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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

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Myocilin, initially denominated trabecular meshwork-inducible glucocorticoid response protein, or TIGR, is an extracellular glycoprotein (55–57 kDa) with a modular design consisting of three domains: (i) the N-terminal leucine zipper-like region; (ii) a central putative linker domain; and (iii) the C-terminal olfactomedin-like domain (encoded by exon 1, 2, and 3, respectively). Mutations in this gene not only cause autosomal dominant juvenile glaucoma but also a subset of adult onset primary open angle glaucoma, generally associated with high intraocular pressure (IOP)⁵ (1, 2). MYOC mRNA is ubiquitously expressed, showing high levels in muscular tissues (i.e. heart and skeletal muscle) and in ocular tissues, such as iris, ciliary body (CB), and trabecular meshwork (TM) (3–8). The protein is also present in aqueous humor (9, 10). Interestingly, most MYOC mutations reported to date in sporadic cases of primary open angle glaucoma are heterozygous and are confined to exon 3 (2, 5, 6, 11–13). Recently, we showed that myocilin undergoes an intracellular endoproteolytic cleavage in the endoplasmic reticulum (ER) between amino acids Arg²²⁶ and Ile²²⁷ located in the putative linker domain (10). This processing predicts the production of two fragments corresponding to the N- and C-terminal domains. The processed C-terminal domain is co-secreted with the full-length protein, but the fate of the processed N-terminal domain has not yet been investigated. We also reported that pathogenic mutations located in the olfactomedin-like domain of myocilin reduce the proteolytic cleavage in the middle of the protein. The highest inhibition of the processing was produced by the myocilin mutation P370L, which is associated with the most severe glaucoma phenotype. Mutations E323K and D380A, found in less severe glaucoma cases, showed a lower proteolytic inhibition.

Despite many efforts made over recent years, the function of this protein in the normal and glaucomatous eye is poorly understood. There is great interest in identifying proteins that interact with myocilin, since it is believed that they could play a key role in its biological function. Several extracellular proteins have been identified to interact with myocilin, including fibronectin (through the HepII domain) (14), the C-terminal regions of fibrilin-1 (15), and hevin, an extracellular matrix protein member of the BM-40/SPARC/osteonectin family (16). Although the functional meaning of the proteolytic processing of myocilin is currently unknown, it has been suggested that controlled changes in the proportion of processed myocilin might contribute to regulate the normal TM structure by differential intermolecular interactions with other extracellular matrix molecules, therefore contributing to modulate aqueous humor outflow and IOP (10).

Calpains comprise a superfamily of 15 mammalian genes (17). They are heterodimeric calcium-dependent intracellular cysteine proteases located in the cytosol, ER, and Golgi apparatus (GA) (18) and are present in most mammalian tissues, including the CB (3, 19–21). Two major types of calpains are known: ubiquitous calpains (calpain I/µ-calpain, calpain II/m-calpain, and calpain 10) and tissue-specific calpains. They are activated by increased intracellular Ca²⁺ at physiologic states and pathologic conditions (22–24) and are implicated in a variety of cellular processes, including cell signaling, attachment, spreading, and migration (25–28). It has been suggested that calpains play an important role in retinal cell death induced by ischemic reperfusion (29) or by hypoxia in cultured retina (30). Common calpain targets include fibronectin (31), cytoskeletal proteins, various muscle proteins, and neurofilament proteins (32–35). The enzymatic activity of calpain is also regulated by calpastatin, an endogenous inhibitory protein (36). Conformational determinants are a significant factor in the cleavage site specificity of this protease (28, 33, 37). Calpain hydrolyzes substrate proteins in a limited manner, producing large fragments retaining intact domains. Calpain is regarded as a biomodulator, since properties of the substrate proteins are often modulated upon cleavage (22, 38, 39).

Our objectives in this work were as follows: (i) to characterize the fate of myocilin fragments released after proteolytic processing; (ii) to analyze the role in the proteolytic processing of different amino acids located around the cleavage point; and (iii) to identify the enzyme responsible for the processing. We show herein that the C-terminal fragment is secreted into the culture medium upon proteolytic processing and that the N-terminal domain remains associated mainly with the cellular fraction. We conclude that myocilin processing is carried out in the lumen of the ER by calpain II.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Myocilin Constructs—Wild-type myocilin cDNA cloned in the mammalian expression vector pcDNA3.1-myc-His (10) was used as a template to produce all myocilin constructs used in this study. A cDNA construct encoding wild-type myocilin, which in addition to the C-terminal Myc epitope incorporates the HA epitope contiguous to the signal peptide sequence (amino acid residue 32), was obtained by amplifying two PCR fragments using primers 1–6 and 3–7, respectively (Table 1). The two PCR products were purified, mixed, and used as PCR templates with primers 1 and 3. The amplified fragment was directly cloned into the EcoRI-BamHI sites of the empty pcDNA3.1-myc-His vector. The N-terminal domain of myocilin (amino acid residues 1–226), cloned into the pcDNA3.1-HA-His vector, was generated by PCR using primers 1 and 2. The C-terminal domain of myocilin (amino acid residues 217–504) was also produced by PCR using primers 3 and 4 (Table 1). The signal peptide of myocilin (amino acid residues 1–32) was incorporated at the 5' end of this PCR fragment using primers 1 and 5 (Table 1). The final PCR product was also cloned into the pcDNA3.1-HA-His vector. Myocilin deletion mutants were obtained as previously described (10). Primer sequences used to generate these mutants are shown in Table 1.

Calpastatin Construct—A cDNA construct encoding the inhibitor domain 1 of calpastatin (Cs-D1) (amino acid residues 143–169), fused to the signal peptide of myocilin (amino acids 1–32) at the N-terminal end, was obtained by reverse transcription-PCR. cDNA synthesized from 293T cell mRNA was used as a template for amplification with primers 32 and 33 (Table 1). The amplified fragment was directly cloned into the EcoRI-BamHI sites of the pcDNA3.1-myc-His vector.

