almansa, no. 14, 02006 Albacete, Spain. Tel.: 34-967-599200 (ext. 2928); Fax: 34-902-204130; E-mail: julio.escribano{at}uclm.es.

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