Skip Navigation


Human Molecular Genetics Advance Access originally published online on February 22, 2007
Human Molecular Genetics 2007 16(6):609-617; doi:10.1093/hmg/ddm001
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrowOA All Versions of this Article:
16/6/609    most recent
ddm001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Google Scholar
Right arrow Articles by Shepard, A. R.
Right arrow Articles by Clark, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shepard, A. R.
Right arrow Articles by Clark, A. F.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure

Allan R. Shepard1,*,{dagger}, Nasreen Jacobson1,{dagger}, J. Cameron Millar1, Iok-Hou Pang1, H. Thomas Steely1, Charles C. Searby3, Val C. Sheffield2,3, Edwin M. Stone2,4 and Abbot F. Clark1

1 Glaucoma Research, Alcon Research, Ltd., 6201 South Freeway, Fort Worth, TX 76134, USA, 2 Howard Hughes Medical Institute, University of Iowa, Iowa City, IA 52242, USA, 3 Department of Pediatrics, University of Iowa, Iowa City, IA 52242, USA and 4 Department of Ophthalmology, University of Iowa, Iowa City, IA 52242, USA

* To whom correspondence should be addressed. Tel: +1 8176152312; Fax: +1 8175687553; Email: allan.shepard{at}alconlabs.com

Received January 3, 2007; Accepted January 9, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glaucoma is a leading cause of worldwide irreversible visual impairment and blindness and is a clinically and genetically heterogenous group of optic neuropathies. Specific mutations in the myocilin (MYOC) gene cause primary open angle glaucoma (POAG) with varying age-of-onset and degree of severity. We show a mutation-dependent, gain-of-function association between human myocilin and the peroxisomal targeting signal type 1 receptor (PTS1R). There was correlation between the glaucoma phenotype and the specific MYOC mutations, with the more severe early-onset POAG mutations having a higher degree of association with PTS1R. Expression of human myocilin glaucomatous mutations in mouse eyes causes elevated intraocular pressure, which is a major phenotype of MYOC glaucoma. This is the first demonstration of a disease resulting from mutation-induced exposure of a cryptic signaling site that causes mislocalization of mutant protein to peroxisomes and the first disease-gene-based animal model of human POAG.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myocilin (MYOC) was the first primary open angle glaucoma (POAG) gene identified and is the most common known genetic cause of glaucoma (1). MYOC maps to the GLC1A locus on chromosome 1q24 and encodes a 504 amino acid protein. Numerous missense and nonsense mutations in MYOC have been shown to be causative for POAG (1) and represent 2–4% (2) of worldwide POAG. Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma, and MYOC glaucoma is associated with elevated IOP. There is a phenotypic correlation of specific MYOC mutations with the age-of-onset, degree of elevated IOP and severity of POAG (3,4). For example, the Y437H, G364V and Q368X mutations have a mean age of diagnosis of 20, 34 and 59 years, with the highest recorded IOP of 44, 36 and 30 mmHg, respectively (4).

Myocilin is a secreted glycoprotein from the trabecular meshwork (TM), a tissue responsible for aqueous humor outflow and therefore the level of IOP. Several lines of evidence indicate that mutant myocilin causes glaucoma by the gain of a deleterious function within specific ocular tissues such as the TM. Patients who are deficient in myocilin protein either by hemizygous interstitial deletion of MYOC on 1q24 [PDB] -q26 (5) or by heterozygous or homozygous R46X truncation mutation of MYOC (6) do not necessarily develop POAG. Mice overexpressing wild-type (WT) or mutant mouse MYOC do not develop elevated IOP. Likewise, IOP is not changed in heterozygous or homozygous MYOC knockout mice (7–9). Therefore, elimination of human or mouse myocilin protein or overexpression of mouse myocilin is not sufficient to cause POAG. In vitro, glaucomatous MYOC mutations lead to inhibition of myocilin secretion (10), detergent insolubility (11) and accumulation of mutant myocilin in the endoplasmic reticulum (ER) (12), further supporting a detrimental role of mutant protein molecules in IOP elevation. In order to better understand the gain-of-function mechanism for mutant myocilin, we identified interacting proteins and the resulting localization of mutant myocilin within TM cells. We found that mutant myocilin interacts with PTS1R leading to co-localization with peroxisomes. PTS1R is a peroxisomal matrix shuttling receptor that recognizes variants of the canonical SKL peroxisomal targeting sequence-1 (PTS1) on the C-terminus of peroxisomal matrix proteins (13). The homologous context of the PTS1 sequence in a protein has been shown to be crucial to its functionality, but substitutions of the SKL motif such as (S/A/C)(K/H/R)(L/M) are tolerated (14). The PTS1R tetratricopeptide repeat (TPR) domain is responsible for recognition of the PTS1 motif. Expression of mutant human myocilin in mouse eyes causes elevated IOP, which is associated with the exposure of a cryptic PTS1 site on the mutant molecule.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of PTS1R as a myocilin interacting partner
Yeast two-hybrid assays are used to identify protein–protein interactions by the use of a bait (myocilin) and prey (cDNA library) coupled to a DNA-binding domain (DBD) or activation domain (AD). Interaction of the bait-DBD and prey-AD drives promoter–reporter gene expression and selectivity in yeast. To detect binding partners for wild-type myocilin (WT MYOC), we performed a yeast two-hybrid screen with full-length human myocilin and a human heart cDNA library. We failed to detect any potential interacting partners (data not shown). We then divided myocilin into two domains containing either the Nterminal half (exons 1–2; aa 1–243) or C-terminal half (exons 2–3; aa 244–504) (Fig. 1A).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. PTS1R associates with the myocilin C-terminus. (A) Schematic representation of myocilin showing the three exon structure, signal peptide region and C-terminal tripeptide. N and C separated by a vertical arrow refer to the N-terminal or C-terminal halves used in the yeast two-hybrid screen. (B) Validated phenotypes for yeast two-hybrid clones. Three reporter genes (HIS3, URA3 and lacZ) with four phenotypic selections were used in the yeast two-hybrid screen. Expected results for negative (Neg) and positive (Pos) interacting clones are indicated. (C) Schematic diagram of the PTS1R clones identified by yeast two-hybrid analysis. All PTS1R clones contained the tetratricopeptide (TPR) PTS1 binding domain. (D) Mammalian two-hybrid analysis of Cos7 cells transfected with plasmids expressing VP16-PTS1R fusion protein and myocilin mutants. Data are reported as mean ± SEM, n = 4 and analyzed statistically by one-way ANOVA followed by Dunnett's test. ** represents statistical significance (P < 0.01) versus the negative control group.