Cell Culture—Human embryonic kidney 293T cells and human retinal pigmented epithelium ARPE-19 cells were bought from the ATCC (American Type Culture Collection). The human cell line 26HCMsv was established from the primary culture of ciliary muscle (CM) cells of a 26-year-old male (cadaver) by viral transformation, as previously described (40). 293T and 26HCMsv cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (Normocin; Invitrogen) at 37 °C in a fully humidified 5% CO₂ atmosphere. ARPE-19 cells were maintained in a 1:1 mixture of DMEM and Ham's F-12 medium, supplemented with 10% fetal bovine serum and antibiotics (Normocin; Invitrogen) at 37 °C in a fully humidified 5% CO₂ atmosphere. Transient plasmid transfections were carried out with 200–500 ng of DNA using the Superfect transfection reagent (Qiagen), following the manufacturer's instructions. Samples were centrifuged at 5,000 x g for 5 min to remove cellular debris from the collected culture medium. The supernatant was stored at -80 °C until used. Adhered cells were washed twice with 1 ml of Dulbecco's phosphate-buffered saline (150 mM NaCl, 3 mM KCl, 1 mM KH₂PO₄, 6 mM Na₂HPO₄, 0.5 mM MgCl₂, 1 mM CaCl₂, pH 7.2), followed by the addition of 200 µl of lysis buffer containing proteinase inhibitors, as previously described (10). Collected cells were vortexed for 30 s at maximum speed, incubated for 30 min on ice, and sonicated for 10 s (cycle 0.5 s). Cell lysates were centrifuged at 15,000 x g for 4 min at 4 °C. The supernatants (cellular soluble fraction) were carefully separated from the pellets (cellular insoluble fraction). Both cellular fractions were stored at -80 °C until used. The efficiency of transfections was estimated in cells transiently transfected with a cDNA construct encoding green fluorescent protein (GFP) by counting the number of GFP-positive cells in a total of 10³ cells in four randomly selected areas per dish. As a negative control, the different cell lines were transfected with 200–500 ng of the nonrecombinant pcDNA3.1-myc-His vector. For time course experiments, the culture medium was replaced by fresh medium every day over a 6-day period.

Tissues—Human eyes were obtained from human cadavers within 24 h after enucleation through the National Disease Research Interchange (Philadelphia, PA). A sample of human skeletal muscle was obtained from a cadaver from the "Servicio de Anatomía Patológica, Complejo Hospitalario Universitario de Albacete," Spain. Eyes were dissected from the posterior pole, both the vitreous and lens were removed, and the CB was microdissected. Homogenization of both the human CB and human skeletal muscle was performed by mechanical grinding with liquid nitrogen using a pestle and mortar. Pulverized frozen tissue was suspended in lysis buffer (10), vortexed for 30 s at maximum speed, and incubated for 30 min on ice. The lysates were centrifuged at 15,000 x g for 4 min at 4 °C. Supernatants were stored at -80 °C until used.

Western Blotting and Antibodies—For Western blot analysis, aliquots of culture medium and intracellular fractions of cultured cell lines (both soluble and insoluble) were fractionated by SDS-PAGE as reported by Laemmli (41), using the Mini-PROTEAN III gel electrophoresis system (Bio-Rad). The samples were normalized for protein content using the Bradford assay. Gels were subsequently transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences) for immunodetection. Commercial mouse monoclonal anti-Myc, anti-HA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-calpain I (Calbiochem) were used as primary antibodies and diluted at 1:1000. Other primary antibodies were anti-calpain II (Calbiochem), anti-calnexin (Sigma), anti-14-3-3 (Sigma), and the anti-myocilin C21A antibody (7), which were diluted at 1:400, 1:3000, 1:2,000, and 1:400, respectively. Horseradish peroxidase-conjugated antibodies against goat (Santa Cruz Biotechnology), mouse, or rabbit IgG (Pierce) were diluted at 1:1,000–1:4,000.

Chemiluminiscence detection was performed with Supersignal Dura Western blot reagents (Pierce). Densitometry for protein band quantification was performed on scanned films using Quantity One 4.1 analysis software (Bio-Rad) in triplicate independent experiments. Data were statistically treated by using SigmaStat 2.0 software (SPSS Science).

Fluorescence Microscopy—26HCMsv cells were seeded in coverslips placed into 24-well plates and transiently transfected with DNA constructs encoding different myocilin-tagged variants. DNA constructs encoding GFP-tagged versions of the first 80 N-terminal amino acids of galactosyltransferase or the signal peptide of calreticulin were used as specific fluorescent GA and ER markers, respectively. These constructs were kindly provided by Dr. Juan Llopis ("Facultad de Medicina/CRIB, Universidad de Castilla-La Mancha, Spain"). All transfections were performed with SuperFect Transfection Reagent (Qiagen). After transfection, cells were washed once with Dulbecco's phosphate-buffered saline and cultured for 24 h. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, followed by incubation with phosphate-buffered solution containing 0.2% Triton X-100, 10% fetal bovine serum, 5% bovine serum albumin for 30 min at room temperature. The recombinant proteins were detected with an anti-HA antibody (Santa Cruz Biotechnology) (at 1:500 dilution) at 4 °C overnight followed by a Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) (1:1000) for 2 h at room temperature. Finally, coverslips were mounted on glass slides using polyvinyl alcohol mounting medium with DABCO (Fluka) and viewed under a fluorescence microscope (Leica DMR/XA) with proper filter sets using a x100 plan objective. Images were captured using a Leica DC500 digital camera.

Protease Inhibitor Assays—In order to find out the protease involved in the endoproteolytic processing of myocilin, different membrane-permeable specific protease inhibitors were used: calpeptin (80 µM), calpain inhibitor IV (50 µM), and N-acetyl-leucyl-leucyl-norleucinal (ALLN) (40 µM) (inhibitors of calpains); lactacystin (0.25 µM) and MG-132 (0.25 µM) (proteosome inhibitors); and ammonium chloride (1 mM) (lysosomal cathepsin inhibitor). Five days after transfecting 293T cells with the wild-type myocilin construct, the culture medium was removed and substituted with fresh culture medium containing the different inhibitors. Cells were incubated for 16 h with the different inhibitors, the culture medium was then collected, and secreted myocilin was analyzed by Western blot. After treatment with the different proteinase inhibitors, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as described (42).

RNA Interference (RNAi)—Two RNAi approaches were assayed to knock down the expression of calpain. In the first one, four shRNA-specific clones for calpain II (MISSION^TM; Sigma; product number SHDNA-NM_001748) were used, and in the second approach a small interfering RNA (siRNA) specific for calpain II (Ambion Inc.; ID 105034) was employed. Calpain I expression was knocked down using five shRNA-specific clones (MISSION^TM; Sigma; product number SHDNA-NM_005186). Transient transfections were carried out using Superfect Transfection Reagent (Qiagen) according to the manufacturer's recommended procedures.

Treatment of Myocilin-transfected 293T Cells with Ionomycin or EGTA—293T cells were transiently transfected with the wild-type myocilin cDNA construct. The culture medium was removed 24 h after transfection, cells were washed with PBS, and fresh culture medium containing 2 µM ionomycin was added. To determine whether chelation of extracellular calcium inhibits the processing, culture conditions were established to enhance myocilin processing. After transfection, the culture medium was changed every day over a 6-day period. Cells were treated with 4 mM EGTA on the last day. Eight hours after treatments, either with ionomycin or EGTA, secreted myocilin was analyzed by Western blot.

