IOVS AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2004;45:3560-3567.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0300
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (25)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gobeil, S.
Right arrow Articles by Raymond, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gobeil, S.
Right arrow Articles by Raymond, V.

Intracellular Sequestration of Hetero-oligomers Formed by Wild-Type and Glaucoma-Causing Myocilin Mutants

Stéphane Gobeil,1 Marc-André Rodrigue,1 Steve Moisan,1,2 Thai D. Nguyen,3 Jon R. Polansky,3 Jean Morissette,1 and Vincent Raymond1

1From the Molecular Endocrinology and Oncology Research Center, Laval University Hospital (CHUL) Research Center, Québec City, Québec, Canada; and the 3Cellular Pharmacology Laboratories, Department of Ophthalmology, School of Medicine, University of California, San Francisco, California.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate mechanism(s) by which mutations in the olfactomedin domain of myocilin (MYOC), also known as the trabecular meshwork–induced glucocorticoid response (TIGR) gene, cause autosomal dominant open-angle glaucoma, the structure and properties of wild-type (WT) MYOC protein were examined, when expressed alone or simultaneously with the Q368X or K423E disease-associated polypeptides.

METHODS. Myocilin was analyzed in human aqueous humor and human trabecular meshwork (HTM) tissues. COS-7 and immortalized human trabecular meshwork (iHTM) cell lines were transfected with expression vectors encoding WT MYOC, mutated, and/or epitope-tagged cDNAs. MYOC proteins were characterized by double-epitope tagging procedures and/or Western blot analysis.

RESULTS. MYOC polypeptides formed highly similar oligomers in aqueous humor, HTM tissues, transfected COS-7, and iHTM cell lines. These complexes ranged in size from 116 kDa to more than 200 kDa. The smallest complex, approximately 116 kDa, resulted from dimerization between two MYOC monomers. Expression of a 150-kDa complex was strongest in aqueous humor. Cotransfections of the WT construct with either the Q368X or K423E cDNA produced MYOCWT/MYOCmutant heterodimers and higher molecular weight hetero-oligomeric complexes. WT homo-oligomeric complexes were secreted in the extracellular media of both cell lines whereas the Q368X and K423E mutant/mutant homomultimers and heteromeric WT/mutant oligomers remained sequestered intracellularly.

CONCLUSIONS. Formation of heteromeric WT/mutant complexes may provide a critical mechanism by which mutant myocilin polypeptides produce autosomal dominant open-angle glaucoma. The intracellular sequestration of abnormal WT/mutant complexes could lead to the malfunction of MYOC-expressing cells and to POAG potentially involving a dominant negative effect.

Glaucoma encompasses a complex of ocular disease entities characterized by optic neuropathy and progressive loss of visual fields. After cataracts, the disorder is the second leading cause of blindness, affecting more than 67 million people worldwide.1 2 Of all the different forms of glaucoma, primary open-angle glaucoma (POAG; Mendelian Inheritance in Man [MIM] 137760)3 occurs most frequently, with approximately 33 million cases in 2000.4 5 POAG shows genetic heterogeneity,6 7 8 and seven loci have been mapped for the disorder: GLC1A to GLC1G. Only two of the GLC1 genes have been characterized: the trabecular meshwork-inducible glucocorticoid response (TIGR) gene,9 also known as myocilin (MYOC),10 at GLC1A, and optineurin (OPTN) at GLC1E.11

More than 40 MYOC mutations have been reported. Most of the disease-causing mutations are located within exon 3 of MYOC12 13 (amino acids [aa]) 246-504) in a domain highly homologous to olfactomedin.14 In affected families, mutations cause autosomal dominant juvenile- (juvenile-onset open-angle glaucoma [JOAG]) and/or autosomal dominant adult-onset open-angle glaucoma.9 12 15 16 17 18 19 20 21 22 These variations were associated with POAG in approximately 2% to 4% of patients of glaucoma in the different populations tested.12 13 22 23 Phenotype-genotype correlation studies have demonstrated that MYOCQ368X is the most frequently observed variation causing late-onset POAG, with an average age at diagnosis of 59 years, whereas, the Y437H mutation induces early-onset glaucoma with an average age of onset of 20 years.13 21 Using a very large French-Canadian pedigree, we also demonstrated that the K423E mutation displays wide variable phenotypic expressivity, ranging from early- to late-onset POAG.20 Four mutant homozygotes were further detected in this kindred; none of them manifested any symptoms of the disorder. As of March 2004, these homozygotes remained normal. Meiotic reversion, parent-of-origin–dependent effects, and reduced penetrance of the mutant allele were ruled out in these asymptomatic mutant homozygotes. MYOCK423E, therefore, caused the first autosomal dominant heterozygote-specific phenotype in humans.20

The MYOC gene encodes a 504-aa polypeptide with a theoretical molecular mass of 56.9 kDa. In ocular tissues, the MYOC protein was mainly localized within the trabecular meshwork, the Schlemm’s canal, the sclera, the ciliary body, the retina, and the optic nerve.24 25 The function of the polypeptide is still unknown, but its colocalization with extracellular matrix proteins such, as fibronectin, laminin, or type IV collagen,26 27 supports that at least part of its role is played out in the extracellular environment. Indeed, myocilin is secreted in aqueous humor (AH),28 29 30 31 and experiences with ocular and nonocular cell lines have shown that the protein is released in the extracellular media.14 30 32 33 34 In contrast, the glaucoma-causing myocilin mutations that have been tested prevent the mutant polypeptides from being secreted30 35 and decrease the expressed protein’s solubility in Triton X-100.36

Molecular mechanisms leading to dominance have been classified into eight categories.37 In humans, one of these categories is dominant negative effects often caused by mutations in multimeric proteins that rely on oligomerization for their activity.37 Two features located in the myocilin NH2-terminal may be involved in protein–protein interactions and may be relevant to the autosomal dominant mode of segregation of glaucoma: a coiled-coil motif between residues 78 and 105 and a leucine zipper containing seven leucine motifs at aa 117-166.14 28 Nine cysteine residues distributed along the polypeptide may also be involved in intermolecular disulfide bonding.