 
Analysis with the N-terminal and C-terminal myocilin domains using human heart and TM cell cDNA libraries identified five clones interacting with the C-terminal half, but none with the N-terminal half (Fig. 1B). All C-terminal interacting clones (A1, A4, A5, B3 and C9) encoded various segments of the peroxisomal targeting signal 1 receptor (PTS1R; RefSeq No. NM_000319 [GenBank] .1) (Fig. 1B and C). All PTS1R clones identified by our yeast two-hybrid screen contained the PTS1 binding domain and encoded two alternative splice variants of PTS1R, a long (PTS1RL) and a short (PTS1RS) form (Fig. 1C).

PTS1R interacts with the myocilin C-terminus
Mammalian two-hybrid assays are similar in concept to yeast two-hybrid assays and use DBD-coupled bait (myocilin) and AD-coupled prey (PTS1R) to drive promoter–reporter gene expression (luciferase), but are performed in a mammalian environment rather than yeast. We verified the myocilin–PTS1R interaction using a mammalian two-hybrid assay. Full-length WT myocilin, N-terminal half WT (aa 1–367), C-terminal half WT (aa 200–504), C-terminal half Y437H mutant and C-terminal half S502P mutant myocilin association with PTS1RS were determined by luciferase reporter activity (Fig. 1D). Full-length and N-terminal half WT myocilin showed control levels of luciferase activity, suggesting a lack of interaction with PTS1R and corroborating the lack of PTS1R interaction seen in the yeast two-hybrid assay. In contrast, the C-terminal half WT myocilin significantly increased luciferase activity (P < 0.01), showing an interaction with PTS1R and confirming the interaction noted in the yeast two-hybrid assay. Introduction of the Y437H mutation into C-terminal-half myocilin had similar but no additional effect on luciferase activity. To determine if the predicted PTS1 sequence (SKM504) on the C- terminus of myocilin is involved in the PTS1R interaction, we mutated serine 502 to proline in C-terminal half myocilin, which eliminated the PTS1 signal. The S502P mutant completely abrogated luciferase activity in the mammalian two-hybrid assay.

Myocilin interacts with PTS1R in a mutation-dependent fashion
We used co-immunoprecipitation assays of transfected TM cells to confirm the interaction of PTS1R with mutant myocilin. Myocilin plasmids were co-transfected into human TM cells along with VP16-tagged PTS1R followed by immunoprecipitation of the cell lysate with antibodies directed against either VP16-PTS1R (Fig. 2A) or myocilin (Fig. 2B). The immunoprecipitated proteins were subjected to western blotting with anti-myocilin antibody (Fig. 2A and B) or anti-PTS1R (Fig. 2B). The interaction of myocilin with PTS1R varied depending on the particular myocilin mutation. Full-length WT myocilin and K398R (a non disease-causing polymorphism) did not appreciably interact with PTS1R (Fig. 2A). In contrast, mutant myocilin interacted with PTS1R either directly or indirectly. The more severe myocilin Y437H and G364V mutants, which contain the PTS1 site, showed the strongest levels of interaction with PTS1R. The Q368X mutation, which lacks the PTS1, did not directly interact with PTS1R. Myocilin is known to form homodimers and multimers via the leucine-zipper domain (Fig. 1A) in the myosin-like amino terminal half (15,16). Specific myocilin mutants that lack a PTS1 signal (e.g. Q368X) may interact indirectly with PTS1R by oligomerization with WT myocilin and exposure of the normally cryptic WT myocilin PTS1 signal. This interaction between WT myocilin, Q368X myocilin and PTS1R was seen in the co-immunoprecipitation experiments (Fig. 2A).