Digestion of Myocilin with Purified Recombinant Calpains—Twenty microliters of conditioned medium containing wild-type myocilin were mixed with 4 µl of 10x digestion buffer (200 mM Tris-HCl, pH 7.5, 30 mM CaCl₂), 15 µl of water, and 1 µl (1 µg/µl) of purified porcine recombinant calpain I or calpain II (Calbiochem). Digestions were carried out at 37 °C in a final volume of 40 µl. Reactions were stopped by the addition of electrophoresis loading buffer. Digestion products were analyzed by Western blot.

Reverse Transcription-PCR Assay—RNA from cultured cells was extracted with the RNeasy minikit (Qiagen), and 1 µg of RNA was reverse transcribed into cDNA using random primers and oligo(dT) with the RevertAid^TM first strand cDNA synthesis kit (Fermentas) in the presence of 5x first strand buffer (250 mM Tris-HCl, 250 mM KCl, 20 mM Mg₂Cl, 50 mM dithiothreitol), 0.25 mM each dNTP (Fermentas), and 200 units of RevertAid^TM Moloney murine leukemia virus reverse transcriptase (Fermentas) for 1 h at 42 °C. For PCR analysis, 1 µl of cDNA was used as a template in a reaction volume of 20 µl containing 10x buffer (750 mM Tris-HCl, pH 9.0, 20 mM MgCl₂, 500 mM KCl, 200 mM (NH₄)₂SO₄), 200 µM dNTPs (Biotools), 10 pmol of primers (see Table 1), and 1 unit of TaqDNA polymerase (Biotools). Thermocycling included an initial denaturation step at 94 °C for 3 min, followed by 25 cycles of 20 s of denaturation at 94 °C, 20 s of annealing at 55 °C, and 20 s of extension at 72 °C. The PCR product was analyzed by agarose gel electrophoresis (1%) and visualized with ethidium bromide staining.

Subcellular Fractionation of 293T Cells Transiently Expressing Recombinant Myocilin—Subcellular fractionation of 293T cells expressing recombinant myocilin was performed using an Optiprep (Axis-Shield, Oslo, Norway) velocity gradient. 293T cells were transiently transfected with a cDNA construct encoding a C-terminal Myc-tagged version of wild-type myocilin (Fig. 1B). After 48 h of expression, cells were washed with PBS, harvested in homogenization buffer (5% Optiprep, 78 mM KCl, 4 mM MgCl₂, 8.4 mM CaCl₂, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM Hepes/KOH, pH 7.0), and broken by 10 repeated strokes in a Dounce homogenizer. Cell debris and nuclei were pelleted by centrifugation at 1,000 x g for 10 min. The postnuclear supernatant, containing cytosol and organelles, was overlaid onto a discontinuous gradient prepared using 1.5-ml layers of 20, 15, 10, and 7% Optiprep solution, from the bottom to the top of the ultracentrifuge tube, respectively. The gradient was allowed to equilibrate vertically at room temperature for 30 min and centrifuged at 100,000 x g for 18 h at 4 °C. After centrifugation, equal fractions of 500 µl were collected by gravity from the bottom of the tube (heavy to light density). Each fraction was divided into two 250-µl aliquots. To check the intraorganular or cytosolic localization of myocilin, calpain I, and calpain II, one of the aliquots of each fraction was treated with proteinase K (0.5 µg/ml) and incubated at 37 °C for 15 min. The digestion was stopped by the addition of 62 µl of 5x Laemmli sample buffer, followed by heating to 100 °C for 10 min. Thirty microliters of each fraction were analyzed by SDS-PAGE. Gels were subsequently transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences) for immunodetection of myocilin, calpain I, and calpain II, as indicated above. The presence of ER and GA microsomes in the different fractions was detected using anticalnexin and anti-14-3-3 protein antibodies, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Analysis of the N-terminal Domain of Myocilin Released after Endoproteolytic Processing—We recently reported that myocilin is intracellularly cleaved at Arg²²⁶ (10). Since antibodies used in the mentioned study recognized epitopes located in the C-terminal region of recombinant myocilin, we were unable to analyze the fate of the processed N-terminal fragment. To detect this myocilin fragment, we generated a myocilin cDNA construct carrying the HA epitope immediately after the signal peptide (at amino acid residue 32) and the Myc epitope at the C terminus (Fig. 1, A and B, HA-myocilin-myc). 293T cells were transiently transfected with this cDNA construct and cultured for 48 h. As expected, upon Western blot of the culture medium using the anti-Myc antibody, myocilin was resolved into a 55-kDa doublet (originated by the full-length protein) and a 35 kDa band (originated by the C-terminal domain) (Fig. 2A, c.m. lane). The N-terminal domain was detected by Western immunoblot (using an anti-HA antibody) mainly in the insoluble cellular fraction as a doublet of around 30 kDa (Fig. 2B, i.c.f. lane). A faint 30-kDa doublet was detected in the culture medium with longer exposure times (data not shown). Since predicted glycosylation sites, in myocilin, are located in the N-terminal region, we tested whether the 30-kDa doublet originated from differences in glycosylation. To that end, transiently transfected cells were treated with tunicamycin (1.5 µgr/ml), a specific inhibitor of N-glycosylation (43). We found that the upper bands of doublets around 55 and 30 kDa disappeared after this treatment (Fig. 2, C and D), showing that they originate from N-glycosylated forms of myocilin.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Scheme of myocilin cDNA constructs used in this study. A, the diagram represents the primary structure of myocilin locating different domains. B, scheme of four different cDNA constructs generated to analyze the fate of the processed N-terminal fragment. The numbers correspond to positions in the myocilin amino acid sequence. C, missense mutations of myocilin cleavage site (consensus sequence of the subtilisin/kexin isozyme-1) used in this study. The amino acid sequence is shown in single-letter code. The scheme above the mutations shows the position of the cleavage site in the putative linker domain of myocilin. D, scheme of different deletion constructs generated around the myocilin cleavage site. The numbers correspond to amino acid positions deleted in the amino acid sequence of myocilin. The dashed lines indicate positions of deleted regions. The boxes placed at the C-terminal end represent Myc (m) or HA epitopes (H), used to detect the recombinant proteins.

View larger version (86K): [in this window] [in a new window]   FIGURE 3. Subcellular distribution of full-length myocilin and the processed N- and C-terminal myocilin fragments in transiently transfected 26HCMsv cells. Two hundred nanograms of cDNA constructs encoding HA-tagged versions of the processed N-terminal fragment (amino acids 1–226; Fig. 1B)(A), the processed C-terminal fragment fused to the peptide signal of myocilin (amino acids 217–504; Fig. 1B)(B), and wild-type myocilin (C) were transfected into 26HCMsv cells and expressed for 24 h. Both the ER and GA were visualized by transfecting cells simultaneously with two DNA constructs encoding either GFP-tagged versions of the signal peptide of calreticulin or the first 80 N-terminal amino acids of galactosyltransferase (D). These two constructs are specific fluorescent markers for both the GA and ER, respectively. The arrows indicate the location of the GA. Original magnification was x1,600.

