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• W2170642535 abstract "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. 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. Myocilin is a glycoprotein of unknown function, originally identified in cultured trabecular meshwork cells upon induction with glucocorticoids (1Polansky J.R. Lutjen-Drecoll Basic Aspects of Glaucoma Research III. Schattauer Verlag, Stuttgart, Germany1993: 307-318Google Scholar, 2Polansky J.R. Fauss D.J. Chen P. Chen H. Lutjen-Drecoll E. Johnson D. Kurtz R.M. Ma Z.D. Bloom E. Nguyen T.D. Ophthalmologica. 1997; 211: 126-139Crossref PubMed Scopus (340) Google Scholar). The cDNA was independently cloned from a subtracted ciliary body cDNA library (3Escribano J. Ortego J. Coca-Prados M. J. Biochem. (Tokyo). 1995; 118: 921-931Crossref PubMed Scopus (72) Google Scholar, 4Ortego J. Escribano J. Coca-Prados M. FEBS Lett. 1997; 413: 349-353Crossref PubMed Scopus (154) Google Scholar) and from a retinal cDNA library (5Kubota R. Noda S. Wang Y. Minoshima S. Asakawa S. Kudoh J. Mashima Y. Oguchi Y. Shimizu N. Genomics. 1997; 41: 360-369Crossref PubMed Scopus (275) Google Scholar). Although the myocilin mRNA has been found to be ubiquitously expressed in many human tissues, its highest abundance appears to be restricted to tissues of the eye such as the iris, ciliary body, and trabecular meshwork (3Escribano J. Ortego J. Coca-Prados M. J. Biochem. (Tokyo). 1995; 118: 921-931Crossref PubMed Scopus (72) Google Scholar, 4Ortego J. Escribano J. Coca-Prados M. FEBS Lett. 1997; 413: 349-353Crossref PubMed Scopus (154) Google Scholar, 5Kubota R. Noda S. Wang Y. Minoshima S. Asakawa S. Kudoh J. Mashima Y. Oguchi Y. Shimizu N. Genomics. 1997; 41: 360-369Crossref PubMed Scopus (275) Google Scholar, 6Huang W. Jaroszewski J. Ortego J. Escribano J. Coca-Prados M. Ophthalmic Genet. 2000; 21: 155-169Crossref PubMed Google Scholar). The gene encoding myocilin is currently referred to as MYOC. It consists of three different sized exons. The amino-terminal region of myocilin, encoded by exon 1, contains a peptide signal sequence (amino acids 1–32) and a leucine zipper-like motif composed of about 50 amino acid residues (amino acids 117–169) with periodic arginine and leucine repeats arranged along an α-helix (4Ortego J. Escribano J. Coca-Prados M. FEBS Lett. 1997; 413: 349-353Crossref PubMed Scopus (154) Google Scholar). The finding of this amphipathic structure suggested that it might participate in molecular interactions (7Coca-Prados M. Escribano J. Ortego J. Prog. Retin. Eye Res. 1999; 18: 403-429Crossref PubMed Scopus (74) Google Scholar). In fact, myocilin-myocilin interactions have been described to occur mainly within amino acids 117–166, in the region containing the leucine zipper-like domain (8Fautsch M.P. Johnson D.H. Invest. Ophthalmol. Vis. Sci. 2001; 42: 2324-2331PubMed Google Scholar). The central region of the protein (amino acids 203–245) is encoded by exon 2, and neither structural nor functional domains have been described in this location so far. The carboxyl-terminal half of myocilin, encoded by exon 3, is homologous to olfactomedin, an extracellular matrix protein of unknown role, that is abundant in the olfactory neuroepithelium (9Bal R.S. Anholt R.R. Biochemistry. 1993; 32: 1047-1053Crossref PubMed Scopus (46) Google Scholar). This domain contains a single disulfide bond connecting cysteine residues 245 and 433 (10Nagy I. Trexler M. Patthy L. Biochem. Biophys. Res. Commun. 2003; 302: 554-561Crossref PubMed Scopus (29) Google Scholar). Interestingly enough, most mutations (missense) reported so far in the MYOC gene in glaucoma patients are heterozygous and are confined to exon 3 (4Ortego J. Escribano J. Coca-Prados M. FEBS Lett. 1997; 413: 349-353Crossref PubMed Scopus (154) Google Scholar, 11Sarfarazi M. Hum. Mol. Genet. 