To investigate the role of myocilin mutations in the pathogenesis of autosomal dominant POAG, we studied potential interactions between the wild-type protein and the Q368X or K423E variants. We report that WT myocilin was detected as oligomers ranging from approximately 116 kDa to more than 200 kDa in human trabecular meshwork (HTM), transiently transfected cell lines, and extracellular media of this tissue and cell lines. The Q368X and K423E glaucoma disease-causing mutants were found to interact with WT myocilin, producing heteromeric complexes that were not exported extracellularly. Mutant Q368X and K423E homomeric complexes also remained sequestered within transfected cell lines. The pathologic role of myocilin mutations may thus be linked to intracellular sequestration of MYOCmutant/MYOCmutant and/or MYOCWT/MYOCmutant complexes.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Trabecular Meshwork Tissues and Aqueous Humor
Pairs of human eyes were obtained less than 24 hours after death from the Centre Hospitalier de l’Université Laval (CHUL) eye bank after approval of the CHUL ethics board in agreement with the tenets of the Declaration of Helsinki. AH was harvested with a 271/2-gauge syringe and frozen at –80°C until needed. HTMs dissected from eyes after enucleation were stored at –80°C in Triton lysis buffer (0.5% Triton X-100, 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, protease inhibitor cocktail tablets [Complete; Roche, Laval, Quebec, Canada], and 0.7 µg/mL pepstatin [Sigma-Aldrich, Oakville, Ontario, Canada]). Before analysis, HTMs were sonicated (Sonic Dismembranator 550; Fisher Scientific, Nepean, Canada), and protein concentrations were measured with a commercial protein assay (Bio-Rad, Mississauga, Ontario, Canada) based on the method of Bradford.38

Construction of Myocilin-Expression Vector, Site-Directed Mutagenesis, and Epitope Tagging
To create a eukaryotic expression vector encoding the human wild-type myocilin cDNA, a 1831-bp HindIII/NotI fragment encompassing 36 bp of the 5' untranslated region, the full-length 1512-bp open reading frame (ORF), and 188 bp of the 3' untranslated region of MYOCWT was subcloned directionally into the HindIII/NotI sites of the plasmid pRcCMV (Invitrogen, Burlington, Ontario, Canada). The myocilin-expression vector was named pRc-MYOC. Tagged MYOC cDNAs were prepared by using the overlap extension method in combination with the polymerase chain reaction.39 Overlapping oligonucleotides used were 5'-TTT-TCC-TTT-TGC-GGC-CGC-TCA-ATT-CAG-ATC-CTC-TTC-TGA-GAT-GAG-TTT-TTG-TTC-CAT-CTT-GGA-GAG-CTT-GAT-GTC-ATA-AGT-3' and 5'-TTT-TCC-TTT-TGC-GGC-CGC-TCA-GTG-ATG-ATG-GTG-GTG-ATG-CAT-CTT-GGA-GAG-CTT-GAT-GTC-ATA-AGT-3' for the C-Myc- and His6-tagged cDNAs, respectively (overlapping sequences are italic). Flanking oligonucleotide used for both constructs was 5'-CCC-ACT-GCT-TAA-CTG-GCT-TAT-CG-3'. The resultant PCR products were cut with the restriction enzymes HindIII and NotI and cloned into pRcCMV digested with the same enzymes. Tags were placed at the carboxyl terminus end of MYOC cDNA, and the tagged plasmids were named pRc-MYOCWT-His and pRc-MYOCWT-Myc. MYOC mutants were generated by site-directed mutagenesis on the pRc-MYOC, pRc-MYOCWT-His, or pRc-MYOCWT-Myc expression vectors using a mutagenesis kit (QuickChange; Stratagene, La Jolla, CA), according to the protocols of the company. All cDNA sequences were verified with a sequencing apparatus (model 3700; Applied Biosystems [ABI], Foster City, CA).

Cell Culture, Transfection, and Cellular Tissue Preparation
COS-7 cells (ATCC, Manassas, VA) were grown in high-glucose DMEM complemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 µM L-glutamine (Invitrogen) and incubated at 37°C in a humidified chamber with 5% CO2. Immortalized human trabecular meshwork (iHTM) cells, established from a 30-year-old individual without glaucoma, as previously described,40 were grown in similar conditions except that low-glucose DMEM was used. This cell line did not produce detectable endogenous myocilin, and the protein was not induced by glucocorticoid treatments, unlike human primary trabecular meshwork cell lines in which MYOC was upregulated by dexamethasone.34 COS-7 and iHTM cells were plated at densities of 1.5 x 105 or 1.2 x 106 cells per 35- or 100-mm culture dishes, respectively. Transient transfections were performed 16 hours later with transfection reagent (FuGene 6; Roche) in conditions recommended by the manufacturer. We used 2 µL of the reagent and 1 µg of total plasmid for COS-7 cells, whereas a ratio of 4 µL for 2 µg was used for iHTM cells (35-mm culture dish). Forty-eight hours after transfection, an aliquot of the extracellular medium was taken before the cells were washed twice with ice-cold PBS and scrapped in Triton X-100 lysis buffer using a rubber policeman. Because myocilin may bind to membranes,14 this procedure efficiently measured the released form of the protein. Cellular extracts were processed as for HTM tissues before protein analysis.

Glycosidases and Tunicamycin Treatments
To prevent N-glycosylation, tunicamycin (1 µg/mL medium) was added to the COS-7 culture medium 12 hours after transfection. To hydrolyze potential O- or N-glycans present on MYOC, O-glycosidase (1 mU/20 µg proteins; incubated at 37°C for 1 hour; Roche) or PNGase F (500 U/20 µg proteins; according to the manufacturer’s protocol; New England BioLabs, Beverly, MA) was added, respectively, to selected cell lysates.

Western Blot Analysis
AH and culture media were directly mixed with LDS sample buffer (final concentration: glycerol 1.09 M, Tris-base 141 mM, Tris-HCl 106 mM, LDS 73 mM, EDTA 0.51 mM, serva blue G250 0.22 mM, and phenol red 0.175 mM; NuPAGE; Invitrogen) as for the cellular extracts (total proteins). Protein samples, heated at 70°C for 10 minutes, were resolved on Tris-acetate 7% or 3% to 8% precast protein gels (NuPAGE; Invitrogen) and transferred onto nitro-cellulose membrane (BioTrace NT; PALL Corporation, Mississauga, Ontario, Canada) with a transblotting module (Mini Trans-Blot; Bio-Rad). Membranes were thereafter blocked 30 minutes with PBSMT (PBS 1x, 5% nonfat milk, and 0.1% Tween-20), incubated overnight with the desired antibody diluted in PBSMT, and washed three times for 10 minutes each in PBST (PBS 1x, 0.1% Tween-20) before the addition of the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (30 minutes; 1/2000 in PBSTM; Amersham Biosciences, Arlington Heights, IL). Finally, membranes were soaked three time for 10 minutes each in PBST and three time for 5 minutes each in PBS. Proteins visualization was accomplished with a chemiluminescence kit (Western Lightning; Perkin Elmer, Markham, Ontario, Canada). The rabbit polyclonal anti-His (1:200) and mouse monoclonal anti-Myc antibodies used (1/200) were bought from Santa Cruz Biotechnologies (Santa Cruz, CA). Specificity of the rabbit polyclonal antibody (1:2000) used against MYOC has been described.14