Figure 2
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Mutant myocilin associates either directly or indirectly with PTS1R. (A–B) Co-immunoprecipitation of myocilin with PTS1R from extracts of TM5 cells transfected with PTS1R and myocilin. Protein extracts were incubated with antibodies to VP16- PTS1R (A) or to myocilin (B). Immunoblots of the proteins were probed with antibody to myocilin (A and B) or PTS1R (B). Minus signs indicate cell lysate controls with no IP performed. Asterisk indicates a no transfection, no IP antibody immunoprecipitation control. (C) Table containing myocilin ortholog C-terminal tripeptide sequences. A PTS1 prediction score is based on the C-terminal 16 amino acids; > 0 = Predicted, –10 to 0 = Marginal, < – 0 = Not Predicted (24).

 
Mutant myocilin localizes to peroxisomes in human TM cells
Myocilin appears to be a stress response protein (17), and the expression of endogenous WT myocilin was upregulated by transfection of TM5 cells (data not shown). This allowed us to examine the mutation-dependent co-localization of myocilin with peroxisomes. TM cells cotransfected with EGFP-PTS1 and WT or mutant myocilin tagged on the C-terminus with DsRed showed mutation-dependent co-localization of DsRed and EGFP fluorescence (Fig. 3A–D). WT myocilin was not co-localized with peroxisomes (Fig. 3A). DsRed tagged WT myocilin was secreted by transfected TM cells, similar to un-tagged WT myocilin (data not shown). Therefore, the intracellular levels of WT myocilin were less than that for mutant myocilins, which were not secreted. The less severe Q368X (MYOC367-DsRed) mutation showed only modest co-localization with peroxisomes (Fig. 3B). In contrast, the more severe MYOC mutants G364V (G364V MYOC504-DsRed) and Y437H (Y437H MYOC504-DsRed) showed increased co-localization with peroxisomes (Fig. 3C and D). The co-localization of mutant myocilin with peroxisomes was even more apparent and unambiguous when examining 3D wide field deconvolution images of the transfected cells. The myocilin-DsRed constructs have internalized, or are missing (MYOC367-DsRed) the myocilin C-terminal PTS1 sequence (SKM). Therefore, any co-localization of myocilin with peroxisomes is due to the indirect interaction of endogenous TM cell WT myocilin with mutant myocilin exposing the cryptic WT myocilin PTS1 site.


Figure 3
View larger version (90K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Wide-field deconvolution microscopic analysis of EGFP-tagged PTS1 and WT or mutant myocilin tagged with DsRed in transiently transfected cells. (A–D) TM5 cells co-transfected with EGFP-PTS1 and either MYOC504 (A), MYOC367 (B), G364V MYOC504 (C) or Y437H MYOC504 (D) tagged on the C-terminus with DsRed. Images are from live, non-fixed cells and represent direct visualization of green and red fluorescence.

 
Myocilin C-terminal PTS1 is required to elevate IOP in mice
To determine whether the myocilin-PTS1R interaction and subsequent peroxisomal localization is necessary for pathogenesis, we evaluated the effects of intravitreal injection of human MYOC expression vectors on mouse IOP. Ad5 expression vectors efficiently transduce TM cells (18). We therefore used Ad5 vectors to express WT or mutant myocilin in individual mouse eyes. Injection of MYOCWT adenovirus had no effect on IOP (Fig. 4A). Likewise, MYOCQ368X (Fig. 4B) and MYOCS502P (Fig. 4D) adenovirus, that lack the PTS1 site, did not elevate IOP. In contrast, injection of MYOCY437H (Fig. 4F) or MYOCG364V (Fig. 4E) mutants significantly elevated IOP (P < 0.01). Changing the PTS1 site in the human Y437H mutant to the mouse LEM sequence (MYOCY437H.LEM), which is no longer a PTS1 site (Fig. 2C), prevented IOP induction (Fig. 4G). Although injection of MYOCQ368X alone had no effect on IOP (Fig. 4B), co-injection of both MYOCWT and MYOCQ368X expression vectors elevated IOP (P < 0.001) (Fig. 4C).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Functional analysis of adenoviral expressed human myocilin in mouse eyes. Adenovirus expressing WT (A), Q368X (B), WT + Q368X (C), S502P (D), G364V (E), Y437H (F) or Y437H containing LEM C-terminus (G) human myocilin (open triangles) was injected into the vitreous of BALB/cJ mice followed 3 days later by measurement of daily IOP. Seven animals were treated per each of the six groups. Contralateral, non-injected eye for each animal served as a control (filled circles). There were no obvious corneal changes in any of the mice or significant clinical signs of inflammation in all but one of the mice. One animal from the Ad.MYOC.Y437H group was eliminated from the analysis due to severe iris hyperemia and profound IOP elevation. Data are reported as mean ± SEM and analyzed statistically by one-way ANOVA followed by Bonferroni's test. *: P < 0.05, **: P < 0.01, ***: P < 0.001 versus the respective baseline IOP values.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Elevated IOP in MYOC glaucoma is a gain-of-function phenotype based on several lines of evidence (5–9). WT myocilin is a secreted multimeric glycoprotein synthesized in the ER-Golgi pathway (10,19–22). In vitro data have shown that myocilin containing glaucomatous mutations is not secreted from TM cells (10), forms heteromultimers that can diminish WT secretion (10), is detergent insoluble (11), is misfolded and retained in the ER (12) and may be retrotranslocated to the cytosol for ubiquitination and proteasomal degradation (23).