1997; 6: 1667-1677Crossref PubMed Scopus (159) Google Scholar, 12Rozsa F.W. Shimizu S. Lichter P.R. Johnson A.T. Othman M.I. Scott K. Downs C.A. Nguyen T.D. Polansky J. Richards J.E. Mol. Vis. 1998; 4: 20-35PubMed Google Scholar, 13Adam M.F. Belmouden A. Binisti P. Brezin A.P. Valtot F. Bechetoille A. Dascotte J.C. Copin B. Gomez L. Chaventre A. Bach J.F. Garchon H.J. Hum. Mol. Genet. 1997; 6: 2091-2097Crossref PubMed Scopus (259) Google Scholar). Myocilin is intracellularly distributed in vesicles (14Stamer W.D. Roberts B.C. Howell D.N. Epstein D.L. Invest. Ophthalmol. Vis. Sci. 1998; 39: 1804-1812PubMed Google Scholar) and processed via the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; GFP, green fluorescent protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; POAG, primary open angle glaucoma. (15Zimmerman C.C. Lingappa V.R. Richards J.E. Rozsa F.W. Lichter P.R. Polansky J.R. Mol. Vis. 1999; 5: 19-24PubMed Google Scholar, 16Caballero M. Rowlette L.L. Borras T. Biochim. Biophys. Acta. 2000; 1502: 447-460Crossref PubMed Scopus (96) Google Scholar). The protein is secreted to the aqueous humor of several species (17Rao P.V. Allingham R.R. Epstein D.L. Exp. Eye Res. 2000; 71: 637-641Crossref PubMed Scopus (49) Google Scholar, 18Jacobson N. Andrews M. Shepard A.R. Nishimura D. Searby C. Fingert J.H. Hageman G. Mullins R. Davidson B.L. Kwon Y.H. Alward W.L. Stone E.M. Clark A.F. Sheffield V.C. Hum. Mol. Genet. 2001; 10: 117-125Crossref PubMed Scopus (247) Google Scholar) and into the culture medium of different cell lines as a doublet of nearly 55–57 kDa. The doublet is caused by glycosylated and nonglycosylated molecules (2Polansky J.R. Fauss D.J. Chen P. Chen H. Lutjen-Drecoll E. Johnson D. Kurtz R.M. Ma Z.D. Bloom E. Nguyen T.D. Ophthalmologica. 1997; 211: 126-139Crossref PubMed Scopus (340) Google Scholar, 18Jacobson N. Andrews M. Shepard A.R. Nishimura D. Searby C. Fingert J.H. Hageman G. Mullins R. Davidson B.L. Kwon Y.H. Alward W.L. Stone E.M. Clark A.F. Sheffield V.C. Hum. Mol. Genet. 2001; 10: 117-125Crossref PubMed Scopus (247) Google Scholar, 19Nguyen T.D. Chen P. Huang W.D. Chen H. Johnson D. Polansky J.R. J. Biol. Chem. 1998; 273: 6341-6350Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Myocilin has also been localized to the Golgi apparatus of both Schlemm's canal cells (21O'Brien E.T. Ren X. Wang Y. Invest. Ophthalmol. Vis. Sci. 2000; 41: 3842-3849PubMed Google Scholar) and corneal fibroblasts (22Wentz-Hunter K. Shen X. Yue B.Y. Mol. Vis. 2003; 9: 308-314PubMed Google Scholar). Glaucoma is a complex and genetically heterogeneous disease characterized by the progressive apoptotic death of retinal ganglion cells that leads to excavation of the optic nerve head and to visual field loss, eventually producing blindness (23Quigley H.A. Nickells R.W. Kerrigan L.A. Pease M.E. Thibault D.J. Zack D.J. Invest. Ophthalmol. Vis. Sci. 1995; 36: 774-786PubMed Google Scholar, 24Quigley H.A. Katz J. Derick R.J. Gilbert D. Sommer A. Ophthalmology. 1992; 99: 19-28Abstract Full Text PDF PubMed Scopus (514) Google Scholar). Mutations in the MYOC gene cause autosomal dominant juvenile glaucoma (GLC1A) and a subset of adult onset primary open angle glaucoma (POAG). The best known risk factor associated with POAG is an increased intraocular pressure. The molecular pathway from the glaucoma genotype to the phenotype is not straightforward, and it probably involves a chain of subtle events. Over recent years, some of these events have been unraveled and have shown, for instance, that mutant forms of myocilin are not secreted in cultured cells; rather, they accumulate intracellularly as misfolded proteins. This leads to endoplasmic reticulum stress and to potential cytotoxicity (16Caballero M. Rowlette L.L. Borras T. Biochim. Biophys. Acta. 2000; 1502: 447-460Crossref PubMed Scopus (96) Google Scholar, 18Jacobson N. Andrews M. Shepard A.R. Nishimura D. Searby C. Fingert J.H. Hageman G. Mullins R. Davidson B.L. Kwon Y.H. Alward W.L. Stone E.M. Clark A.F. Sheffield V.C. Hum. Mol. Genet. 