title: Characterization of the intracellular proteolytic cleavage of myocilin and identification of calpain II as a myocilin-processing protease.
abstract: 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.
authors.affiliation: Area de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Avda. de Almansa, no. 14, 02006 Albacete, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Sánchez
authors.forename: Francisco
authors.initials: F
authors.lastname: Martínez-Redondo
authors.forename: J Daniel
authors.initials: JD
authors.lastname: Aroca-Aguilar
authors.forename: Miguel
authors.initials: M
authors.lastname: Coca-Prados
authors.forename: Julio
authors.initials: J
authors.lastname: Escribano
mesh_terms: D002135:Calcium-Binding Proteins / Q000737:chemistry; D002154:Calpain / Q000737:chemistry*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry / Q000494:pharmacology; D019286:Endopeptidase K / Q000737:chemistry; D005136:Eye Proteins / Q000737:chemistry*; D005901:Glaucoma / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D020125:Mutation, Missense; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection; D000099179:Myocilin
chemical_list: D002135:Calcium-Binding Proteins; D002154:Calpain; D003598:Cytoskeletal Proteins; D004151:Dipeptides; D004533:Egtazic Acid; D019286:Endopeptidase K; D005136:Eye Proteins; D006023:Glycoproteins; D015759:Ionomycin; D000099179:Myocilin; C039304:calpain inhibitors; C071482:calpeptin; C033668:calpastatin