We have identified an unexpected binding partner, PTS1R, for misfolded myocilin. PTS1R was identified by yeast two-hybrid analysis using TM or heart cell cDNA libraries as binding partners with C-terminal half WT or mutant myocilin (Fig. 1B and C), and this interaction was confirmed by mammalian two-hybrid analysis (Fig. 1D). Myocilin itself was not detected in our two-hybrid assays likely due to the inefficiency of large multimeric myocilin to enter the nucleus, which is required for detection in two-hybrid assays. Co-immunoprecipitation studies showed that mutant myocilin interacts either directly (e.g. Y437H, G364V mutants) or indirectly (e.g. Q368X mutant) with PTS1R via mutation-induced misfolding and exposure of the normally cryptic C-terminal PTS1 sequence (SKM) in human myocilin (Fig. 2). Our co-immunoprecipitation data also suggest that early onset, severe myocilin mutants such as Y437H interact with PTS1R more effectively than later onset, milder myocilin mutants such as Q368X. Evidence for PTS1R shuttling of mutant myocilin to peroxisomes was seen by direct visualization of fluorescent protein-tagged myocilin and PTS1 in TM cells (Fig. 3).

The expression of human mutant myocilin in the eyes of mice caused the relevant glaucoma phenotype of elevated IOP, and the degree of IOP elevation was mutation specific (Fig. 4). In our previous studies, injection of certain, specific adenoviral vectors either intracamerally or intravitreally affected IOP in mice. However, intravitreal injection, as done in this study, typically produced a more pronounced and consistent response. We speculate that the vitreous serves as a depot for the vector resulting in a slow release and longer duration of transduction. Studies using an adenoviral vector encoding the green fluorescence protein (GFP) as a reporter gene delivered by intravitreal injection induced expression of GFP mainly in TM cells, with much lower expression in some anterior iris epithelial cells, corneal endothelial cells and rarely in the lens epithelium (data not shown). Injection of adenovirus expressing MYOC mutations, G364V (Fig. 4E) and Y437H (Fig. 4F) caused significant IOP elevation, whereas WT MYOC did not (Fig. 4A). Constructs that lacked the PTS1 signal, including Q368X (Fig. 4B), S502P (Fig. 4D) and Y437H-LEM (Fig. 4G), also had no effect on IOP. Although transduction with Q368X did not elevate IOP, support for indirect interaction of mutant myocilin with PTS1R was shown by the WT + Q368X co-infection, where only the two viruses expressed together caused elevated IOP (Fig. 4C versus A and B). Of the myocilin orthologs identified to date, mouse, cat and rat are not predicted to encode peroxisomal targeting signals on their C-terminus (Fig. 2C) (24). Therefore, substituting the human C-terminus (SKM) with the mouse myocilin C-terminus (LEM) in the Y437H-LEM mutant (Fig. 4G) showed that the human C-terminus is necessary for IOP elevation. Mutation of the human C-terminal SKM to PKM (S502P mutant) also verified the importance of the human C-terminal PTS1 for IOP elevation. S502P is the myocilin variant reported in a British family with juvenile open angle glaucoma (25) and is not secreted from transfected TM cells using a western blot assay previously shown to distinguish pathogenic from non-pathogenic myocilin polymorphisms (data not shown) (10), supporting S502P as a disease-causing mutation. The lack of a PTS1 signal on mouse myocilin explains why IOP was unchanged in mice overexpressing mouse WT myocilin (7) and in knock-in mice expressing the mouse ortholog of human Y437H myocilin (9). These data support a unique gain-of-function role for MYOC glaucoma contingent on the interaction of mutant human myocilin with PTS1R. Lack of glaucoma seen by Zillig et al. (26) in mice expressing human Y437H myocilin is likely due to the lens-targeted expression and retention of mutant myocilin within the lens. Based on our results, transgenic mice expressing human mutant myocilin in the aqueous outflow pathway may prove a valuable model of human glaucoma.