Immunoprecipitation
COS-7 cells, plated at a density of 1.2 x 106 cells per 100-mm culture dish, were harvested 48 hours after cDNA transfections. Cells were washed twice with ice-cold PBS and lysed in 1 mL lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, protease inhibitor cocktail tablets [Complete; Roche], and 0.7 µg/mL pepstatin; Sigma-Aldrich). The homogenized suspensions were centrifuged 10 minutes at 12,000g and supernatants precleared at least 3 hours with 50 µL protein A-agarose (Roche). Agarose beads were then pelleted and supernatants transferred to fresh microcentrifuge tubes. Precleared samples were subsequently incubated for 1 hour on a rocking platform with 5 µg of the anti-His antibody (Santa Cruz Biotechnologies) before 50 µL of protein A-agarose was added for at least 3 hours. Protein complexes were collected by centrifugation and the supernatants removed. Beads were washed twice with lysis buffer, twice with washing buffer 2 (50 mM Tris-HCl [pH 7.4], 300 mM sodium chloride, 0.1% Nonidet P-40, and 0.05% sodium deoxycholate) and finally once with washing buffer 3 (50 mM Tris-HCl [pH 7.4], 0.1% Nonidet P-40, and 0.05% sodium deoxycholate) before analysis of the immunoprecipitated proteins by Western blot analysis.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Myocilin Oligomerization Status
To test for interactions compatible with dominant negative effects, we first investigated the oligomerization status of myocilin protein in human AH and HTM tissues, using Western blot analysis under nonreducing conditions. We optimized visualization of the bands of more than 150 kDa by resolving proteins through Tris-Acetate 3% to 8% protein gels. MYOC protein was detected with a well-characterized rabbit polyclonal antibody developed against the whole protein.14 As depicted in Figure 1A (lanes 1 and 4), MYOC immunoreactivity displayed migration patterns that were highly similar in HTM tissues and AH. Immunoreactive bands were detected at approximately 116, 150, and 180 kDa and as several complexes migrating at/and above 200 kDa. AH and HTM obtained from five additional donors, aged 67 to 83 years, were also tested (data not shown). The samples produced migration patterns identical with those shown in Figure 1A , with the 150-kDa band consistently stronger in AH than in HTM (Fig. 1A , compare lane 4 to lane 1). Because the MYOC gene encodes a 504-aa polypeptide with a theoretical molecular mass of 56.9 kDa, our experiments suggested that the different complexes observed in nonreducing conditions may be oligomers formed by two or more MYOC polypeptide(s) and/or by one MYOC polypeptide associated with other proteins.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1. Western blot analysis of wild-type myocilin. (A) Myocilin detection pattern under nonreducing conditions in different tissues and media. Five micrograms total protein from COS-7 and iHTM cells transiently expressing MYOCWT and 2.5 µg of HTM tissues were resolved on Tris-acetate 3% to 8% protein gels, and 10 µL of both culture media (COS-7 and iHTM cells) and 5 µL of AH were analyzed. (B) Myocilin protein from AH and HTM tissues was analyzed under reducing conditions using 5 µL AH and 2.5 µg HTM tissues, both treated with 100 mM DTT. Reduced proteins were resolved on Tris-Acetate 7% protein gel, and MYOC was detected using our polyclonal antibody.

 
We next investigated the stability of the HTM and AH myocilin complexes after various denaturing conditions. Boiling and SDS concentration up to 2% had no effect on complex patterns (data not shown). On the contrary, addition of 100 mM of the reducing agent dithiothreitol (DTT) to protein extracts completely disrupted the complexes, showing that disulfide bonds are essential for stabilization of myocilin protein complexes. As depicted in Figure 1B , myocilin was detected in HTM and AH as a doublet migrating at approximately 55 and 57 kDa after DTT treatment. This doublet probably represents glycosylated and unglycosylated forms of the protein.32 33 41

To establish an in vitro system in which we could assess the nature of the oligomers as well as that of the doublet, the pRc-MYOC expression vector encoding WT myocilin was transiently transfected into the COS-7 and iHTM cell lines. These two cell lines were chosen because they did not produce detectable endogenous immunoreactive MYOC protein. Forty-eight hours after transfection, cellular extracts and culture media from COS-7 and iHTM cells were analyzed by immunoblot assay. MYOC migration patterns in cellular extracts and culture media from both cell lines were found to be almost identical with those observed in AH and HTM tissues, with the exception of the 150-kDa band, which was not detected in iHTM cells (Fig. 1A , lanes 3 and 6). The COS-7 and iHTM cell lines were therefore selected to investigate further the nature of MYOC immunoreactive complexes in vitro.

Myocilin Glycosylation Status
Because myocilin polypeptide contains several potential sites for N- and O- glycosylation,14 we determined whether one of these posttranslational modification mechanisms was involved in generating the distinct myocilin isoforms observed as doublets in denaturing conditions. COS-7 cells were transfected with MYOCWT cDNA for 12 hours before a 36-hour incubation period with the antibiotic tunicamycin (an inhibitor of the dolichol-dependent N-glycoside sugar chain biosynthesis). Western blot analysis under reducing conditions revealed that the inhibitor prevented the synthesis of the 57-kDa monomer (Fig. 2A , lane 2), suggesting that oligosaccharides were linked to the 55-kDa isoform. The nature of this (these) link(s) was then investigated by transfecting the cells with the WT construct and treating the cellular extracts with specific glycosidases before protein analysis. As depicted in Figure 2A (lane 3), treatment with the N-glycosidase PNGase F, which releases asparagine-linked oligosaccharides, removed all carbohydrates from the polypeptide backbone of the 57-kDa monomer, leaving intact the 55-kDa isoform. In contrast, treatment with O-glycosidase did not alter the migration pattern of either isoform (Fig. 2A , lane 4), supporting that oligosaccharides were linked to the 55-kDa isoform solely by an N-glycoside chain. The sensitivity of the 57-kDa monomer to PNGase F digestion was also observed in HTM and AH (Figs. 2B 2C , respectively), demonstrating that, in vivo, myocilin also formed an N-glycosylated polypeptide. Because MYOC harbored only one potential site for N-glycosylation at Asn57 (57NESS), we then mutated this amino acid to a tyrosine residue. Transfection and analysis of this recombinant revealed that MYOCN57Y cDNA generated only one 55-kDa isoform, the secretion of which was not altered by its lack of glycosylation (Fig. 2D , lane 2). These results established that the 57-kDa monomer was generated by the addition of an N-amino glycan to the Asn57 residue of the 55-kDa monomer.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 2. Glycosylation status of myocilin. All samples were treated with 100 mM DTT before migration. (A) COS-7 cells transiently expressing MYOCWT were treated with tunicamycin, an inhibitor of the dolichol-dependent N-glycoside sugar chain biosynthesis. The nature of the oligosaccharide link was determined by treating protein extracts with specific glycosidases. Western blot analysis was performed using 2.5 µg of proteins from each extract. (B) HTM tissues were treated with the N-glycosidase PNGase F and 2 µg of proteins were used for analysis. (C) AH was also submitted to PNGase F digestion and 2.5 µL were migrated. (D) COS-7 cells were transiently transfected with MYOCWT or MYOCN57Y cDNA and 48 hours post-transfection, 5 µL of extracellular media from each transfection were analyzed by Western blot analysis.