We also analyzed by immunocytochemistry the microscopic distribution of full-length myocilin and the two terminal domains, resulting after proteolytic cleavage, in transiently transfected 26HCMsv cells. Fluorescence from cells expressing the N-terminal leucine zipper-like domain was localized in the cytoplasm with a pattern that suggested ER staining and a weak perinuclear GA-like signal (Fig. 3A). Fluorescence of the olfactomedin-like domain showed a cytoplasmic distribution, indicating that it was localized in both the ER and GA (Fig. 3B). As expected, wild-type myocilin was detected in the secretory pathway, including a reticular network signal located around the nucleus and cytoplasm and an intense GA-like signal in the perinuclear region (Fig. 3C), resembling those signals produced by specific fluorescent markers for the ER and GA (Fig. 3D). These data are also in accordance with our Western immunoblot analyses. Similar results were obtained with 293T cells (data not shown). Collectively, these results consistently show that the C-terminal domain of myocilin resulting upon myocilin processing is secreted, whereas the N-terminal domain remains retained in the ER.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Analysis of the endoproteolytic processing of myocilin in transiently transfected 293T cells. A cDNA construct encoding wild-type myocilin carrying the Myc epitope at the C terminus and the HA epitope at the N terminus (Fig. 1B, HA-myocilin-myc) was transfected into 293T cells. Forty-eight hours after transfection, myocilin was analyzed in the culture medium (20 µl) and in the soluble and insoluble intracellular fractions (20 µg of total protein) by SDS-PAGE (10% polyacrylamide). Myocilin detection was carried out by Western blot using anti-Myc (A) or anti-HA (B) monoclonal antibodies. The arrows indicate the position of the 55-kDa myocilin doublet. The black arrowhead indicates the position of the 35-kDa myocilin band corresponding to the processed olfactomedin-like domain. The white arrowheads indicate the position of the N-terminal domain. This fragment was detected in the insoluble cellular fraction. Shown is Western blot analysis of full-length myocilin (C) and the N-terminal domain (amino acids 1–226; Fig. 1B)(D), expressed in transfected 293T cells treated with tunicamycin (1.5 µg/ml) for 24 h. Recombinant proteins were detected in the culture medium (C) or in the insoluble cellular fraction (D) using an anti-HA monoclonal antibody. E, expression of two cDNA constructs encoding the N-terminal (amino acids 1–226) (N-t) and C-terminal (amino acids 217–504) (C-t) proteolytic fragments of myocilin (Fig. 1B). The cDNAs were transfected into 293T cells, and 48 h after transfection, distribution of these domains among the cell culture medium, soluble cellular fraction, and insoluble cellular fraction was analyzed by Western blot using an anti-HA monoclonal antibody. c.m., culture medium; s.c.f., soluble cellular fraction; i.c.f., insoluble cellular fraction; Non trans., nontransfected; Tunic., tunicamycin.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Time course analysis of the endoproteolytic processing of myocilin in transiently transfected cells. A cDNA construct encoding wild-type myocilin carrying the Myc epitope at its C terminus (Fig. 1B, myocilin-myc) was used to transiently transfect either 293T (200 ng of cDNA) (A), or 26HCMsv cells (500 ng of cDNA) (B). After transfection, the culture medium was removed, cells were washed with PBS, and fresh medium was added for 24 h. The culture medium was replaced with fresh medium every day, and aliquots (20 µl) were analyzed by SDS-PAGE (10% polyacrylamide) under reducing conditions. Recombinant myocilin was detected by Western blot using an anti-Myc monoclonal antibody. The arrows indicate the position of the 55-kDa myocilin band. The arrowheads indicate the position of the 35-kDa myocilin band corresponding to the processed olfactomedin-like domain.

Site-directed and Deletion Mutational Analysis of Myocilin Processing—We have reported that the cleavage site of myocilin (²²⁶RILKE²³⁰) contains the predicted consensus sequence of the subtilisin/kexin isozyme-1 and that deletion of this sequence inhibits myocilin processing after 48 h of expression (10). We analyzed the role of the amino acid sequence of this site in the cleavage of the protein by site-directed and deletion mutational analysis (Fig. 1, C and D). Mutants were transiently expressed into 293T cells. Western blot analysis of the culture medium showed that mutations K229A and E230A did not significantly affect proteolytic processing 48 h after transfection (Fig. 5A), indicating that these two residues are not essential for myocilin cleavage. On the other hand, mutations R226A and I227G reduced processing 48 h after transfection (Fig. 5A). Interestingly, the mutation R226Q slightly increased myocilin processing, in accordance with the fact that the bovine protein has a Gln residue at the equivalent position and is efficiently processed (10). Two double mutants (R226A/K229A and R226A/E230A) and deletion of the consensus sequence of the subtilisin/kexin isozyme-1 (226–230del) completely blocked endoproteolytic processing after 48 h of expression (Fig. 5A).

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Characterization of the cleavage site of myocilin by site-directed mutagenesis. Two hundred nanograms of cDNA constructs encoding either wild-type or different missense mutations of myocilin located in the predicted subtilisin/kexin isozyme-1 cleavage site (Fig. 1C) were used to transiently transfect 293T cells. The culture medium was replaced with fresh medium every day, and at 2 (A) and 6 (B) days after transfection, recombinant myocilin in the culture medium (20 µl) was analyzed by SDS-PAGE (10% polyacrylamide) and Western blot using an anti-Myc monoclonal antibody. The arrows and arrowheads indicate the positions of 55-kDa full-length and 35-kDa processed myocilin C-terminal domain, respectively.

To check the hypothesis of alternative processing sites that are located close to the one already identified, we performed four deletions around the cleavage site (Fig. 1D, 226–230del, 216–230del, 213–230del, and Exon2del). It was observed that these deletions blocked the cleavage after 48 h of expression (Fig. 6A). However, only deletion of the whole peptide encoded by exon 2 significantly reduced myocilin cleavage after 6 days of expression (Fig. 6B), suggesting that the complete linker domain is required for the efficient proteolytic processing of myocilin.

A consensus furin cleavage site is predicted at amino acid residues 179–182 (RLRR). To find out whether it was involved in the possible alternative cleavage of myocilin, we removed this site from the full-length protein (Fig. 1D, delFur) as well as from the deletion mutants mentioned above (Fig. 1D, 213–230delFur, 216–230delFur, and Exon2delFur). Six days after transfection, all furin deletion mutants were processed like the corresponding proteins containing the furin cleavage site (Fig. 6C), showing that the predicted furin consensus site is not an alternative myocilin cleavage site.

