- Research article
- Open Access
Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage
© Shepard et al; licensee BioMed Central Ltd. 2003
- Received: 6 December 2002
- Accepted: 2 April 2003
- Published: 2 April 2003
Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. In addition, myocilin has been associated with glucocorticoid-induced ocular hypertension and steroid-induced glaucoma. To better understand the role myocilin plays in steroid-induced glaucoma and open-angle glaucoma, we examined rabbit myocilin for use in the rabbit animal model of steroid-induced glaucoma.
We have cloned the rabbit ortholog of human MYOC. Rabbit MYOC consists of three exons and an open reading frame encoding a 490 amino acid, 54,882-Da protein, which is 14 amino acids shorter at the N-terminus than human myocilin but 84% identical overall. Rabbit myocilin migrates as a single electrophoretic band, vs. double-banded human myocilin, by SDS-PAGE/immunoblot analysis. We determined that the differential migration exhibited is due to an N-glycosylation site that is present in human (Asn57), monkey and mouse myocilin but absent in rabbit (Ser43), rat and bovine myocilin. Rabbit myocilin is secreted in vitro in trabecular meshwork cell culture and in vivo in aqueous humor. Secretion of human myocilin is shown to be dependent on the signal peptide and independent of the extra 14 amino acids not found in rabbit myocilin. Many of the amino acids in myocilin that are mutated in glaucoma patients are conserved across species.
We have cloned the rabbit MYOC cDNA and determined that rabbit myocilin is secreted but not N-linked glycosylated. Knowledge of the rabbit MYOC cDNA sequence will facilitate future studies in the rabbit animal model examining the role of myocilin in steroid-induced glaucoma and the gain-of-function hypothesis in open-angle glaucoma.
- Aqueous Humor
- Primary Open Angle Glaucoma
- Primary Open Angle Glaucoma
- Leucine Zipper Motif
Mutations in MYOC have been shown to be causative for primary open angle glaucoma (POAG) [1–3]. Glucocorticoids have been implicated in ocular hypertension and glaucoma due to increased plasma cortisol levels [4, 5] and altered cortisol metabolism [6, 7] in POAG patients and from the increased risk of developing POAG in steroid-responsive patients [8, 9]. Myocilin has been shown to be upregulated in glucocorticoid-treated human trabecular meshwork (TM) cells [10, 11], and there is variable evidence for increased MYOC expression in glaucomatous individuals [12–14]. In addition, glaucoma patients have a higher chance of responding to steroids than non-glaucomatous individuals. However, an extensive study of mutations in the proximal promoter and coding region of MYOC found no sequence polymorphisms associated with steroid responsiveness .
Myocilin has been shown to be secreted both in human TM cell cultures and in vivo in aqueous humor (AH) . Glaucomatous mutations, however, alter myocilin Triton solubility  and prevent myocilin from being secreted . The glycosylation status of myocilin is somewhat controversial. Several groups have shown that myocilin is glycosylated [10, 16–21] whereas one group suggests that myocilin is not glycosylated . In our hands, both an N-linked glycosylated (≈57-kDa) and unglycosylated (≈55-kDa) form of myocilin is detected in TM cell culture media  and aqueous humor . The basis for both secretion and N-linked glycosylation of myocilin is part of the focus of this paper.
Rabbits have been successfully used as a model of glucocorticoid-induced ocular hypertension [23–27]. In order to study the association of MYOC gene expression with steroid-induced ocular hypertension, it will be helpful to know the sequence of rabbit myocilin. We report here the cloning, sequence comparison and expression analysis of rabbit MYOC. We show that an N-terminal signal peptide is necessary for myocilin secretion and we identify the N-linked glycosylation site found in human but not rabbit myocilin.