 
Wild-type–Mutant Myocilin Protein Interactions
To investigate whether wild-type myocilin interacts with its disease-causing mutant counterparts, we designed double-epitope tagging procedures that detected complexes made of at least two MYOC polypeptides. Briefly, different combinations of His-tag and Myc-tag MYOC cDNAs (MYOCWT-His, MYOCWT-Myc, MYOCK423E-Myc, or MYOCQ368X-Myc) were transfected into COS-7 cells. Forty-eight hours after transfection, newly generated proteins were immunoprecipitated with an antibody raised against a six histidines (His-tag) component added to the C-terminal of myocilin protein, resolved on a 7% Tris-acetate protein gel under nonreducing conditions and visualized by immunoblotting using an anti-Myc antibody specific to the 11-aa Myc-tag epitope added to the C-terminal of a second MYOC polypeptide. When detected, MYOC complexes thus contained at least one His-tag and one Myc-tag myocilin polypeptide. Tagging the C terminus of MYOC polypeptides with a His-tag or a Myc-tag antigenic component did not interfere with their secretion or alter their migration patterns when analyzed with our anti-myocilin polyclonal antibody (Fig. 3A) . To validate the specificity of our double-epitope tagging procedures, we tested for cross-reactivity protein extracts obtained from COS-7 cells transfected with either His-tag or Myc-tag MYOC cDNA alone (Fig. 3B , lanes 4 and 5, respectively). In these conditions, no immunoreactive signal was detected when anti-His-tag immunoprecipitation was performed followed by anti-Myc-tag immunoblot analysis.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 3. Wild-type/mutant myocilin protein interaction analysis. (A) Forty-eight hours after transfection, 10 µL of extracellular media from COS-7 cells expressing WT, WT-His, or WT-Myc MYOC were examined by Western blot (Wb) analysis using the polyclonal anti-MYOC antibody. (B) COS-7 cells were transfected with equal quantities of WT-His/WT-Myc, WT-His/K423E-Myc, WT-His/Q368X-Myc, WT-His, alone or WT-Myc alone myocilin plasmids. Forty-eight hours after transfection, His-tagged proteins were precipitated with a rabbit polyclonal anti-His antibody, migrated under nonreducing conditions on a Tris-acetate 7% gel, and transferred onto a nitrocellulose membrane. Complexes containing Myc-tagged proteins were visualized using a mouse monoclonal anti-Myc antibody (1/200 dilution). (C) Protein samples from COS-7 cells transiently expressing MYOCWT, MYOCK423E, or MYOCQ368X were treated with 100 mM DTT and 2.5 µg of total proteins were migrated and transferred onto a nitrocellulose membrane. Myocilin was detected using our polyclonal anti-MYOC antibody. (D) Five micrograms of total proteins from COS-7 cells cotransfected with different ratios of MYOCWT and MYOCQ368X cDNAs were migrated and analyzed by Western blot analysis under nonreducing conditions. WT and mutant proteins were visualized with the polyclonal anti-MYOC antibody.

 
We first examined whether WT myocilin interacts with itself. COS-7 cells were transfected with equal quantities of MYOCWT-His and MYOCWT-Myc cDNAs, and our double-epitope tagging procedures was performed on cellular extracts. As depicted in Figure 3B , lane 1, several WT-His/WT-Myc MYOC complexes were detected. They ranged from approximately 116 kDa to more than 200 kDa. Shorter time exposures of the autoradiograms (<20 seconds) revealed immunoreactive migration patterns similar to those displayed in Figure 1 (data not shown). Because the WT and K423E proteins migrated, under reducing conditions, at approximately 55 to 57 kDa (Fig. 3C , lanes 1 and 3), the 116-kDa complex (Fig. 3B , lane 1) thus resulted from homodimerization between two MYOCWT polypeptides, whereas complexes of more than 180 kDa were generated by two MYOC polypeptides interacting together with an additional myocilin entity(ies) and/or in association with other proteins.

We next investigated whether interactions also occur between wild-type MYOC and glaucoma-causing mutants. We first cotransfected COS-7 cells with different ratios of untagged MYOCWT and MYOCQ368X cDNAs. The MYOCQ368X variant is the most common mutation associated with adult-onset primary open-angle glaucoma, carried by more than 1% of all patients with POAG.12 22 Because this mutant cDNA yielded two truncated polypeptides of approximately 42 and 44 kDa (Fig. 3C , lane 2) harboring an intact leucine zipper, we hypothesized that, on simultaneous transfection of MYOCWT and MYOCQ368X cDNAs in COS-7 cells, interaction between the WT construct and the Q368X recombinant would generate novel heterodimers of sizes intermediate between MYOCWT and MYOCQ368X homodimers. Indeed, as depicted in Figure 3D , lanes 1 and 5, when MYOCWT and MYOCQ368X recombinants were transfected alone, they formed homodimers migrating at approximately 116 and 84 kDa, respectively. In cells transfected simultaneously with both cDNAs, a novel immunoreactive complex of intermediate size was produced at approximately 96 kDa (Fig. 3D , lane 3). This newly generated complex most likely resulted from heterodimerization of one MYOCWT monomer with its MYOCQ368X counterpart. These results show that MYOC polypeptides underwent oligomerization in cultured cells without the need for extraneous protein. Moreover, because MYOCQ368X did not encode cysteine 433, these data suggested that this C-terminal cysteine was not essential in heterodimerization.

To confirm heterodimerization between WT and mutant myocilin polypeptides, protein extracts obtained from COS-7 cells simultaneously transfected with equal quantities of MYOCWT-His and MYOCK423E-Myc or of MYOCWT-His and MYOCQ368X-Myc were tested with our double-epitope procedures. Several WT/K423E and WT/Q368X heterocomplexes (Fig. 3B , lanes 2 and 3, respectively) were detected as multiple bands migrating at and more than 200 kDa and as heterodimers at approximately 116 and 96 kDa, respectively (Fig. 3B , lanes 2 and 3). Taken together, our results demonstrate that WT MYOC interacted with itself and with the K423E and Q368X glaucoma-causing mutants generating homo- and heterodimers as well as multiple high molecular weight hetero-oligomers, alone and/or in association with other proteins.