2001; 10: 117-125Crossref PubMed Scopus (247) Google Scholar, 25Caballero M. Borras T. Biochem. Biophys. Res. Commun. 2001; 282: 662-670Crossref PubMed Scopus (74) Google Scholar, 26Liu Y. Vollrath D. Hum. Mol. Genet. 2004; 13: 1193-1204Crossref PubMed Scopus (182) Google Scholar). Although certain molecular mechanisms such as homoallelic complementation, haploinsuffiency, or negative dominant effect have been proposed to explain the pathogenesis of myocilin glaucoma, most experimental evidence supports the gain of function theory (27Morissette J. Clepet C. Moisan S. Dubois S. Winstall E. Vermeeren D. Nguyen T.D. Polansky J.R. Cote G. Anctil J.L. Amyot M. Plante M. Falardeau P. Raymond V. Nat. Genet. 1998; 19: 319-321Crossref PubMed Scopus (122) Google Scholar, 28Lam D.S. Leung Y.F. Chua J.K. Baum L. Fan D.S. Choy K.W. Pang C.P. Invest. Ophthalmol. Vis. Sci. 2000; 41: 1386-1391PubMed Google Scholar, 29Kim B.S. Savinova O.V. Reedy M.V. Martin J. Lun Y. Gan L. Smith R.S. Tomarev S.I. John S.W. Johnson R.L. Mol. Cell. Biol. 2001; 21: 7707-7713Crossref PubMed Scopus (223) Google Scholar). Here we show for the first time that wild type myocilin is endoproteolytically processed, probably in the ER, and that pathogenic glaucoma mutations significantly inhibit this proteolytic processing. This work provides new insights not only into the physiological function of myocilin but also into the role it plays in the pathogenesis of POAG. Myocilin Constructs—Total RNA was extracted from 20 mg of frozen human skeletal muscle mechanically homogenized using Tri-Reagent (Sigma). Primers 5′-CGGAATTCTGATGAGGTTCTTCTGTGCACG-3′ (primer 1) and 5′-GCGGGATCCATCTTGGAGAGCTTGATGTC-3′ (primer 2) were employed to amplify human myocilin cDNA from the RNA preparation using the AccessQuick™ RT-PCR System (Promega). The sequence of primers was designed so that the PCR product incorporated EcoRI and BamHI restriction cleavage sites. The obtained cDNA, which contained the full-length myocilin sequence, including its signal peptide, was cloned into the EcoRI-BamHI sites of the mammalian expression vector pcDNA3.1(–) (Invitrogen). This vector was previously adapted by inserting coding sequences for Myc and His epitopes or green fluorescent protein (GFP) in the BamHI-HindIII sites. DNA from the wild type myocilin cloned in the pcDNA 3.1 vector containing Myc-His epitopes was used as a template to produce different point mutations with the QuikChange site-directed mutagenesis kit (Stratagene). The specific PCR primers used for mutagenesis were as follows: 5′-CCTAGGCCACTGAAAAGCACGGGTGCTGTG-3′ and 5′-CACAGCACCCGTGCTTTTCAGTGGCCTAGG-3′ for E323K; 5′-CCACGGACAGTTCCTGTATTCTTGGGGTGGC-3′ and 5′-GCCACCCCAAGAATACAGGAACTGTCCGTGG-3′ for P370L; and 5′-GGCTACACGGATATCGCCTTGGCTGTGGATG-3′ and 5′-CATCCACAGCCAAGGCGATATCCGTGTAGCC-3′ for D380A. The truncated version of myocilin Q368X was also produced by PCR using the following primers: 5′-CGGAATTCTGATGAGGTTCTTCTGTGCACG-3′ and 5′-CGGGATCCCCGTGGTAGCCAGCTCC-3′. The mutated cDNAs were subcloned into the EcoRI-BamHI sites of the mammalian expression vector pcDNA3.1(–) adapted as described above. To prepare the deletion construct R226-E230del, two PCR fragments were first amplified from each side of the deletion using as template the full-length myocilin cDNA cloned as described above. The 