Characterization of Rabbit MYOC
Rabbit and Human MYOC primers used for PCR and Cloning
5'-GCAGGATCC CCAAGGTTTTTCCCTGCACATTGCTGC-3'; BamH1 underlined
5'-AGTGCGGCCGC GTTGTCTCCCTCTTCACCC-3'; Not1 underlined
5'-pCTTCCCTGGAGCCTGCAGGCACAT-3'; p indicates phosphorylation
5'-pCGTTGTCTCCCTCTTCACCCCTGG-3'; p indicates phosphorylation
5'-CCCAAGCTT ATGCCAGCTGTCCAGCTGCT-3'; HindIII underlined
5'-CCCAAGCTT ATGAGGACAGCTCAGCTCAGGAAG-3'; HindIII underlined
5'-CCGCCTCAGG GTGCCCAGCT-3'; Bsu36I underlined
5'-GGCCAGTCCCAG TGAATCCAGCTGCCC-3'; Mutation underlined
5'-GGGCAGCTGGATTCAC TGGGACTGGCC-3'; Mutation underlined
Myocilin ortholog identity comparison
Secretion of Rabbit MYOC
Next we created a human myocilin expression construct where the N-terminal 14aa were deleted (pcDNA3.hMYOC.ΔN14) forcing translation to begin at the second methionine codon (Fig. 2). GTM66 cells were chosen for this study based on their ability to grow and transfect well and are a glaucomatous TM cell line derived from a 92-year-old female donor [11, 17]. Transfection of GTM66 cells with the N-terminal deletion mutant resulted in a distribution pattern similar to wild-type human myocilin (Fig. 3B). The fact that we see no discernable difference in the relative mobility between transfected human wild-type myocilin and 14 amino acid amino-terminal deleted myocilin (Fig. 3B) and human vs. monkey myocilin in AH (Fig. 3A) may be due to the use of small format gels or possibly to the usage of the second N-terminal methionine by the cell for synthesizing human myocilin, making human and monkey molecular weights comparable.
Nonetheless, an N-terminal signal peptide is likely necessary for directing synthesis of myocilin, via the ER-Golgi pathway, for secretion outside the cell. To determine what actually constitutes the signal sequence for myocilin, we conducted an analysis of signal peptide cleavage sites using the Center for Biological Sequence Analysis SignalP v1.1 Database http://www.cbs.dtu.dk/services/SignalP/. A signal peptide cleavage site was predicted to exist between rabbit aa 18 and 19 (human aa 32 and 33) . We directly tested the importance of the signal peptide in myocilin secretion by replacing the first 32 aa of human myocilin with a single methionine (pcDNA3.hMYOC.ΔN32). Transfection of GTM66 cells with pcDNA3.hMYOC.ΔN32 resulted in intracellular but not extracellular myocilin distribution (Fig. 3B). This is the result expected if the signal peptide were necessary for secretion. This suggests that the first 18 aa of rabbit myocilin may function as a signal peptide and are necessary for myocilin secretion.
Human myocilin has been shown to migrate as a 55/57-kDa doublet by SDS-PAGE/immunoblot analysis [10–12, 16, 17]. The modification accounting for this molecular weight difference is hypothesized to be due to differential glycosylation. N-linked glycosylation sites were predicted using the Center for Biological Sequence Analysis NetNGlyc 1.0 Prediction Server http://www.cbs.dtu.dk/services/NetNGlyc/. Human, monkey and mouse myocilin contain a consensus asparagine (N)-linked glycosylation site (NES) near the N-terminus, whereas rabbit, rat and bovine code for SES at this position (Fig. 2). Having a serine present at this position would eliminate N-linked glycosylation and would be predicted to result in a difference in relative gel mobility between human/monkey and rabbit myocilin.
To test this hypothesis we analyzed human, monkey, and rabbit AH myocilin side-by-side in SDS-PAGE/immunoblots. The anti-human myocilin antibody 129 used in these studies (generated to human amino acids 156–171[12, 17]) cross-reacts with rabbit and monkey myocilin. With human and monkey myocilin we see a clear doublet, whereas rabbit myocilin migrates as a single band with the same relative mobility as the lower band of human and monkey myocilin (Fig. 3A), suggesting that the asparagine vs. serine amino acid difference is responsible for the differential glycosylation.