Myocilin Secretion Studies
Several studies demonstrated that WT myocilin protein was secreted from ocular and nonocular cells in culture, whereas variations affecting the olfactomedin domain, associated or not with glaucoma, remained sequestered intracellularly.30 32 33 35 In these experiments, protein analysis was mostly performed in denaturing conditions and did not investigate whether disease-causing mutants, including the Q368X and K423E mutations, blocked the secretion of the WT protein by interacting with it. To assess whether myocilin mutants block secretion of their wild-type counterpart when forming hetero-oligomers, COS-7 cells were transfected with increasing concentrations of pRc-MYOCK423E-Myc in the presence of decreasing amounts of pRc-MYOCWT-His. Forty-eight hours after transfection, Myc-tag or His-tag immunoreactivities were measured in extracellular media and in cellular extracts. Using specific epitopes for each transfected cDNA allowed us to assess the contribution of each protein to the oligomers. As expected, when WT and mutant proteins were transfected in the absence of their partners, both were highly expressed in COS-7 cellular extracts (Figs. 4A , lane 1; 4B, lane 5). On the contrary, when extracellular media of this experiment were tested, no expression of the K423E-Myc mutant polypeptide was detected outside the cell (Fig. 4D , lane 5), whereas high levels of wild-type MYOCWT-His protein were observed in the media samples (Fig. 4C , lane 1). The K423E-Myc mutant polypeptide therefore remained sequestered within the cells. Indeed, when we investigated the presence of the Myc epitope attached to the K423E mutant, no mutant protein was detected in the extracellular medium in any of the samples tested (Fig. 4D , lanes 1–5). To demonstrate that the failure to detect the Myc epitope outside the cells was not caused by inefficient transfection, protein transfer or visualization procedures, culture media containing MYOCWT-Myc was processed along with the WT-His/K423E-Myc samples (Fig. 4D , lane 6). Increasing the ratios of the K423E-Myc proteins while decreasing that of the WT-His progressively hampered secretion of the latter (Fig. 4C , lanes 2, 3, 4). However, it is noteworthy that high expression levels of the K423E-Myc protein did not totally prevent the secretion of MYOCWT-His, as the wild-type protein was still detected in extracellular media even at a WT-His to K423E-Myc cDNA ratio of 25% to 75% (Fig. 4C , lane 4).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 4. Wild-type/K423E myocilin secretion analysis. Indicated ratios of MYOCWT-His/MYOCK423E-Myc cDNAs were transiently transfected in COS-7 cells. Five micrograms of total proteins or 10 µL of culture media were migrated on a 7% Tris-acetate gel. Tagged proteins were detected using the indicated antibody. (A) Cellular extracts and (C) culture medium were analyzed using the polyclonal anti-His antibody. (B) Cellular extracts and (D) culture medium were analyzed using the monoclonal anti-Myc antibody. (D, lane 6) Control culture medium from MYOCWT-Myc transfected in COS-7 cells.

 
As depicted in Figures 3B and 3D , dimerization between one WT polypeptide and one Q368X molecule generated an intracellular immunoreactive complex migrating at approximately 96 kDa. To assess whether WT/Q368X heterodimers remain sequestered within the cellular compartment as observed for the WT/K423E complexes, we searched for these characteristic 96-kDa protein bands in extracellular media of COS-7 cells transfected simultaneously with different ratios of MYOCWT and MYOCQ368X cDNAs. As previously observed in Figure 3D (lane 5), the mutant polypeptide was efficiently produced by COS-7 cells but was not detected in extracellular media (Fig. 5 , lane 5). Furthermore, the 96-kDa WT/Q368X heterodimers were not detected outside the cells when both proteins were coexpressed at various ratios (Fig. 5 , lanes 2, 3, 4). As for the K423E mutant, we also noticed that high expression level of MYOCQ368X progressively hampered, but did not totally block, the secretion of MYOCWT (Fig. 5 , lane 4). These results clearly indicate that WT/mutant complexes were not secreted, in a similar fashion as the mutant/mutant, whereas the WT/WT oligomers still retained the capacity to be released in the medium.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 5. Myocilin/Q368X secretion analysis. Indicated ratios of MYOCWT/MYOCQ368X cDNAs were transiently transfected in COS-7 cells. Ten microliters of culture media were migrated on a Tris-acetate 7% gel. Myocilin was detected using the rabbit polyclonal anti-MYOC. The WT/Q368X 96-kDa and Q368X/Q368X 84-kDa dimer immunoreactivities were not observed.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Our studies showed that wild-type myocilin protein in its native form displayed multiple complexes ranging from approximately 116 kDa to more than 200 kDa. These complexes were highly similar in HTM and in transfected COS-7 and iHTM cell lines. In AH, an additional 150-kDa immunoreactive band was also consistently observed, increasing the total number of AH complexes to seven migrating at approximately 116, 150, 180, and 200 kDa and above 250 kDa. Our data were in close agreement with gel filtration experiments performed by Fautsch and Johnson28 who reported in human AH, myocilin immunoreactive complexes ranging in size from 120 to 180 kDa with a peak of activity at 150 kDa. In contrast, in bovine and monkey AH, gel filtration studies performed by Russel et al.31 demonstrated that most myocilin immunoreactivity migrates in protein fractions above 250 kDa. Such discrepancies may be related to species-specific forms of myocilin or to the less disruptive conditions used by Russel et al. during their gel filtration experiments.

Several studies have suggested that myocilin protein may associate with itself.14 28 31 33 Other investigations also demonstrated that the protein may interact with specific extracellular matrix proteins26 27 or with optimedin, an olfactomedin-related protein.42 Our experiments clearly showed that two myocilin monomers preferentially interacted to generate homodimers that migrated at approximately 116 kDa. We also showed that complexes at or above 180 kDa contained at least two myocilin moieties, which may then interact with different polypeptides and/or with themselves to generate larger homo- and/or hetero-oligomers. At present, we cannot differentiate between these processes, but further experiments using immunoprecipitation and/or purification columns combined with mass spectrometry analysis should help to elucidate their compositions.