5′ portion of this construct was amplified using primer 1 and the deletion-specific antisense primer 5′-CCTGAGATAGCCAGATGGGCTGGAAGCAGGAACTTCAGTTAGC-3′. The 3′ portion was amplified using the deletion-specific sense primer 5′-GCTAACTGAAGTTCCTGCTTCCAGCCCATCTGGCTATCTCAGG-3′ and primer 2. The primary PCR products were mixed and used as a template in a secondary PCR with primers 1 and 2. The PCR fragment containing the deleted region was directly cloned into the EcoRI-BamHI sites of the pcDNA3.1-Myc-His vector. All of the DNA constructs obtained were sequenced to confirm both the correct insertion and absence of undesirable mutations. Tissues and Cell Lines—Bovine and human eyes were obtained from a local slaughterhouse or from human cadavers within 24 h after enucleation through the National Disease Research Interchange (Philadelphia, PA), respectively. Human aqueous humor samples were collected from patients undergoing intraocular surgery after obtaining informed consents. Investigation of the human subjects was approved by the Human Subjects Committee of Yale University and followed the tenets of the Declaration of Helsinki, as far as it applies. Eyes were microdissected from the posterior pole, both the vitreous and lens were removed, and the ciliary body and iris were microdissected. Homogenization of ciliary body and iris was performed by mechanical grinding with liquid nitrogen using a mortar and pestle. Pulverized frozen tissue was suspended in 300 μl of lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% (v/v) Triton X-100, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mm Na3VO4, and 1 mm NaF), vortexed for 30 s at maximum speed, and incubated for 30 min on ice. The lysates were centrifuged at 20,000 × g for 5 min at 4 °C. Supernatants were stored at –80 °C until used. Monkey kidney COS1 and human embryonic kidney 293T cell lines were bought from the ATCC (American Type Culture Collection). The human cell lines 26HCMsv and 59HIsv were established from primary cultures of ciliary muscle cells of a 26-year-old male (cadaver) and from the iris pigmented epithelial cells of a 59-year-old male (cadaver) respectively, by viral transformation, as previously described (30Coca-Prados M. Wax M.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8754-8758Crossref PubMed Scopus (60) Google Scholar). The four cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Normocin; Invitrogen) at 37 °C, in a fully humidified 5% CO2 atmosphere. Cell Transfections—Cells growing in 6-well plates at 70–80% cell confluence were transiently transfected with 0.5–1.0 μg of plasmid DNA using the Superfect Transfection Reagent (Qiagen), according to the manufacturer's instructions. In order to avoid proteolytic degradation of secreted recombinant myocilin, a commercial mixture of proteinase inhibitors for specific use in culture media (Sigma) was used. Forty-eight h after transfection, the culture medium was collected. To remove cellular debris from the collected culture medium, samples were centrifuged at 5,000 × g for 5 min. 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 an addition of 200 μl of lysis buffer containing proteinase inhibitors. 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). Cells lysates were centrifuged at 20,000 × g for 5 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 myocilin-GFP, by counting the number of GFP-positive cells in a total of 103 cells in four randomly selected areas per dish. The average transfection efficiency was 20–25%. As negative control, the different cell lines were transfected with 0.5 μg of the nonrecombinant pcDNA3.1-vector. Western Blotting and Antibodies—Analytical 10% polyacrylamide slab gel electrophoresis in the presence of SDS was performed as reported by Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), using the Mini-PROTEAN III gel electrophoresis system (Bio-Rad). Gels were stained with Coomassie Brilliant Blue R250 (32Wilson C.M. Methods Enzymol. 