To directly test this theory, we mutated human myocilin amino acid 57 from Asn to Ser, as found in rabbit myocilin. Media and cell lysate from transfected GTM66 cells contained a single immunoreactive myocilin band that migrated with the same relative mobility as the lower wt myocilin band (Fig. 3B). This suggests that the 55/57-kDa human myocilin doublet is due to differential glycosylation and that the site of N-linked glycosylation on human myocilin is Asn 57.
To determine the effects of deglycosylation on myocilin, we treated human, monkey, and rabbit AH with a cocktail of glycosidases (N- and O-linked). Our results show that the upper band of human and monkey myocilin is eliminated whereas the lower band remains unaltered (Fig. 3A). This indicates that the upper human and monkey myocilin band is glycosylated. In addition, the single rabbit myocilin band shifted downward slightly with deglycosylation. This may be due to removal of O-linked glycosylation.
We report the cloning and characterization of rabbit myocilin. Rabbit myocilin is highly similar to the other myocilin orthologs (Table 2) and contains a leucine zipper motif encoded by exon 1 (Fig. 2). This conservation likely reflects the importance of the leucine zipper motif in directing protein-protein interactions [34, 35]. Many amino acid mutations encoded in the olfactomedin homology domain of exon 3 that are known to cause glaucoma in humans are also conserved in rabbit myocilin. In fact, all 31 amino acids (34 missense/nonsense mutations total) for human myocilin documented in the Human Gene Mutation Database http://uwcmml1s.uwcm.ac.uk/uwcm/mg/search/5584221.html to cause glaucoma are conserved in rabbit myocilin (Fig 2).
Myocilin has been shown to be secreted in vitro and in vivo by several investigators [10–12, 20, 21, 34, 36–38]. Secreted proteins typically contain a 16–30 aa signal sequence on their N-terminus which directs the completion of protein synthesis across the ER membrane. A signal peptidase cleaves the signal sequence and the trimmed protein is shuttled through the Golgi for secretion. We demonstrate the functionality of the N-terminal signal peptide in directing myocilin secretion. Deletion of the predicted N-terminal signal peptide results in loss of secretion but not intracellular appearance (Fig. 3B).
There are conflicting reports on whether myocilin is glycosylated. Differential glycosylation of myocilin has been shown in this report and by others [17, 20, 21] to account for the appearance of a 55/57-kDa doublet by Western immunoblot analysis. In contrast, Huang et al.  report that myocilin is not glycosylated. This discrepancy may be due to technical reasons. Huang et al. did not resolve the 55/57-kDa doublet using their SDS-PAGE system and only detected a 57-kDa human myocilin band, so any deglycosylation (PNGase F, NANase II, and O-Glycosidase) occurring would be difficult to resolve. A positive control was not reported in their deglycosylation experiment, so their conclusion that myocilin is not glycosylated may simply be due to inactive glycosidase. The lack of glycosylation seen in the in vitro canine pancreatic microsomal membrane translation experiment may again be due to poor 55/57-kDa myocilin doublet resolution or the inability of this artificial system to faithfully glycosylate myocilin. However, Caballero et al. was able to detect deglycosylation affects using similar methodology .
Nguyen et al.  identified a 66-kDa band as the glycosylated and major extracellular form of human myocilin and not the upper band in the 55/57-kDa doublet. These authors claim that the lower band of the unglycosylated 55/57-kDa doublet is due to cleavage of the N-terminal 32 amino acid signal peptide. In our hands, we do not see a 66-kDa band on our anti-myocilin Western blots, even using human aqueous humor as a source of myocilin. We provide evidence that the upper band of the 55/57-kDa doublet is the glycosylated form of myocilin (Fig. 3A). Another group has also reported the 66-kDa band using an anti-myocilin antibody different from the Nguyen et al. antibody . The reason for the appearance of this 66-kDa is unclear, but may be due to cross-reaction of the primary or secondary antibody with another protein such as albumin, which has a molecular weight of 65-kDa (GenPept accession # NP_000468). To date, protein sequence data identifying the 66-kDa band as myocilin has not been reported.