Myocilin glycosylation studies revealed that the protein was partially N-glycosylated at asparagine 57, leading to the formation of two isoforms migrating at 55 and 57 kDa, as already observed.32 33 41 Different combinations between these two isoforms may explain the formation of several dimeric forms migrating at slightly different positions, as observed for the wild-type 116 to 120 kDa (Fig. 3D , lane 1) and for the Q368X 82- to 86-kDa (Fig. 3D , lane 5) dimer bands. The reason for this incomplete glycosylation state of myocilin may be competition between N-glycosylation and disulfide bond formation, as previously observed with some proteins—for example, the proteins Wingless,43 carboxypeptidase Y44 or hemagglutinin-neuraminidase glycoprotein of the Newcastle disease virus.45

MYOC self-interaction is likely to be mediated through its N-terminal region, which is coiled-coil rich.14 28 32 33 In agreement with this hypothesis, we clearly demonstrated that myocilin oligomerization was not impaired by the C-terminal Q368X or K423E mutations, because these proteins were detected as several homo-oligomers migrating near and over 200 kDa and as homodimers of ~82 and 116 kDa, respectively (Figs. 3D , lane 5; 4B lane 5). Furthermore, we found that these disease-causing mutants interacted with the wild-type protein to yield numerous hetero-oligomers and heterodimers. That MYOCQ368X did not encode the phylogenetically conserved cysteine 433 suggests that this C-terminal cysteine is not essential for the homo- and hetero-oligomerization process. Our findings may be extended to other olfactomedin domain variations, associated or not with glaucoma. Indeed, preliminary experiments in our laboratory have demonstrated that several mutations in this domain, for instance the P370L and Y437H glaucoma-causing mutant polypeptides,9 12 form heterodimers with their wild-type counterpart, which remain sequestered intracellularly (Gobeil S, et al. manuscript in preparation).

Several studies have reported that mutations located within the olfactomedin-like domain of myocilin inhibit its secretion.30 33 35 However, these reports did not investigate whether these disease-causing mutants hamper secretion of their wild-type counterpart by directly interacting with it. Using our double-epitope strategy and protein migration under nonreducing conditions, we observed that the Q368X and K423E homo-oligomers remained sequestered within transfected COS-7 cells. More important, heteromeric complexes formed between WT and mutant polypeptides also remained sequestered within the cells. Intracellular sequestration of wild-type proteins by its mutant counterparts has been demonstrated for several disease-causing proteins. For example, coexpression of the human anion exchanger (AE1), a membrane glycoprotein, with a truncated variant missing 11 amino acids of the carboxyl end results in the formation of heterocomplexes and in the retention of these within the intracellular compartment. This mechanism leads to an autosomal dominant form of the distal renal tubular acidosis (dRTA) disease.46 Furthermore, aquaporin (AQP)-2, a protein implicated in water reabsorption interacts with its counterpart E258K to produce complexes not exported to the membranes, causing an autosomal dominant form of diabetes insipidus.47

Mechanisms leading to glaucoma once the WT/mutant and mutant/mutant myocilin oligomers remained sequestered within the cell were not investigated in the present study. However, it is well recognized that mutant membrane and extracellular proteins that fail to fold and/or to oligomerize correctly are often retained within the endoplasmic reticulum (ER) compartment.48 49 In agreement with this model, Caballero and Borras32 presented evidence that a truncated form of myocilin (aa 1-344) was not processed correctly in the ER and accumulated in insoluble aggregates, and Joe et al.50 demonstrated that mutant myocilin was concentrated into fine punctate aggregates in the ER. More recently, Liu and Vollrath51 have shown that several disease-causing myocilin mutants also accumulate in the ER and are prone to aggregate, leading to cell toxicity.

Several genetic20 52 53 54 and biochemical lines of evidence30 32 50 55 have suggested that autosomal dominant POAG-linked myocilin mutations may act through a pathologic gain-of-function mechanism caused by the intracellular accumulation of mutant proteins. Our results, showing for the first time that WT and mutant myocilin proteins interact and that these heterocomplexes are not secreted, support this hypothesis. However, the asymptomatic condition of the K423E homozygote carriers remains puzzling. As determined by us and Jacobson et al.,30 the K423E protein remained sequestered within cells when expressed in an homozygotic fashion. Thus, the unaffected condition of the K423E/K423E carriers does not result from homoallelic complementation,56 a process that would have restored the normal secretion of this mutant protein, but may be explained by metabolic interference. Indeed in 1980, Johnson57 considered dominant negative effects to propose a mechanism called metabolic interference that accounted for hypothetical forms of simple inheritance in which the heterozygote alone was affected. Metabolic interference assumed a one-locus mechanism in which a wild-type allele A and a mutant allele A' interact so that homozygosity for either allele has no phenotypic consequence, but the heterozygous state AA' leads to a deleterious defect, due to interference between the protein products of the two different alleles. To account for metabolic interference, we thus hypothesize that the K423E homomeric complexes may be degraded by the ER-associated degradation (ERAD) pathway in the cytosol.58 In contrast, the WT/K423E complexes may not be subjected to this degradation pathway. Over the years, accumulation of these abnormal hetero-oligomers could lead to the malfunction of myocilin-expressing cells and finally to POAG through a dominant negative effect. This mechanism could involve a variety of processes including an activation of the stress apoptotic pathways. Experiences investigating the turnover rates of the homo- and heteromeric mutant complexes should further our understanding of the mechanisms underlying myocilin-associated glaucoma.


   Acknowledgements
 
The authors thank Eric Winstall for critical reading of the manuscript and Ghislaine Chamberland, Francine Simard, and Ide Dubé of the CHUL Eye Bank for generous contributions to this work. VR also thanks Claude Dion for helpful discussions.


   Footnotes
 
2 Present affiliation: DiaTech Oncology, Montréal, Québec, Canada. Back

Supported by Canadian Institutes of Health Research (CIHR) Grant MOP-53232; Canadian Foundation for Innovation Grant 548; The Glaucoma Research Foundation, San Francisco, CA; The Glaucoma Foundation, New York, NY; La Fondation des Maladies de l’Oeil, Québec City, Québec Canada; and the Fonds de la Recherche en Santé du Québec (FRSQ) Health Vision Research Network. SG is supported by a CIHR KM Hunter doctoral studentship. VR is an FRSQ National Investigator.

Submitted for publication March 16, 2004; revised June 23, 2004; accepted June 25, 2004.