Effect of Different Protease Inhibitors on the Proteolytic Processing of Myocilin Expressed in Cells in Culture—Collectively, the above data indicate that the subtilisin/kexin isozyme-1 is not involved in myocilin processing. They also show that although certain amino acid substitutions in the cleavage site affect myocilin proteolysis, the protease carrying out the processing has no strict requirements in the amino acid sequence of the cleavage site. This is a known feature of calpains (44, 45). Thus, we set out experiments to identify the protease involved in the endoproteolytic processing of myocilin, considering calpains as the main candidates. 293T cells transiently transfected with myocilin-myc cDNA were incubated with different membrane-permeable protease inhibitors, including three well characterized calpain inhibitors (calpain inhibitor IV, calpeptin, and ALLN), two specific proteosome inhibitors (lactacystin and MG-132), and one inhibitor of lysosomal cathepsins (ammonium chloride). Myocilin cleavage was significantly prevented by the three calpain inhibitors (Fig. 7, A and C). In contrast, the other protease inhibitors did not affect myocilin processing. These data indicate that intracellular calpains are responsible for the proteolytic processing of myocilin. We analyzed cell viability to rule out any processing differences due to the cytotoxicity of inhibitors. No significant cell viability differences among treatments, at the given concentrations, were found (Fig. 7D). Higher concentrations of the protease inhibitors used in these experiments reduced protein synthesis and cell viability (data not shown). To determine whether myocilin processing is also carried out by calpains in ocular cell lines, 26HCMsv and ARPE-19 cells were transiently transfected with myocilin and treated with calpeptin. We observed that the processing was also significantly inhibited in these cell lines (Fig. 7B).

Effect of EGTA and Ionomycin on the Proteolytic Processing of Myocilin Expressed in Cells in Culture—Since calpains are intracellular calcium dependent proteases, we tested whether calcium concentration variations affect myocilin processing. We chelated extracellular calcium by adding EGTA to the culture medium of 293T cells transfected with myocilin. In another set of experiments, we induced calcium uptake with the calcium ionophore ionomycin in cells expressing recombinant myocilin. Myocilin processing was inhibited by EGTA (Fig. 8A) and up-regulated by ionomycin (Fig. 8B). These results show that myocilin processing is a calcium-dependent process, further supporting the role of calpains in this process.

Myocilin Is Cleaved in Vitro by Calpain I and Calpain II—To confirm the cleavage of myocilin by calpains, a sample of culture medium containing unprocessed recombinant myocilin (55 kDa) was obtained from 293T cells cultured for 24 h after transfection. The culture medium was incubated with purified recombinant calpain I or calpain II. As shown in Fig. 10A (lanes 2 and 3), the 55-kDa doublet disappeared, and a band of around 35 kDa was detected by SDS-PAGE after 15 min of digestion, showing that calpains produce in vitro the same C-terminal fragment obtained when myocilin is expressed in 293T cells (Fig. 9A, lane 9). Moreover, myocilin proteolysis by calpains I and II was inhibited when calcium in the digestion buffer was chelated with EGTA (Fig. 9A, lanes 4 and 5), further supporting that the cleavage is mediated by calpains. We used the recombinant pigment epithelium-derived factor produced in the same cell line as a control. Pigment epithelium-derived factor was not cleaved by purified recombinant calpains, showing that the cleavage of myocilin by calpains is specific (Fig. 9A, lanes 6–8). To detect the N-terminal fragment resulting after calpain digestion, we used culture medium of 293T cells transiently transfected with a cDNA construct encoding myocilin tagged with HA and Myc epitopes at the N and C terminus, respectively (Fig. 1B, HA-myocilin-myc). Bands of 35 and 30 kDa were detected with the anti-Myc and anti-HA antibodies, respectively (Fig. 9B), supporting that calpain I and calpain II splits myocilin into two fragments.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Analysis of the endoproteolytic processing of myocilin by deletion mutagenesis around the cleavage site. cDNA constructs encoding myocilin with different deletions around the consensus subtilisin/kexin isozyme-1 cleavage site (Fig. 1D) were used to transfect 293T cells. After transfection of cells, the culture medium was replaced with fresh medium every day for a 6-day period. Secretion and processing of these deleted versions of myocilin (20 µl of culture medium) were analyzed by Western blot, using an anti-Myc monoclonal antibody, 2 (A) and 6 days (B) after transfection. C, three deletion mutants (Fig. 1D) were generated to determine whether a furin cleavage site located at amino acid residues 179–182 is an alternative cleavage site in myocilin. Cells were transfected as indicated in A and B, and 6 days after transfection, myocilin in the culture medium (20 µl) was analyzed by Western blot.

View larger version (50K): [in this window] [in a new window]   FIGURE 7. Inhibition of myocilin processing by calpain inhibitors. A, a cDNA construct (200 ng) encoding wild-type myocilin (Fig. 1B, myocilin-myc) was transiently transfected into 293T cells for 5 days and then cultured for 16 h with DMEM containing either 0.5% Me2SO (vehicle control) or different membrane-permeable proteinase inhibitors: ALLN (40 µM), calpeptin (80 µM), calpain inhibitor IV (50 µM), ammonium chloride (1 mM), MG-132 (0.25 µM), or lactacystin (0.25 µM). Secreted myocilin in the culture medium (20 µl) was analyzed by Western blot with an anti-Myc monoclonal antibody. B, 500 ng of the same cDNA construct were transiently transfected into 26HCMsv and ARPE-19 cells and then cultured in the presence of vehicle control or calpeptin (80 µM). Myocilin in the culture medium (20 µl) was analyzed by Western blot, as indicated in A. C, quantitation by densitometry of the olfactomedin-like domain released into the culture medium as a result of the proteolytic processing in the presence of the different protease inhibitors used in A. Values are expressed as a percentage of myocilin processing. D, cell viability was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at the end of these experiments. Error bars, S.E. of triplicate experiments. Statistical significance was calculated by using Student's t test. ***, p < 0.001.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. Regulation of the endoproteolytic processing of myocilin by ionomycin and EGTA in cells in culture. A cDNA construct encoding wild-type myocilin (Fig. 1B, myocilin-myc) was transiently transfected into 293T cells and cultured in the presence of 4 mM EGTA (A) or 2 µM ionomycin (B), as indicated under "Experimental Procedures." Secreted myocilin in the culture medium (20 µl) was analyzed by Western blot using an anti-Myc monoclonal antibody. Myocilin cleavage was up-regulated by ionomycin and inhibited by EGTA. The arrows and arrowheads indicate the mobility of 55-kDa full-length myocilin and 35-kDa processed myocilin C-terminal domain.