Human myocilin Asn57 is part of an N-linked glycosylation consensus (NES) and is the only residue on myocilin predicted to be N-glycosylated. We provide both site-directed mutagenesis (Fig. 3B) and deglycosylation (Fig. 3A) evidence that the upper band of the 55/57-kDa human and monkey myocilin doublet is the glycosylated form. The increase in mobility of rabbit myocilin with deglycosylation (Fig. 3A) is likely due to removal of O-linked glycosylation. Analysis of O-linked glycosylation by the CBS NetOGlyc 2.0 Prediction Server http://www.cbs.dtu.dk/services/NetOGlyc/ identified two unique (rabbit Thr93 and Thr267) and three common (rabbit Thr66, Thr263, and Ser45) O-linked glycosylation sites in rabbit vs. human/monkey myocilin. The additional O-linked glycosylation sites in rabbit may be responsible for the mobility shift seen in the deglycosylation experiment and the relative diffusiveness of the rabbit myocilin band.
A naturally occuring Asn57Asp polymorphism may be associated with JOAG  which, if truly disease-associated, would suggest the functional importance of N-linked glycosylation. N-linked glycosylation is known to confer stability on many extracellular proteins . Myocilin is similar to fibronectin (an extracellular matrix protein) in that both a glycosylated and unglycosylated form is secreted from the cell. The unglycosylated form of fibronectin is more susceptible to proteolytic degradation than the glycosylated form . It will be interesting to determine why two forms of myocilin are secreted in humans and what functionality, if any, N-linked glycosylation imparts on human vs. rabbit myocilin.
It is still unknown how myocilin functions normally and pathophysiologically. However, recent evidence suggests that disease-causing myocilin mutations are gain of function. Disease-causing myocilin mutants are less soluble in Triton  and are retained by the cell . Other studies have shown that a truncated form of myocilin does not get secreted [20, 21] and that mouse MYOC knockouts are without discernable phenotype . Furthermore, a human hemizygous individual containing an interstitial deletion of chromosome 1, which encodes MYOC, was without evidence of glaucoma  and another patient homozygous for a severly truncated form of myocilin had no evidence of glaucoma  or other discernable phenotypes. This suggests that MYOC haploinsufficiency is not causative for glaucoma and that myocilin may even be dispensable or redundant in normal human development or only important when ocular stress occurs.
Rabbit MYOC encodes a protein highly similar to human MYOC. Rabbit myocilin is secreted and is detected in rabbit aqueous humor. Secretion of myocilin is dependent on an 18 amino acid N-terminal signal peptide. Unlike human and monkey AH myocilin, rabbit myocilin does not undergo canonical N-linked glycosylation due to the substitution of a serine in the analogous human N-linked glycosylation site (N57). Rabbits may provide an in vivo system for modeling some aspects of glaucoma, such as steroid induced ocular hypertension [23–27] and the gain-of-function hypothesis in POAG .
Cloning of rabbit MYOC
Total RNA was extracted from rabbit eye and heart tissue (Pel Freez) using Trizol (Invitrogen). Primers derived from conserved regions of human and mouse MYOC (Table 1, primers Exon1F/Exon1R) were used to amplify a 250-bp section of rabbit MYOC exon 1 (Fig. 1, nts 184–433) by PCR as described . 5' and 3' RACE (Invitrogen) was used to obtain the 5' and 3' cDNA ends. Essentially, two micrograms rabbit total RNA was converted into first strand cDNA with adapter primer and Superscript II reverse transcriptase. Primary and nested 5' and 3' RACE primers were all designed within the 250-bp region of rabbit MYOC exon 1 (Table 1). PCR products were subcloned into TOPO™ TA cloning vectors (Invitrogen) and subjected to cycle sequencing (Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center).