Disclosure: S. Gobeil, None; M.-A. Rodrigue, None; S. Moisan, None; T.D. Nguyen, InSite Vision (F); J.R. Polansky, InSite Vision (F); J. Morissette, None; V. Raymond, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Vincent Raymond, Molecular Endocrinology and Oncology Research Center, CHUL Research Center, Room T3-67, 2705 Laurier Boulevard, Québec City, Québec G1V 4G2, Canada; vincent.raymond{at}crchul.ulaval.ca.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol. 1996;80:389–393.[Abstract/Free Full Text]
  2. Quigley HA. Reappraising the risk and benefits of aggressive glaucoma therapy. Ophthalmology. 1997;104:1985–1986.[ISI][Medline][Order article via Infotrieve]
  3. McKusick VA. Mendelian Inheritance in Man: a Catalog of Human Genes and Genetic Disorders.. 1994; 11th ed. Johns Hopkins University Press Baltimore.
  4. Leske MC. The epidemiology of open-angle glaucoma: a review. Am J Epidemiol. 1983;118:166–191.[Free Full Text]
  5. Thylefors B, Negrel AD. The global impact of glaucoma. Bull World Health Org. 1994;72:323–326.[ISI][Medline][Order article via Infotrieve]
  6. Raymond V. Molecular genetics of the glaucomas: mapping of the first five "GLC" loci. Am J Hum Genet. 1997;60:272–277.[ISI][Medline][Order article via Infotrieve]
  7. Sarfarazi M. Recent advances in molecular genetics of glaucomas. Hum Mol Genet. 1997;6:1667–1677.[Abstract/Free Full Text]
  8. Wirtz MK, Samples JR, Rust K, et al. GLC1F, a new primary open-angle glaucoma locus, maps to 7q35–q36. Arch Ophthalmol. 1999;117:237–241.[Abstract/Free Full Text]
  9. Stone EM, Fingert JH, Alward WL, et al. Identification of a gene that causes primary open angle glaucoma. Science. 1997;275:668–670.[Abstract/Free Full Text]
  10. Kubota R, Noda S, Wang Y, et al. A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping. Genomics. 1997;41:360–369.[CrossRef][ISI][Medline][Order article via Infotrieve]
  11. Rezaie T, Child A, Hitchings R, et al. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science. 2002;295:1077–1079.[Abstract/Free Full Text]
  12. Fingert JH, Héon E, Liebmann JM, et al. Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum Mol Genet. 1999;8:899–905.[Abstract/Free Full Text]
  13. Fingert JH, Stone EM, Sheffield VC, Alward WL. Myocilin glaucoma. Surv Ophthalmol. 2002;47:547–561.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Nguyen TD, Chen P, Huang WD, Chen H, Johnson D, Polansky JR. Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J Biol Chem. 1998;273:6341–6350.[Abstract/Free Full Text]
  15. Adam MF, Belmouden A, Binisti P, et al. Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma. Hum Mol Genet. 1997;6:2091–2097.[Abstract/Free Full Text]
  16. Mansergh FC, Kenna PF, Ayuso C, Kiang AS, Humphries P, Farrar GJ. Novel mutations in the TIGR gene in early and late onset open angle glaucoma. Hum Mutat. 1998;11:244–251.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Kennan AM, Mansergh FC, Fingert JH, et al. A novel Asp380Ala mutation in the GLC1A/myocilin gene in a family with juvenile onset primary open angle glaucoma. J Med Genet. 1998;35:957–960.[Abstract]
  18. Wiggs JL, Allingham RR, Vollrath D, et al. Prevalence of mutations in TIGR/Myocilin in patients with adult and juvenile primary open-angle glaucoma. Am J Hum Genet. 1998;63:1549–1552.[CrossRef][ISI][Medline][Order article via Infotrieve]
  19. Richards JE, Ritch R, Lichter PR, et al. Novel trabecular meshwork inducible glucocorticoid response mutation in an eight-generation juvenile-onset primary open-angle glaucoma pedigree. Ophthalmology. 1998;105:1698–1707.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Morissette J, Clépet C, Moisan S, et al. Homozygotes carrying an autosomal dominant TIGR mutation do not manifest glaucoma. Nat Genet. 1998;19:319–321.[CrossRef][ISI][Medline][Order article via Infotrieve]
  21. Alward WL, Fingert JH, Coote MA, et al. Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A). N Engl J Med. 1998;338:1022–1027.[Abstract/Free Full Text]
  22. Faucher M, Anctil JL, Rodrigue MA, et al. Founder TIGR/myocilin mutations for glaucoma in the Québec population. Hum Mol Genet. 2002;11:2077–2090.[Abstract/Free Full Text]
  23. Pang CP, Leung YF, Fan B, et al. TIGR/MYOC gene sequence alterations in individuals with and without primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2002;43:3231–3235.[Abstract/Free Full Text]
  24. Karali A, Russell P, Stefani FH, Tamm ER. Localization of myocilin/trabecular meshwork–inducible glucocorticoid response protein in the human eye. Invest Ophthalmol Vis Sci. 2000;41:729–740.[Abstract/Free Full Text]
  25. Swiderski RE, Ross JL, Fingert JH, et al. Localization of MYOC transcripts in human eye and optic nerve by in situ hybridization. Invest Ophthalmol Vis Sci. 2000;41:3420–3428.[Abstract/Free Full Text]
  26. Filla MS, Liu X, Nguyen TD, et al. In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin. Invest Ophthalmol Vis Sci. 2002;43:151–161.[Abstract/Free Full Text]
  27. Ueda J, Wentz-Hunter K, Yue BY. Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci. 2002;43:1068–1076.[Abstract/Free Full Text]
  28. Fautsch MP, Johnson DH. Characterization of myocilin-myocilin interactions. Invest Ophthalmol Vis Sci. 2001;42:2324–2331.[Abstract/Free Full Text]
  29. Rao PV, Allingham RR, Epstein DL. TIGR/myocilin in human aqueous humor. Exp Eye Res. 2000;71:637–641.[CrossRef][ISI][Medline][Order article via Infotrieve]
  30. Jacobson N, Andrews M, Shepard AR, et al. Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor. Hum Mol Genet. 2001;10:117–125.[Abstract/Free Full Text]
  31. Russell P, Tamm ER, Grehn FJ, Picht G, Johnson M. The presence and properties of myocilin in the aqueous humor. Invest Ophthalmol Vis Sci. 2001;42:983–986.[Abstract/Free Full Text]
  32. Caballero M, Borras T. Inefficient processing of an olfactomedin-deficient myocilin mutant: potential physiological relevance to glaucoma. Biochem Biophys Res Commun. 2001;282:662–670.[CrossRef][ISI][Medline][Order article via Infotrieve]
  33. Caballero M, Rowlette LL, Borras T. Altered secretion of a TIGR/MYOC mutant lacking the olfactomedin domain. Biochim Biophys Acta. 2000;1502:447–460.[Medline][Order article via Infotrieve]