View larger version (26K): [in this window] [in a new window]   FIGURE 9. Myocilin is cleaved in vitro by calpains I and II. A, culture medium (20 µl) from 293T cells containing unprocessed recombinant wild-type myocilin (obtained in 293T cells cultured for 24 h after transfection) or wild-type pigment epithelium-derived factor (negative control) was incubated at 37 °C for 15 min with 1 µg of calpain I or calpain II in the presence of 3 mM CaCl2. EGTA (4 mM) was used to test the calcium-dependent calpain activity. Digestion products were analyzed by Western blot using an anti-Myc monoclonal antibody. B, culture medium (20 µl) from 293T cells containing wild-type myocilin tagged with HA and Myc epitopes at the N and C termini, respectively, was incubated at 37 °C for 15 min with calpain I or calpain II in the presence of 3 mM CaCl2. Digestion products were analyzed by Western blot using either anti-Myc (left) or anti-HA (right) monoclonal antibodies. The arrows and black arrowheads indicate positions of 55 and 35 kDa myocilin bands corresponding to full-length and processed olfactomedin-like domain, respectively. The white arrowhead shows the N-terminal domain generated by the calpain endoproteolytic cleavage of myocilin. C, in vitro calpain digestion was also performed in the presence of protease inhibitors (80 µM calpeptin, 50 µM calpain inhibitor IV, 40 µM ALLN, 0.25 µM lactacystin) to confirm the specificity of this cleavage. D, lysates from the human skeletal muscle (S.M.) and human CB (C.B.) were incubated at 37 °C for 1 h with 1 µg of calpain I or calpain II in the presence of 3 mM CaCl2. Digestion products were analyzed by Western blot using the anti-myocilin polyclonal antibody C21A (7).

To test whether native myocilin from human tissues is processed by calpains in a similar fashion to that of recombinant myocilin, protein extracts of skeletal muscle and CB, two tissues where the protein is abundantly expressed (5), were incubated with purified calpain I or calpain II as already described. A fragment of around 35 kDa was released after this treatment (Fig. 9D). This fragment was similar to that obtained with the recombinant protein, since it was detected by the anti-myocilin antibody C21A, which recognizes amino acid residues 468–488 (7). This fragment was also recognized by another myocilin antibody, R14T, raised against amino acid residues 272–285 (7), located at the beginning of the olfactomedin-like domain (data not shown). These data show that the fragment released from native myocilin, present in these two human tissues, corresponded to the complete olfactomedin domain of myocilin.

Effect of Calpain I or Calpain II RNAi on the Proteolytic Cleavage of Myocilin Expressed in Cells in Culture—To finally support the role of calpain in myocilin processing, we attempted to specifically knock down calpain expression using RNAi. Initially, we selected calpain II as a target, because it has previously been reported that a subset of this protease is located within the lumen of the ER (18), the place where myocilin is probably processed (10). We assayed four calpain II shRNA clones and selected clone 4 for further experiments, because it produced the highest reduction of calpain II expression (data not shown). shRNA clone 1 had no detectable effect either on calpain II levels or on myocilin processing and was used as a negative control (Fig. 10, A and B, Cp II shRNA-1 lanes). Transient co-expression (48 h) in 293T cells of recombinant myocilin and calpain II shRNA clone 4 resulted in a 40% reduction of myocilin processing, relative to the proteolysis obtained in the control (Fig. 10, A and B, Cp II shRNA-4 lanes). A similar result was obtained with an siRNA specific for calpain II (Fig. 10, A and B, Cp II siRNA lanes).

To corroborate the specific involvement of calpain in myocilin processing, we decided to analyze the effect of its endogenous specific inhibitor calpastatin. In order to target this inhibitor to the ER, we made a cDNA construct that encoded Cs-D1 coupled to the signal peptide of myocilin. Once this cDNA construct was transiently co-expressed with myocilin, we observed that myocilin processing was reduced by 55%, relative to the processing observed in the control (Fig. 10, A and B, Cs-D1 lanes). Quantitation of calpain II in cells treated with calpain II RNAi showed that the expression of this enzyme was reduced by 50–60% (Fig. 10C, lanes Cp II shRNA-4 and Cp II siRNA), whereas, as expected, the expression of calpain II was not affected by Cs-D1 (Fig. 10C). These data indicate that residual calpain activity in both RNAi- and calpastatin-treated cells account for the incomplete inhibition of myocilin processing.

View larger version (40K): [in this window] [in a new window]   FIGURE 10. Myocilin processing is down-regulated by both calpain II RNAi and calpastatin but not by calpain I RNAi. A, 293T cells were transiently co-transfected with 200 ng of a cDNA construct encoding wild-type myocilin (Fig. 1B, myocilin-myc) and 300 ng of calpain II shRNA clone 1 (Cp II shRNA-1) (negative control), 300 ng of calpain II shRNA clone 4 (Cp II shRNA-4), or 200 pmol of a calpain II siRNA (Cp II siRNA). In a second approach, 293T cells were transiently co-transfected with 500 ng of a cDNA construct encoding wild-type myocilin and 500 ng of a cDNA construct encoding the inhibitor domain 1 of calpastatin (Cs-D1) targeted to the ER. Twenty-four hours after transfection, the culture medium was replaced by fresh medium, and 24 h later, secreted myocilin in the culture medium was analyzed by Western blot with an anti-Myc monoclonal antibody. B, densitometric quantitation of the olfactomedin-like domain released into the culture medium after the different treatments in A. C, densitometric quantitation of calpain II detected by Western blot in the cell lysates from A. Aliquots of the cell lysates from A were analyzed by Western blot using an anti-calpain II antibody, and specific signals were quantitated by densitometry. D, 293T cells were transiently co-transfected with 200 ng of a cDNA construct encoding wild-type myocilin and 300 ng of calpain I shRNA clone 1 (Cp I shRNA-1) (negative control), 300 ng of calpain I shRNA clone 2 (Cp I shRNA-2), or 500 ng of calpain II shRNA clone 4 (positive control). Secreted myocilin in the culture medium was analyzed by Western blot as described in A. E, densitometric quantitation of the olfactomedin-like domain released into the culture medium and detected by Western blot after the different treatments in D. F, densitometric quantitation of calpain I in the cell lysates from D. Aliquots of cell lysates were analyzed by Western blot using an anti-calpain I antibody, and specific signals were quantitated by densitometry. The error bars represent the S.E. of triplicate experiments. Asterisks indicate statistical significance calculated by using Student's t test. **, p < 0.01; ***, p < 0.001. The arrows indicate the position of the 55-kDa myocilin band. The arrowheads indicate the position of the 35 kDa myocilin band corresponding to the cleaved olfactomedin-like domain.

Expression of Calpains in Cells in Culture—We verified, by reverse transcription-PCR, that both calpains I and II were co-expressed in all three cell lines (293T, 26HCMsv, and ARPE-19) used in the present study (supplemental Fig. 1), further supporting that myocilin proteolytic processing is also carried out by these enzymes in cultured cells.

