Perfusion with the Olfactomedin Domain of Myocilin Does Not Affect Outflow Facility

Goldwich, Andreas; Ethier, C. Ross; Chan, Darren; Tamm, Ernst R.

doi:10.1167/iovs.02-0863

Cited by 36 publications

(35 citation statements)

References 19 publications

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“…Therefore, it will be important to identify factors influencing this processing. In accordance with our results and as mentioned above, Goldwich et al (55) found that long term myocilin production in 293 EBNA cells resulted in significant increase of N-and C-terminal fragments of the protein.…”

Section: Discussionsupporting

confidence: 94%

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

Sánchez-Sánchez

Martínez-Redondo

Aroca-Aguilar

et al. 2007

Journal of Biological Chemistry

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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 calciumactivated 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.

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Section: Discussionsupporting

confidence: 94%

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

Sánchez-Sánchez

Martínez-Redondo

Aroca-Aguilar

et al. 2007

Journal of Biological Chemistry

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“…Four findings sustain that the 35-kDa myocilin fragment is not a product of nonspecific degradation: (i) general proteinase inhibitors do not affect its production, as Goldwich et al also observed (40); (ii) deletion of the cleavage site inhibits the production of the 35-kDa fragment; (iii) the 35-kDa fragment originates intracellularly, probably in the ER and; (iv) different pathogenic mutations also inhibit the endoproteolytic cleavage. Interestingly, one such reported fragment was produced by 293 EBNA cells as a doublet, and it was determined that it matched the C-terminal region of myocilin, starting at the amino acid residue Leu 215 (40), in a place close to the cleavage site reported here. These data raise the possibility that proteolysis might occur at other nearby sites.…”

Section: Discussionmentioning

confidence: 57%

“…Several studies have reported the existence of myocilin fragments of ϳ30 -35 kDa in samples of recombinant myocilin expressed in cell lines and in tissue extracts (36,40,42). These fragments were usually interpreted as products of nonspecific myocilin proteolysis.…”

Section: Discussionmentioning

confidence: 99%

Myocilin Mutations Causing Glaucoma Inhibit the Intracellular Endoproteolytic Cleavage of Myocilin between Amino Acids Arg226 and Ile227

Aroca-Aguilar

Sánchez-Sánchez

Ghosh

et al. 2005

Journal of Biological Chemistry

114

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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 Arg 226. 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.

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“…1A). Published data suggest that the C-terminal fragment of myocilin is secreted (3,23,66), while data concerning secretion of the N-terminal fragment are controversial (23,66). To test whether N-and C-terminal fragments of myocilin are (52) and anti-FLAG antibodies, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Myocilin Is a Modulator of Wnt Signaling

Kwon

Lee

et al. 2009

Molecular and Cellular Biology

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It is well documented that mutations in the MYOCILINGlaucoma is a leading cause of blindness in the world. Primary open-angle glaucoma is the most common form of glaucoma. It will affect more than 60 million and blind about 4.5 million people worldwide by the year 2010 (62). It is now well established that a genetic component may contribute to glaucoma, and several glaucoma-associated genes have been identified. The first-identified and the most-studied gene is MYOCILIN (MYOC), which is highly expressed in and secreted by the trabecular meshwork (1,19,72,75,77,78), one of the key components of the eye aqueous humor outflow system. Dominant mutations in MYOC may lead to juvenile openangle glaucoma and are found in 3 to 4% of patients with primary open-angle glaucoma (18,19). The encoded protein, myocilin, belongs to a family of glycosylated proteins containing a C-terminal olfactomedin domain (57,90,91). Olfactomedin domain was originally identified in a glycoprotein isolated from the olfactory epithelium of frogs (71) and subsequently was found in a group of proteins forming a family of olfactomedin domaincontaining proteins. The family of olfactomedin domain-containing proteins includes both secreted and membrane-bound proteins that each exhibit a characteristic distribution in different tissues (4,10,27,30,44,51,58,78,79). Most of the glaucomacausing mutations in myocilin are located in the olfactomedin domain (1,2,18,19,24,72).Besides the trabecular meshwork and sclera (1, 77), high levels of MYOC were observed in the ciliary body (1, 13, 39), iris (89), retinal pigment epithelium/choroid (78, 88), and optic nerve (74). Low levels of MYOC expression were detected in several nonocular tissues (75). Myocilin, being a secreted protein, was also found in the aqueous humor, an intraocular fluid responsible for the supply of nutrients and for removal of metabolites from the avascular tissues of the eye anterior segment (63, 65). Some mutations in the MYOC gene lead to the inhibition of mutated myocilin secretion. Secretion of wildtype myocilin also can be reduced or blocked in the presence of mutated myocilin (22,36,52). It has been suggested that the intracellular accumulation of myocilin aggregates is deleterious to the trabecular meshwork cells, resulting in the deterioration of their function and subsequent elevation of intraocular pressure (IOP) (38,49).Although the MYOC gene has been studied for more than 10 years, the functions of myocilin protein are still not well understood (19,75). Biochemical data indicate that myocilin may interact with several intracellular and extracellular matrix proteins (16,37,48,61,73,78), although the biological significance of such interactions is not clear. The absence of openangle glaucoma in an elderly woman homozygous for the Arg46Stop mutation (45), as well as the absence of glaucoma in people hemizygous for MYOC (87), suggests that the loss of functional myocilin is not critical for the development of glaucoma or for normal eye functioning. These observations are supporte...

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Perfusion with the Olfactomedin Domain of Myocilin Does Not Affect Outflow Facility

Cited by 36 publications

References 19 publications

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

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

Myocilin Mutations Causing Glaucoma Inhibit the Intracellular Endoproteolytic Cleavage of Myocilin between Amino Acids Arg226 and Ile227

Myocilin Is a Modulator of Wnt Signaling

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