  34. Lo WR, Rowlette LL, Caballero M, Yang P, Hernandez MR, Borras T. Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma. Invest Ophthalmol Vis Sci. 2003;44:473–485.[Abstract/Free Full Text]
  35. Izumi K, Mashima Y, Obazawa M, et al. Variants of the myocilin gene in Japanese patients with normal-tension glaucoma. Ophthalmic Res. 2003;35:345–350.[CrossRef][ISI][Medline][Order article via Infotrieve]
  36. Zhou Z, Vollrath D. A cellular assay distinguishes normal and mutant TIGR/myocilin protein. Hum Mol Genet. 1999;8:2221–2228.[Abstract/Free Full Text]
  37. Wilkie AO. The molecular basis of genetic dominance. J Med Genet. 1994;31:89–98.[Abstract]
  38. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[CrossRef][ISI][Medline][Order article via Infotrieve]
  39. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77:51–59.[CrossRef][ISI][Medline][Order article via Infotrieve]
  40. Polansky JR, Weinreb RN, Baxter JD, Alvarado J. Human trabecular cells. I. Establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci. 1979;18:1043–1049.[Abstract/Free Full Text]
  41. Shepard AR, Jacobson N, Sui R, et al. Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage. BMC Genet. 2003;4:5.[CrossRef][Medline][Order article via Infotrieve]
  42. Torrado M, Trivedi R, Zinovieva R, Karavanova I, Tomarev SI. Optimedin: a novel olfactomedin-related protein that interacts with myocilin. Hum Mol Genet. 2002;11:1291–1301.[Abstract/Free Full Text]
  43. Allen S, Naim HY, Bulleid NJ. Intracellular folding of tissue-type plasminogen activator: effects of disulfide bond formation on N-linked glycosylation and secretion. J Biol Chem. 1995;270:4797–4804.[Abstract/Free Full Text]
  44. Holst B, Bruun AW, Kielland-Brandt MC, Winther JR. Competition between folding and glycosylation in the endoplasmic reticulum. EMBO J. 1996;15:3538–3546.[ISI][Medline][Order article via Infotrieve]
  45. McGinnes LW, Morrison TG. Disulfide bond formation is a determinant of glycosylation site usage in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. J Virol. 1997;71:3083–3089.[Abstract]
  46. Quilty JA, Cordat E, Reithmeier RA. Impaired trafficking of human kidney anion exchanger (kAE1) caused by hetero-oligomer formation with a truncated mutant associated with distal renal tubular acidosis. Biochem J. 2002;368:895–903.[CrossRef][ISI][Medline][Order article via Infotrieve]
  47. Kamsteeg EJ, Wormhoudt TA, Rijss JP, van Os CH, Deen PM. An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus. EMBO J. 1999;18:2394–2400.[CrossRef][ISI][Medline][Order article via Infotrieve]
  48. Sidrauski C, Chapman R, Walter P. The unfolded protein response: an intracellular signalling pathway with many surprising features. Trends Cell Biol. 1998;8:245–249.[CrossRef][ISI][Medline][Order article via Infotrieve]
  49. Bross P, Corydon TJ, Andresen BS, Jorgensen MM, Bolund L, Gregersen N. Protein misfolding and degradation in genetic diseases. Hum Mutat. 1999;14:186–198.[CrossRef][ISI][Medline][Order article via Infotrieve]
  50. Joe MK, Sohn S, Hur W, Moon Y, Choi YR, Kee C. Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem Biophys Res Commun. 2003;312:592–600.[CrossRef][ISI][Medline][Order article via Infotrieve]
  51. Liu Y, Vollrath D. Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum Mol Genet. 2004;13:1193–1204.[Abstract/Free Full Text]
  52. Lam DS, Leung YF, Chua JK, et al. Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2000;41:1386–1391.[Abstract/Free Full Text]
  53. Kim BS, Savinova OV, Reedy MV, et al. Targeted disruption of the myocilin gene (Myoc) suggests that human glaucoma-causing mutations are gain of function. Mol Cell Biol. 2001;21:7707–7713.[Abstract/Free Full Text]
  54. Wiggs JL, Vollrath D. Molecular and clinical evaluation of a patient hemizygous for TIGR/MYOC. Arch Ophthalmol. 2001;119:1674–1678.[Abstract/Free Full Text]
  55. Sohn S, Hur W, Joe MK, et al. Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities. Invest Ophthalmol Vis Sci. 2002;43:3680–3685.[Abstract/Free Full Text]
  56. Rawls JM, Jr. Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster. Mol Gen Genet. 1981;184:174–179.[ISI][Medline][Order article via Infotrieve]
  57. Johnson WG. Metabolic interference and the +/– heterozygote: a hypothetical form of simple inheritance which is neither dominant nor recessive. Am J Hum Genet. 1980;32:374–386.[ISI][Medline][Order article via Infotrieve]
  58. Kopito RR. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 2000;10:524–530.[CrossRef][ISI][Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 IOVSHome page
J. Kuchtey, M. E. Kallberg, K. N. Gelatt, T. Rinkoski, A. M. Komaromy, and R. W. Kuchtey
Angiopoietin-like 7 Secretion Is Induced by Glaucoma Stimuli and Its Concentration Is Elevated in Glaucomatous Aqueous Humor
Invest. Ophthalmol. Vis. Sci., August 1, 2008; 49(8): 3438 - 3448.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
Y. Zhou, O. Grinchuk, and S. I. Tomarev
Transgenic Mice Expressing the Tyr437His Mutant of Human Myocilin Protein Develop Glaucoma
Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 1932 - 1939.
[Abstract] [Full Text] [PDF]

Home page
 Arch OphthalmolHome page
F. W. Rozsa, K. Scott, H. Pawar, S. Moroi, and J. E. Richards
Effects of Timolol on MYOC, OPTN, and WDR36 RNA Levels
Arch Ophthalmol, January 1, 2008; 126(1): 86 - 93.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
G. H.-F. Yam, K. Gaplovska-Kysela, C. Zuber, and J. Roth
Sodium 4-Phenylbutyrate Acts as a Chemical Chaperone on Misfolded Myocilin to Rescue Cells from Endoplasmic Reticulum Stress and Apoptosis
Invest. Ophthalmol. Vis. Sci., April 1, 2007; 48(4): 1683 - 1690.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. R. Shepard, N. Jacobson, J. C. Millar, I.-H. Pang, H. T. Steely, C. C. Searby, V. C. Sheffield, E. M. Stone, and A. F. Clark
Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure
Hum. Mol. Genet., March 15, 2007; 16(6): 609 - 617.
[Abstract] [Full Text] [PDF]

Home page
 Arch OphthalmolHome page
J. L. Wiggs
Genetic Etiologies of Glaucoma
Arch Ophthalmol, January 1, 2007; 125(1): 30 - 37.
[Abstract] [Full Text] [PDF]