Subcellular Localization of Myocilin, Calpain I, and Calpain II by Velocity Gradient Centrifugation—To determine the subcellular localization of myocilin, calpain I, and calpain II, 293T cells were transiently transfected with a myocilin cDNA construct, and lysates of these cells were subjected to velocity gradient centrifugation. Aliquots (30 µl) of the fractions obtained from the gradient were analyzed by Western blot to detect myocilin, calpain I, calpain II, and proteins specifically associated with the ER (calnexin (46)) or GA (trans-GA-associated 14-3-3 protein (47)). The results show that full-length myocilin localizes in the ER fractions (Fig 11A, fractions 1–6), whereas the C-terminal processed fragment eluted separately in fractions containing GA microsomes (Fig. 11A, fractions 8–12), as demonstrated by reactivity to antibodies against calnexin and 14-3-3 protein, respectively (Fig. 11A). The 80-kDa form of calpain II was distributed through the gradient, and its 78-kDa form was detected in fractions 1, 2, 9, and 14. In contrast, calpain I was restricted to fractions 1–3 and 14 (Fig. 11A). These data showed that full-length myocilin mainly co-eluted with calpain II and also with calpain I in three fractions.

Analysis of the Cytosolic or Intraorganular Localization of Myocilin, Calpain I, and Calpain II—To determine the cytosolic or intraorganular localization of myocilin, calpain I, and calpain II, aliquots of the fractions obtained by velocity gradient centrifugation were treated with proteinase K, which digests proteins on the cytoplasmic side of microsomes (18). After proteinase K digestion, a significant portion of myocilin signal was detected in fractions 2–5 (full-length myocilin) and 8–12 (C-terminal myocilin fragment) (Fig. 11B), supporting that myocilin is located in the lumen of both the ER and GA, as expected for a secreted protein. Treatment with proteinase K eliminated all calpain I signal (Fig. 11B), indicating that it is associated with the cytosolic side of organelles, according to previous studies (18). In contrast, calpain II signal was restricted to fractions 2 and 3 (Fig. 11B), suggesting that a subpopulation of this isoform is located inside the ER. These data are in accordance with those of Hood and co-workers (18). Finally, significant signals of calnexin and 14-3-3 protein were detected after proteinase K digestion, showing, as expected, their intraluminal localization. Overall, these data show that myocilin and a subpopulation of calpain II, but not calpain I, are located in the lumen of the ER and strongly support the possibility that calpain II cleaves myocilin in this organelle in cells in culture.

View larger version (17K): [in this window] [in a new window]   FIGURE 11. Subcellular co-localization of myocilin and calpain II to the lumen of ER in transfected 293T cells using Optiprep gradient centrifugation. 293T cells were cultured to 50% confluence and transiently transfected with a cDNA construct encoding wild-type myocilin (Fig. 1B, myocilin-myc). After 48 h of expression, cells were broken by repeated strokes in a Dounce homogenizer. The cell lysate was separated on a discontinuous Optiprep velocity gradient. Gradient fractions (fractions 1–14) were incubated without (A) or with proteinase K (0.5 µg/ml) (B) for 15 min at 37 °C to check the possible intraorganullar localization of the proteins. The collected fractions were analyzed by SDS-PAGE and Western blot, using antibodies to Myc (myocilin), calpain II, calpain I, calnexin (ER marker), and 14-3-3 protein (GA marker). The arrows and arrowheads indicate the position of 55-kDa full-length and 35-kDa processed myocilin, respectively. Fraction 1 was high density; fraction 14 was low density.

It is interesting to point out that a cDNA construct encoding the C-terminal region of myocilin, starting at position 217 (amino acid residues 217–504; Fig. 1B), produced a secreted doublet of around 35 kDa when expressed in 293T cells (Fig. 12C). The upper band disappeared after 7 days of culture, and only the lower band remained, which showed the same electrophoretic mobility as the C-terminal fragment released from full-length myocilin (amino acids 227–504). This finding indicates that it was cleaved at the same position as full-length myocilin and suggests that the leucine zipper domain is not required for calpain processing.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The biological function of myocilin has remained unknown for more than 10 years. Elucidating its role would help to understand how MYOC gene mutations cause glaucoma. Myocilin is expressed in many tissues, and its biological role is not restricted to the eye. Within the eye, MYOC is highly expressed in the iris and CB and probably secreted by the ciliary epithelium into the aqueous humor (5, 10), where it can reach tissues of the anterior chamber, including the TM. In addition, it has been proposed that myocilin might regulate both TM and uveoscleral (through the CM) outflow (51), as indicated by the presence of extracellular myocilin around individual CM cells (8, 52). In this work, therefore, we used a well characterized human embryonic kidney cell line (293T) and two cell lines derived from human CM (26HCMsv) and retinal pigment epithelium (ARPE-19) to approach the study of both the general and the ocular function of myocilin. Here we have characterized a recently described intracellular proteolytic processing of myocilin. Similarly, photomedin-1 and gliomedin, two olfactomedin-like domain containing proteins, are also proteolytically processed in the middle of the molecule, releasing the olfactomedin-like domain (53, 54). Likewise, Goldwich and co-workers (55) reported the presence of similar N- and C-terminal fragments of recombinant myocilin in the culture medium of 293 EBNA cells. They identified the cleavage point at amino acid 215. Since calpain, the enzyme that cleaves myocilin according to our data, does not show strict cleavage site sequence requirements, this discrepancy could be due to differences in the cell line and/or in the culture conditions used in each study. Our results show that the C-terminal domain is secreted upon processing, whereas the N-terminal fragment mainly remains intracellularly retained in the ER. Other groups have also reported the nonsecretion of this region of myocilin. Caballero et al. (56) found that a truncated myocilin containing the entire N-terminal domain and lacking most of the olfactomedin-like domain (amino acid residues 1–344) accumulated inside the cell. Recently, Stamer et al. (57) found substantial intracellular accumulation of the N-terminal region of myocilin (amino acids 1–186) fused at its C-terminal end with GFP. Moreover, the glaucoma-associated truncated myocilin Q368X is not secreted (10, 58, 59). Whether the released N-terminal fragment is intracellularly degraded upon processing or interacts with other intracellular proteins remains to be investigated.

View larger version (16K): [in this window] [in a new window]   FIGURE 12. Analysis of structural requirements for the endoproteolytic processing of myocilin by calpains. A, culture medium aliquots from 293T cells transfected with a cDNA construct encoding wild-type myocilin (Fig. 1B, myocilin-myc) were denatured at 95 °C for 5 min and then incubated at 37 °C for 15 min with recombinant calpain I or calpain II in the presence of 5 mM CaCl2. Digestion products were analyzed by Western blot using an anti-Myc monoclonal antibody. Controls consisted of nondenatured culture medium containing recombinant myocilin (RT). B, 293T cells were transfected with cDNA constructs (200 ng) encoding either wild-type myocilin-myc or the D380A mutant myocilin-myc. After transfection, cells were grown at either 37 or 30 °C for 4 days. Secreted myocilin (20 µl of culture medium) was analyzed by Western blot using an anti-Myc monoclonal antibody. C, cDNA constructs encoding either wild-type myocilin-myc or the processed C-terminal domain (amino acids 217–504; Fig. 1B, C-terminal) were transiently transfected into 293T cells and then cultured for either 2 or 7 days with DMEM. Secreted myocilin in the culture medium (20 µl) was analyzed by Western blot, as indicated in B. The arrows and arrowheads indicate positions of 55-kDa full-length and 35-kDa processed myocilin C-terminal domain, respectively.