There is a phenotype–genotype correlation for many of the glaucomatous MYOC mutations (4). In our study, interaction of mutant myocilin with PTS1R and the degree of IOP elevation in MYOC transduced mouse eyes correlated well with the clinical IOP phenotypes of MYOC glaucoma patients. WT myocilin is secreted from TM cells and is found in the aqueous humor. In contrast, mutant myocilin is misfolded, retained in the ER and not secreted from TM cells. Within the ER, misfolded myocilin is likely recognized by the ERAD pathway and dislocated to the cytoplasm for ubiquination and proteosome mediated degradation. However, exposure of the normally cryptic PTS1 signal in mutants would allow interaction of myocilin with PTS1R (Fig. 5). Therefore, there is likely to be competition between ubiquitin–proteasome degradation and PTS1R interaction of the mutant myocilin proteins. Several mechanisms may be responsible for the mutation specific interaction with PTS1R and degree of IOP elevation. Myocilin normally exists as homodimers and higher order multimers, which in MYOC glaucoma patients would lead to WT myocilin-mutant myocilin heterodimers. Interaction of WT myocilin with the misfolded mutant myocilin may or may not expose the normally cryptic PTS1 signal on WT myocilin. The higher degree of interaction between Y437H myocilin and PTS1R may be due to exposure of the cryptic PTS1 sites on both the mutant and WT myocilin molecules, essentially doubling the chance for binding PTS1R. Other MYOC mutations may expose the PTS1 site only on the mutant myocilin molecule. Interaction of WT myocilin with mutant myocilin proteins that lack a PTS1 site (S502P, Q368X) may expose the cryptic signal on WT myocilin (Fig. 5). These latter two scenarios may lead to weaker interactions with PTS1R. In addition, specific myocilin mutations may lead to varying degrees of myocilin misfolding, some of which are better recognized by the ubiquitin–proteasome degradation pathway causing decreased opportunity to interact with PTS1R and a milder IOP phenotype.


Figure 5
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Proposed model for mutant myocilin-induced PTS1R interaction. (A) Properly folded WT myocilin containing a cryptic C-terminal PTS1 motif is normally synthesized, oligomerized and transported through the ER-Golgi pathway and secreted from the cell. (B) Misfolded mutant (Mut) myocilin (e.g. Y437H or G364V) with an exposed PTS1 motif, is synthesized and oligomerized in the ER, retro-translocated from the ER into the cytosol, and either ubiquitylated and degraded by the proteosome or bound by PTS1R and shuttled to the peroxisome. Misfolded mutant myocilin that lacks the PTS1 motif (e.g. Q368X or S502P), associates by heteroligomerization with WT myocilin causing misfolding and exposure of its partner WT myocilin PTS1 motif.

 
Accumulation of misfolded mutant myocilin within cells may lead to activation of the unfolded protein response (UPR) or segregation of misfolded myocilin into aggresomes. Several reports (12,23), as well as our own research, suggest that mutant myocilin does not form the classical aggresomes as often seen in neurodegenerative conformational disorders such as Huntington's, Alzheimer's and Parkinson's disease (27). Alternatively, the intracellular accumulation of mutant myocilin may cause ER stress and induction of UPR. Mice expressing mutant mouse myocilin did not develop elevated IOP and did not have activated UPR despite non-secretion of myocilin (9). However, transduction of cultured TM cells with mutant human myocilin caused ER stress and TM dysfunction (12), suggesting that in addition to or in response to dislocation of mutant human myocilin to peroxisomes, ER stress may play a role in the pathogenic process. The abnormal accumulation of misfolded protein may also impair the ubiquitin–proteasome system (28), resulting in a viscous cycle of protein accumulation and ER stress.

In summary, these experiments identify a novel mechanism for IOP elevation in glaucoma. Our results are the first to explain why mutations in human but not mouse MYOC cause IOP elevation. We show that mutations in human MYOC induce exposure of a cryptic peroxisomal targeting sequence whose interaction with the PTS1R is necessary for IOP elevation. Importantly, mutants that lead to stronger interactions with the PTS1R induced earlier and more severe IOP elevation compared to more weakly interacting mutants. This is the first disease-gene-based mouse model of human POAG that may be useful for the further dissection and understanding of glaucoma pathophysiology. Mutation-induced exposure of a cryptic signaling site in a protein may be a more general event in gain-of-function diseases than is currently recognized.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Constructs for mammalian cell expression
Plasmid pEGFP.SKL was constructed essentially as described (29). Myocilin mutants G364V, Q368X, K398R, Y437H and S502P were created from WT myocilin in the pcDNA3 vector (Invitrogen, Carlsbad, CA, USA), as previously described (10). DsRed-tagged myocilin was created by subcloning WT or mutant myocilin from pcDNA3.MYOC into pDsRed-N1 (Clontech, Palo Alto, CA, USA). DsRed-tagged mutant myocilin was created using Stratagene Quick Change Mutagenesis (Stratagene, Cedar Creek, Texas, USA). The MYOC367-DsRed fusion was created by replacing the WT MYOC504DsRed sequence with an amplified fragment containing amino acids 1–367 from the Q368X mutant with an additional BamH1 site incorporated to allow inframe fusion with DsRed. All clones were verified by restriction digestion and sequencing.

Cell culture
Human TM5 cells were cultured as previously described (30). COS-7 cells were maintained in DMEM (ATCC, Manassas, VA, USA). All cells were supplemented with 10% FBS (Hyclone, Logan, UT, USA), 100 units/ml Penicillin G, 100 µg/ml Streptomycin sulfate, 2 mm L-glutamine (Invitrogen) and incubated in a humidified 5% CO2 atmosphere at 37°C.