1983; 91: 236-247Crossref PubMed Scopus (141) Google Scholar). For Western blot analysis aliquots of culture medium, intracellular fractions of cultured cell lines (both soluble and insoluble) or tissue extracts and aqueous humor were fractionated by SDS-PAGE. The samples were normalized for protein content using the Bradford assay. Gels were subsequently transferred onto Hybond ECL nitrocelulose membranes (Amersham Biosciences) for immunodetection or onto Immobilon-P (Millipore Corp.) for N-terminal microsequencing. Anti-myocilin antibodies R14T, C21A, or a commercial mouse monoclonal anti-Myc antibody (9E10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used as primary antibodies diluted at 1:400–1:500. Antibodies R14T and C21A were raised in rabbits immunized with synthetic myocilin peptides corresponding to amino acid residues 272–285 and 468–488, respectively (Supplementary Fig. 1) (6Huang W. Jaroszewski J. Ortego J. Escribano J. Coca-Prados M. Ophthalmic Genet. 2000; 21: 155-169Crossref PubMed Google Scholar). Horseradish peroxidase-conjugated antibodies against either mouse or rabbit IgG (Pierce) were diluted at 1:1000. Chemiluminiscence was performed with Supersignal Dura Western blot reagents (Pierce). Densitometry for protein band quantitation was performed on scanned films using Quantity One 4.1 analysis software (Bio-Rad) on triplicate independent experiments. Data were statistically treated by using SigmaStat 2.0 software (SPSS Science). Inhibition of Protein Secretion with Brefeldin A (BFA) or Monensin— Following transfection with different myocilin constructs, cells were washed with Dulbecco's phosphate-buffered saline and further cultured for 3 h with culture medium containing either BFA (Fluka) (1 μg/ml) or monensin (Sigma) (0.32 μg/ml). To remove the recombinant protein secreted before adding the inhibitors, the culture medium was eliminated, and adhered cells were briefly washed with Dulbecco's phosphate-buffered saline and cultured for 24 h with fresh Dulbecco's modified Eagle's medium containing BFA or monensin. The culture medium and cells were processed as described above. Nickel-chelating Chromatography of Recombinant Myocilin—Recombinant wild type myocilin was isolated by nickel-chelating chromatography using a Hi-Trap Chelating HP 1-ml column (Amersham Biosciences). Two ml of culture medium from transiently transfected 293T cells were equilibrated in binding buffer (20 mm Tris-HCl, pH 7.9, 0.5 m NaCl) containing 5 mm imidazole, using Amicon Ultra-4 Centrifugal filter devices (Millipore Corp.) with a nominal molecular mass limit of 30 kDa. The column was charged with 1 ml of 50 mm NiSO4 and washed with 5 ml of binding buffer to remove any excess of NiSO4. Two ml of equilibrated culture medium were loaded in the column. The column was eluted with 5 ml of binding buffer followed by 5 ml of the same buffer containing 75 mm imidazole. The retained molecules were eluted with 3 ml of binding buffer containing 1 m imidazole. The presence of myocilin in the collected samples was tested by Western blot analysis with the anti-Myc 9E10 antibody. N-terminal Amino Acid Sequence Analysis and Matrix-assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) Peptide Mass Fingerprint Analysis—The 35-kDa myocilin band isolated by nickel chelating chromatography was characterized by both N-terminal sequencing and MALDI-TOF peptide mass fingerprinting. N-terminal sequencing by Edman degradation was performed using a sample separated by SDS-PAGE and electrotransferred onto a polyvinylidene fluoride membrane (Immobilon-P; Millipore Corp.), as described above. The membrane was stained with Coomassie Blue, and the 35-kDa band was cut and loaded into a Procise 494 protein sequencer (Applied Biosystems). For MALDI-TOF analysis, another aliquot was separated by SDS-PAGE with silver staining. The 35-kDa band was excised with a scalpel and subjected to in-gel digestion as reported elsewhere (33Li G. Waltham M. Anderson N.L. Unsworth E. Treston A. Weinstein J.N. Electrophoresis. 