Unexpectedly, the mutational analysis performed to characterize the place of proteolysis showed no strict amino acid sequence requirements at the cleavage site, suggesting that calpain may possibly carry out the cleavage. Although a wide variety of proteins have in fact been shown to be cleaved by calpains, no common amino acid sequences have been defined (44, 45); rather, calpains probably recognize secondary or higher order structures and then cut somewhere in the vicinity, in disordered regions between structured domains (22, 28, 35, 60).

Our conclusion about the calpain-dependent cleavage of myocilin is supported by several findings: (i) this processing is selectively prevented by calpain inhibitors (including the inhibitor domain 1 of calpastatin; (ii) it is calcium-dependent; and (iii) in vitro incubation of both full-length recombinant and native myocilin with purified calpain I or calpain II produces the same proteolytic pattern found in cells expressing myocilin. Moreover, RNAi-mediated knockdown of calpain II, but not of calpain I, reduced myocilin processing, showing that only calpain II is involved in the cleavage of myocilin in cells.

Calpain I and calpain II are regarded as cytosolic proteins, but it has been reported that they also associate with the cytosolic surface of both the ER and Golgi apparatus and that a subset of calpain II is also present within the lumen of these organelles (18). In accordance with this idea, our subcellular fractionation analysis shows that full-length myocilin and a subset of calpain II reside in the lumen of the ER, strongly suggesting that calpain II cleaves myocilin in the lumen of the ER and explaining why calpain RNAi knock-down of calpain I does not affect myocilin processing. The production of the processed C-terminal domain of myocilin by 26HCMsv and ARPE-19 cells and its presence in the human CB and aqueous humor (10) support the physiologic role of this proteolytic processing.

To address whether native myocilin from human tissues is also processed by calpains, we digested the skeletal muscle and CB extracts with purified calpains. Our results clearly show that myocilin from these sources is processed by calpains in a way similar to that of the recombinant protein. However, we observed that the digestion of native myocilin was not complete. It could be due to the conditions used for the digestion, or alternatively, this result might indicate that calpain digestion of a fraction of myocilin present in these tissues could be protected by macromolecular interactions with other proteins.

To check whether the native conformation of myocilin is required for calpain cleavage, we incubated heat-denatured myocilin with purified calpains. Our results support the possibility that conformational determinants rather than amino acid sequence motifs are responsible for the recognition and/or cleavage of myocilin by these proteases, as has been described for other calpain substrates (35, 60). It is interesting to observe that glaucoma-associated myocilin mutations, which have been shown to reduce proteolytic cleavage, are located in the olfactomedin-like domain, which does not form part of the cleavage site. Reduction of proteolytic processing induced by the pathogenic mutation D380A was reverted when the protein was expressed at 30 °C, indicating that an adequate conformation of the olfactomedin-like domain is required for myocilin proteolysis by calpain. These results might indicate that amino acid positions mutated in glaucoma could influence the structure of the myocilin binding site to calpain. We have observed in this study that the recombinant C-terminal domain of myocilin (amino acid residues 217–504) transiently expressed by cells in culture also appears to be cleaved, suggesting that the N-terminal domain is not required for the proteolytic processing. These data suggest that two different regions of myocilin are involved in its processing by calpains: the olfactomedin-like domain, which could be recognized by the protease, acting as a substrate binding site, and the cleavage site, located in the linker domain (Fig. 13).

It is believed that myocilin interactions play a key role in its biological function. We suggested that the proteolytic processing of myocilin might contribute to regulate its interaction with other proteins (10). Full-length myocilin may form high molecular weight aggregates by cross-linking proteins through two binding sites. However, the two fragments released by calpain cleavage probably have a reduced capability to aggregate. In fact, it has been described that calpain cleavage decreases the cross-linking ability of other proteins, such as troponins T and I, tropomiosin, C-protein (28), and vinculin (61). Therefore, it is tempting to speculate that an increase of myocilin processing could reduce formation of its complexes in the extracellular space (i.e. aqueous humor, TM, and CM extracellular matrix), contributing to facilitate aqueous outflow. Supporting this hypothesis, it has been reported that infusion of full-length recombinant myocilin partially purified from both prokaryotic and eukaryotic expression systems increased outflow resistance in cultured human anterior segments (62, 63). However, perfusion of the C-terminal domain of myocilin (containing the olfactomedin-like region) did not influence outflow resistance (55). In addition, perfusion of human anterior eye segments in culture with adenovirus expressing a truncated version of myocilin containing the N-terminal region of the protein caused a reduction in IOP that was in turn associated with a decline of endogenous myocilin in the effluent of the infected organ cultures (56). These results support the possibility that only unprocessed full-length myocilin increases resistance of aqueous outflow. Further work is required to confirm this hypothesis.

View larger version (42K): [in this window] [in a new window]   FIGURE 13. Model of the endoproteolytic processing of myocilin by calpain II. According to this model, the proteolytic cleavage of myocilin is carried out by a subpopulation of calpain II in the lumen of the ER, producing two myocilin fragments: one containing the leucine zipper-like domain (LZ) and another one containing the olfactomedin-like domain (OLF). It is proposed that two different regions of myocilin are involved in this processing. The olfactomedin-like domain could act as the substrate binding site to calpains, whereas the putative linker domain (LINK) contains the cleavage site. Full-length myocilin and the olfactomedin-like domain resulting from processing by calpain are secreted, and the processed N-terminal region mainly remains in the ER. The proportion of secreted full-length myocilin/olfactomedin-like domain is controlled by factors that regulate calpain activity, such as culture time. Physiologic factors that regulate the processing rate remain to be identified. Notice that full-length myocilin contains two interaction sites, whereas the processed domains contain only one.

To summarize, we conclude that myocilin is processed in the lumen of the ER by calpain II in different cell lines. These results provide new clues to unravel the elusive function of myocilin and its role in IOP regulation, and they could contribute to understanding the role of myocilin mutations in glaucoma pathogenesis.

	FOOTNOTES

 
^* 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.

⁵ The abbreviations used are: IOP, intraocular pressure; ALLN, N-acetyl-leucyl-leucyl-norleucinal; CB, ciliary body; CM, ciliary muscle; Cs-D1, inhibitor domain 1 of calpastatin; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; GA, Golgi apparatus; GFP, green fluorescent protein; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA; TM, trabecular meshwork; HA, hemagglutinin.

	ACKNOWLEDGMENTS

 
We thank Carmen Cifuentes and Ana María Alonso for technical assistance.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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