Yeast two-hybrid analysis
Human heart and TM yeast two-hybrid cDNA libraries were screened with human MYOC cDNA clones using the ProquestTM Y2H system (Life Technologies, Rockville, MD, USA). The TM cDNA library was constructed by Life Technologies from a pool of 22 TM cell lines. False positives were controlled in this assay by using four phenotypes for selecting interacting proteins: HIS3 and URA3, cell growth on histidine- or uracil-lacking plates; URA3, conversion of non-toxic 5- fluoroorotic acid to toxic 5-fluoroacil; and lacZ, blue color when assayed with X-gal.

Mammalian two-hybrid analysis
The CheckMateTM mammalian two-hybrid system (Promega, Madison, WI, USA) was used according to the manufacturer's directions. pACT and pBIND plasmids were altered by insertion of a single nucleotide upstream of the Sal1 site in order to facilitate direct, in-frame subcloning of the Y2H clones. These altered plasmids were termed pACT + 1 and pBIND + 1. Full-length WT myocilin, N-terminal 1–367, and mutant C-terminal half Y437H or S502P constructs were tagged on their N-terminus with the yeast GAL4 DNA binding domain (DBD; amino acids 1–147) in the pBIND + 1 vector. PTS1RS_N163 (clone A1.1 encoding N-terminal truncated PTS1RS) was fused to the C-terminus of the herpes simplex virus VP16 AD (amino acids 411–456) in the pACT + 1 vector. Vectors pBIND-Id and pACT-MyoD, supplied in the kit, were used as positive controls. Empty pBIND and pACT vectors were used as negative controls. Control or test pBIND and pACT plasmids were cotransfected into Cos7 cells along with pG5luc and assayed for firefly and renilla luciferase expression using the Dual-Luciferase Reporter Assay System (Promega) and a Turner-TD20/20 luminometer (Turner Instruments). Firefly luciferase activity was normalized to Renilla luciferase activity expressed from the pBIND plasmid.

Co-immunoprecipitation and immunobloting
TM5 cells were transiently transfected with Lipofectamine 2000 (Invitrogen) and various plasmids. Cells were harvested 48 h after transfection and lysed in M-PER (Pierce, Rockford, IL, USA) buffer containing protease inhibitors (CompleteTM mini protease inhibitor cocktail, Roche, Mannheim, Germany). Protein concentrations in all lysates were determined using a Coomassie Plus protein assay (Pierce). Immunoprecipitation was performed with 80 µg protein and either anti-VP16 agarose or anti-myocilin antibody 3.1 (21), respectively. Myocilin-antibody complexes were collected with Protein A Sepharose (Pharmacia), precipitates washed, size-fractionated on a 10% NuPage gel (Invitrogen) using MOPS buffer, and transferred to Immobilon-P PVDF (Amersham Biosciences, Piscataway, NJ, USA). Membranes were blocked with 2.7% gelatin in PBS for 2 h at RT. Blots were probed with either rabbit polyclonal anti-PTS1R antisera raised against a 45-kDa C-terminal PTS1R fragment (1 : 5000; gift of Dr Suresh Subramani, UCSD) (31) or with anti-myocilin antibody 129 (1 : 5000) (10) antibody overnight at RT. Blots were washed and stained with HRP-conjugated anti-rabbit IgG secondary antibody for 1 h at RT. Membranes were developed using ECL Plus reagent (Amersham Biosciences) and Kodak BioMax film.

Immunofluorescence microscopy
TM5 cells were grown on no. 1.5 LabTek II cover glass chambers (Nunc, Rochester, NY, USA) and transiently transfected with constructs encoding EGFP-PTS1 and either WT myocilin-DsRed or mutant myocilin-DsRed, using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, myocilin and PTS1 tagged GFP were detected by direct fluorescence of myocilin-DsRed at 555/28 excitation and 617/73 emission and EGFP-PTS1 (green) at 490/20 excitation and 528/38 emission. Fixed cells were imaged using a Deltavision Spectris wide-field deconvolution system (Applied Precision, Issaquah, WA, USA) equipped with a precision motorized XYZ stage; an Olympus IX70 inverted fluorescence microscope with a PLANAPO 60X oil, NA1.4 objective, mercury arc lamp and Sony ICX285ER CCD camera; and operated with SoftWoRx DV 3.1.4 Linux software.

Adenovirus preparation
Plasmids expressing WT or mutant myocilin were subcloned into the Ad5.RSV.K-N.pA shuttle vector and adenovirus particles prepared by the U. of Iowa Gene Transfer Vector Core (GTVC), as previously described (10). Each virus was tested by the GTVC for WT revertants and for titer by PCR and A549 plaque assay.