1997; 18: 391-402Crossref PubMed Scopus (75) Google Scholar). The digest was analyzed in a MALDI-TOF/TOF 4700 Proteomics Analyzer (Applied Biosystems) in the proteomics facility of the “Parque Científico de Madrid.” External calibration was performed using a mixture of angiotensin II, ACTH/CLIP, bombesin, and somatostatin. Data base searching was performed with ProteinProspector MS-Fit (available on the World Wide Web at prospector.ucsf.edu). Fluorescence Microscopy—293T cells were seeded in coverslips placed into 6-well plates. They were transfected with DNA constructs encoding different myocilin-GFP variants. DNA constructs encoding GFP-tagged versions of the first 80 N-terminal amino acids of galactosyl-transferase or the signal peptide of calreticulin were used as specific fluorescent Golgi 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. The cells were fixed with 4% paraformaldehyde for 10 min. Finally, the coverslips were mounted on glass slides using polyvinyl alcohol mounting medium with 1,4-diazabicyclo[2.2.2]octane (Fluka) and viewed under a fluorescence microscope (Leica DMR/XA) with proper filter sets, using a ×100 plan objective. Images were captured using a Leica DC500 digital camera. Affinity Purification of Myocilin—The anti-peptide antibody (IgG) C21A was immobilized to a commercially available resin support according to the manufacturer's instructions (AminoLink Plus Activated Support) (Pierce). The soluble fraction (1 mg of protein) of a human ciliary body extract was incubated with the resin to capture the target protein (myocilin). The resin was washed and eluted following the manufacturer's recommendations. Fifty μg of eluted protein were separated on SDS-PAGE, transferred onto a membrane, and probed with either R14T or C21A anti-peptide antibodies directed against the N- and C-terminal regions of the olfactomedin-like domain of myocilin, respectively (6Huang W. Jaroszewski J. Ortego J. Escribano J. Coca-Prados M. Ophthalmic Genet. 2000; 21: 155-169Crossref PubMed Google Scholar). Bioinformatics—A search for myocilin domains was performed in the collection of protein families and domains contained in the Pfam data base (34Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) (available on the World Wide Web at www.sanger.ac.uk/Software/Pfam/). Prediction for proteinase cleavage sites was performed using the Web-based data base of eukaryotic linear motifs (ELM) (available on the World Wide Web at elm.eu.org/) (35Puntervoll P. Linding R. Gemund C. Chabanis-Davidson S. Mattingsdal M. Cameron S. Martin D.M. Ausiello G. Brannetti B. Costantini A. Ferre F. Maselli V. Via A. Cesareni G. Diella F. Superti-Furga G. Wyrwicz L. Ramu C. McGuigan C. Gudavalli R. Letunic I. Bork P. Rychlewski L. Kuster B. Helmer-Citterich M. Hunter W.N. Aasland R. Gibson T.J. Nucleic Acids Res. 2003; 31: 3625-3630Crossref PubMed Scopus (520) Google Scholar). Expression of Wild Type Myocilin in Cultured Cell Lines and Ocular Mammalian Tissues—In a first approach to characterize the synthesis and secretion of wild type myocilin, COS1 and 293T cells were chosen, since they are reliable models for producing recombinant eukaryotic proteins. We also established the cell lines 26HCMsv