Animal studies
All animals were treated in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and all protocols were approved and monitored by the Animal Care and Use Committee of Alcon Research, Ltd. The BALB/c mice (1–4 months old) (Jackson Laboratories, Bar Harbor, ME, USA) used in this study were housed and handled as described (32). For adenovirus injections, mice were anesthetized followed by injection through the sclera into the vitreous. Each mouse had one eye injected with an adenovirus (2E7 pfu in 2 µl injection volume, Hamilton microsyringe fitted with sterile 33G needle) while the uninjected contralateral eye served as a control. Each injection was made over the course of 1 min. The needle was then left in place in the vitreous for a further minute before being rapidly withdrawn. Eyes to be injected were randomized and investigators were masked to the identity of the injected adenovirus. IOP measurements were taken in conscious mice using a rebound tonometer as described (32). IOP measurements were taken 96 h post-injection, to allow for viral gene expression, and then every day until sacrifice. Mice were sacrificed at typical peak IOP on day 7 post-injection.


   ACKNOWLEDGEMENTS
 
We thank the Oregon Health and Science University MMI Research Core Facility (http://www.ohsu.edu/research/core) for help in obtaining some of the images presented. We thank Dr Suresh Subramani (UCSD) for supplying us with PTS1R antibody and for helpful discussions and Dr Paul Watkins (Kennedy Krieger) for technical support and helpful discussions. We also thank Dr Simon John (HHMI and The Jackson Laboratory) for helpful discussions and suggestions on the manuscript. VCS and EMS are investigators of the Howard Hughes Medical Institute. This work was supported by Alcon Research, Ltd. and by NIH grant RO1 EY10564 (VCS and EMS).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Stone E.M., Fingert J.H., Alward W.L.M., Nguyen T.D., Polansky J.R., Sunden S.L.F., Nishimura D., Clark A.F., Nystuen A., Nichols B.E., et al. (1997) Identification of a gene that causes primary open angle glaucoma. Science 275:668–670.[Abstract/Free Full Text]

  2. Fingert J.H., Heon E., Liebmann J.M., Yamamoto T., Craig J.E., Rait J., Kawase K., Hoh S.T., Buys Y.M., Dickinson J., et al. (1999) Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum. Mol. Genet. 8:899–905.[Abstract/Free Full Text]

  3. Shimizu S., Lichter P.R., Johnson A.T., Zhou Z., Higashi M., Gottfredsdottir M., Othman M., Moroi S.E., Rozsa F.W., Schertzer R.M., et al. (2000) Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma. Am. J. Ophthalmol. 130:165–177.[CrossRef][ISI][Medline]

  4. Alward W.L., Fingert J.H., Coote M.A., Johnson A.T., Lerner S.F., Junqua D., Durcan F.J., McCartney P.J., Mackey D.A., Sheffield V.C., et al. (1998) Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A). N. Engl. J. Med. 338:1022–1027.[Abstract/Free Full Text]

  5. Wiggs J.L. and Vollrath D. (2001) Molecular and clinical evaluation of a patient hemizygous for TIGR/MYOC. Arch. Ophthalmol. 119:1674–1678.[Abstract/Free Full Text]

  6. Lam D.S., Leung Y.F., Chua J.K., Baum L., Fan D.S., Choy K.W., Pang C.P. (2000) Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 41:1386–1391.[Abstract/Free Full Text]

  7. Gould D.B., Miceli-Libby L., Savinova O.V., Torrado M., Tomarev S.I., Smith R.S., John S.W. (2004) Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma. Mol. Cell Biol. 24:9019–9025.[Abstract/Free Full Text]

  8. Kim 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. (2001) Targeted Disruption of the Myocilin Gene (Myoc) suggests that human glaucoma-causing mutations are gain of function. Mol. Cell Biol. 21:7707–7713.[Abstract/Free Full Text]

  9. Gould D.B., Reedy M., Wilson L.A., Smith R.S., Johnson R.L., John S.W. (2006) Mutant myocilin non-secretion in vivo is not sufficient to cause glaucoma. Mol. Cell Biol 26:8427–8436.[Abstract/Free Full Text]

  10. Jacobson N., Andrews M., Shepard A.R., Nishimura D., Searby C., Fingert J.H., Hageman G., Mullins R., Davidson B.L., Kwon Y.H., et al. (2001) Nonsecretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor. Hum. Mol. Genet. 10:117–125.[Abstract/Free Full Text]

  11. Zhou Z. and Vollrath D. (1999) A cellular assay distinguishes normal and mutant TIGR/myocilin protein. Hum. Mol. Genet. 8:2221–2228.[Abstract/Free Full Text]

  12. Joe M.K., Sohn S., Hur W., Moon Y., Choi Y.R., Kee C. (2003) Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem. Biophys. Res. Commun. 312:592–600.[CrossRef][ISI][Medline]

  13. Subramani S., Koller A., Snyder W.B. (2000) Import of peroxisomal matrix and membrane proteins. Annu. Rev. Biochem. 69:399–418.[CrossRef][ISI][Medline]