and 59HIsv, derived from the human ciliary muscle and the human iris pigmented epithelial cells, respectively, to set up an expression model closer to ocular tissues where the MYOC gene is physiologically transcribed. Transient cell transfections were optimized in all of the cell lines by using varying amounts of DNA (0.5–4.0 μg). In these analyses, secreted and intracellular myocilin was detected by Western immunoblot at 6, 12, 24, and 48 h after transfection by using the anti-Myc 9E10 antibody. The most appropriate production of wild type myocilin was accomplished at 48 h post-transfection with 0.5–1.0 μg of DNA (data not shown). These conditions were used in subsequent experiments. Western blot analysis employing the anti-myocilin R14T antibody, which binds to a peptide located at the beginning of the olfactomedin-like domain (Supplementary Fig. 1), showed that myocilin was present in the culture medium of the four transfected cell lines (293T, COS1, 59HIsv, 26HCMsv) as two major bands of nearly 55 and 35 kDa (Fig. 1A, culture medium lanes). The 55-kDa band was resolved as a doublet when films were shortly exposed. This doublet is due to differential glycosylation of secreted myocilin. Overexposed films revealed that the 35-kDa band was also present intracellularly, as seen in Fig. 1B (see arrow), where the overexposed intracellular fraction of 293T cells is shown as a representative example. The 35-kDa band was also seen in human aqueous humor as well as in bovine iris and ciliary body whole extracts (Fig. 1A, H. AH and Tissues lanes; see arrow). Nonetheless, it was detected in whole extracts from human iris and ciliary body only after overexposing the blots (data not shown). Full-length myocilin present in bovine tissues displayed an apparent molecular weight higher than that of myocilin visualized in human tissues or in cell lines. This is in accordance with previous studies (36Russell P. Tamm E.R. Grehn F.J. Picht G. Johnson M. Invest. Ophthalmol. Vis. Sci. 2001; 42: 983-986PubMed Google Scholar). Since lengths of human and bovine myocilin polypeptide chains are approximately the same, this difference can be explained by distinct posttranslational modifications of the protein in these two species. The specificity of the anti-myocilin R14T antibody was shown by the absence of bands in control samples of culture medium and cell lysates from the four cell lines transfected with the nonrecombinant vector. Fig. 1A ((–) lanes) shows a representative result obtained with 26HCMsv cells. Moreover, in previous experiments, we determined that the use of preimmune serum or preincubation of the antibody with the immunizing peptide in Western blots did not recognize any band (data not shown), further supporting the specificity of the R14T antibody. To confirm that the human ciliary body produces the myocilin fragment, we isolated this polypeptide by affinity chromatography. The soluble protein from a ciliary body extract was fractionated using the resin-coupled C21A anti-myocilin antibody. Western immunoblot analysis with the R14T anti-m" @default.
• W2170642535 created "2016-06-24" @default.
• W2170642535 creator A5013436965 @default.
• W2170642535 creator A5024057820 @default.
• W2170642535 creator A5039993206 @default.
• W2170642535 creator A5057672243 @default.
• W2170642535 creator A5071087634 @default.
• W2170642535 date "2005-06-01" @default.
• W2170642535 modified "2023-09-30" @default.
• W2170642535 title "Myocilin Mutations Causing Glaucoma Inhibit the Intracellular Endoproteolytic Cleavage of Myocilin between Amino Acids Arg226 and Ile227" @default.
• W2170642535 cites W1586765487 @default.
• W2170642535 cites W1974043792 @default.
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