  14. Elgersma Y., Vos A., van den Berg M., van Roermund C.W., van der Sluijs P., Distel B., Tabak H.F. (1996) Analysis of the carboxyl-terminal peroxisomal targeting signal 1 in a homologous context in Saccharomyces cerevisiae. J. Biol. Chem. 271:26375–26382.[Abstract/Free Full Text]

  15. Fautsch M.P. and Johnson D.H. (2001) Characterization of myocilin–myocilin interactions. Invest. Ophthalmol. Vis. Sci. 42:2324–2331.[Abstract/Free Full Text]

  16. Gobeil S., Rodrigue M.A., Moisan S., Nguyen T.D., Polansky J.R., Morissette J., Raymond V. (2004) Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants. Invest. Ophthalmol. Vis. Sci. 45:3560–3567.[Abstract/Free Full Text]

  17. Polansky 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. (1997) Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica 211:126–139.[ISI][Medline]

  18. Borras T., Gabelt B.T., Klintworth G.K., Peterson J.C., Kaufman P.L. (2001) Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo. J. Gene. Med. 3:437–449.[CrossRef][ISI][Medline]

  19. O'Brien E.T., Ren X., Wang Y. (2000) Localization of myocilin to the golgi apparatus in Schlemm's canal cells. Invest. Ophthalmol. Vis. Sci. 41:3842–3849.[Abstract/Free Full Text]

  20. Sohn S., Hur W., Joe M.K., Kim J.H., Lee Z.W., Ha K.S., Kee C. (2002) Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities. Invest. Ophthalmol. Vis. Sci. 43:3680–3685.[Abstract/Free Full Text]

  21. Clark A.F., Steely H.T., Dickerson J.E. Jr, English-Wright S., Stropki K., McCartney M.D., Jacobson N., Shepard A.R., Clark J.I., Matsushima H., et al. (2001) Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest. Ophthalmol. Vis. Sci. 42:1769–1780.[Abstract/Free Full Text]

  22. Nguyen T.D., Chen P., Huang W.D., Chen H., Johnson D., Polansky J.R. (1998) Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J. Biol. Chem. 273:6341–6350.[Abstract/Free Full Text]

  23. Liu Y. and Vollrath D. (2004) Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum. Mol. Genet. 13:1193–1204.[Abstract/Free Full Text]

  24. Neuberger G., Maurer-Stroh S., Eisenhaber B., Hartig A., Eisenhaber F. (2003) Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J. Mol. Biol. 328:581–592.[CrossRef][ISI][Medline]

  25. Stoilova D., Child A., Brice G., Desai T., Barsoum-Homsy M., Ozdemir N., Chevrette L., Adam M.F., Garchon H.J., Pitts Crick R., et al. (1998) Novel TIGR/MYOC mutations in families with juvenile onset primary open angle glaucoma. J. Med. Genet. 35:989–992.[Abstract]

  26. Zillig M., Wurm A., Grehn F.J., Russell P., Tamm E.R. (2005) Overexpression and properties of wild-type and Tyr437His mutated myocilin in the eyes of transgenic mice. Invest. Ophthalmol. Vis. Sci. 46:223–234.[Abstract/Free Full Text]

  27. Kopito R.R. (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10:524–530.[CrossRef][ISI][Medline]

  28. Bence N.F., Sampat R.M., Kopito R.R. (2001) Impairment of the ubiquitin proteasome system by protein aggregation. Science 292:1552–1555.[Abstract/Free Full Text]

  29. Wiemer E.A., Wenzel T., Deerinck T.J., Ellisman M.H., Subramani S. (1997) Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules. J. Cell Biol. 136:71–80.[Abstract/Free Full Text]

  30. Shepard A.R., Jacobson N., Fingert J.H., Stone E.M., Sheffield V.C., Clark A.F. (2001) Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 42:3173–3181.[Abstract/Free Full Text]

  31. Wiemer E.A., Nuttley W.M., Bertolaet B.L., Li X., Francke U., Wheelock M.J., Anne U.K., Johnson K.R., Subramani S. (1995) Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders. J. Cell. Biol. 130:51–65.[Abstract/Free Full Text]

  32. Wang W.H., Millar J.C., Pang I.H., Wax M.B., Clark A.F. (2005) Noninvasive measurement of rodent intraocular pressure with a rebound tonometer. Invest. Ophthalmol. Vis. Sci. 46:4617–4621.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
J. M. Skarie and B. A. Link
The Primary open-angle glaucoma gene WDR36 functions in ribosomal RNA processing and interacts with the p53 stress-response pathway
Hum. Mol. Genet., August 15, 2008; 17(16): 2474 - 2485.
[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
 IOVSHome page
W.-H. Wang, L. G. McNatt, I.-H. Pang, P. E. Hellberg, J. H. Fingert, M. D. McCartney, and A. F. Clark
Increased Expression of Serum Amyloid A in Glaucoma and Its Effect on Intraocular Pressure
Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 1916 - 1923.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrowOA All Versions of this Article:
16/6/609    most recent
ddm001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (7)
Google Scholar
Right arrow Articles by Shepard, A. R.
Right arrow Articles by Clark, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shepard, A. R.
Right arrow Articles by Clark, A. F.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?