Due to difficulties in amplifying the rabbit MYOC coding region from cDNA, we pieced together the entire contiguous open-reading frame using rabbit genomic DNA (Clontech; New Zealand White) as a template with a multi-step PCR/ligation process. In the first step, exons 1 and 2 were amplified from genomic DNA with primers P357F/P368R to exon 1 and P355F/P365R to exon 2. Primers P368R and P355F directly abut each other at the exon 1/2 boundary. PCR amplification was performed in a 50 ul volume with the PfuTurbo Hotstart DNA Polymerase system (Stratagene) and included 0.2 mM dNTPs, 100 ng each primer, 2.5 units PfuTurbo DNA polymerase, 100 ng genomic DNA, and 1X cloned Pfu DNA polymerase reaction buffer. Cycling conditions consisted of 2 min at 95°C followed by 15 cycles at 95°C 30 sec, 60°C 30 sec, and 72°C 1 min in a Perkin Elmer 9700 thermalcycler. PCR products were restriction digested with BamH1 (exon 1) or Not1 (exon 2), purified from an agarose gel by Qiaquick extraction (Qiagen), and simultaneously subcloned into a BamH1/Not1 digested pcDNA3 (Invitrogen) vector to create a directional clone of rabbit MYOC exons 1 and 2 spliced together (pcDNA3.RbMYOCex1–2). Sequence integrity of the cloned DNA was verified by cycle sequencing and restriction digestion.
Exons 1–2 of plasmid pcDNA3.RbMYOCex1–2 was then amplified with primers P357F/P370R and exon 3 was amplified from genomic DNA with primers P356F/P356R. Primers P370R and P356F directly abut each other at the exon 2/3 boundary. PCR was performed as above and PCR products digested with BamH1 (exons 1–2) or Not1 (exon 3) and processed/cloned as above to create a directional clone of rabbit MYOC exons 1–2 and 3 spliced together (pcDNA3.RbMYOCex1–3). Plasmid DNA integrity was confirmed by restriction analysis and cycle sequencing.
Sequence analysis and comparison
Alignment of DNA sequence contigs, protein sequences, and plasmid DNA sequence/construction was performed with Vector NTI 7.1 software (Informax).
Construction of human MYOC mutants
Human MYOC cDNA was cloned into the pcDNA3 (Invitrogen) mammalian expression vector as described . Human myocilin signal sequence deletions were generated by PCR. A 5' PCR primer, with a 5' HindIII site added, was designed to eliminate either the first 42- (P235; ΔN14) or 96- (P236; ΔN32) nucleotides of the human MYOC ORF and used in conjunction with a 3' PCR primer (P237) covering the internal Bsu36I restriction site (Table 1). Primer P236 replaced the first 32 amino acid codons with a methionine codon. PCR and processing were as described above with the exception that the 5' human MYOC deletion products were cloned into a HindIII/Bsu36I digested pcDNA3.hMYOC wild-type vector.
Substitution of human myocilin Asn 57 with Ser was accomplished using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primer pair P316F/P316R was designed to replace the Asn57 AA T codon with the Ser AG T codon (Table 1). The entire mutated cDNA was sequence verified and subcloned into a fresh pcDNA3 vector to avoid non-specific Taq DNA polymerase errors. Proper cloning was verified by restriction digestion analysis and sequencing.
Cell culture and transfection
GTM66 cells were derived from a 92-year old woman with glaucoma and were cultured in low glucose DMEM (HyClone Laboratories) with 10% FBS (HyClone Laboratories) and antibiotics (Invitrogen) as described [11, 17]. Cells were transfected with Lipofectamine 2000 reagent according to the manufacturers recommendations (Invitrogen). Transfected plasmid DNA included wild-type human MYOC (pcDNA3.hMYOC; ), wild-type rabbit MYOC (pcDNA3.RbMYOC.ex1–3), N-terminal 14 amino acid deleted human MYOC (pcDNA3.hMYOC.ΔN14), N-terminal 32 amino acid deleted human MYOC (pcDNA3.hMYOC.ΔN32), or N57S human MYOC (pcDNA3.hMYOC.N57S). Cells were switched to serum-free media 24 h post-transfection and media or cell lysates were collected after overnight incubation. Equivalent cell lysate protein amounts (5 μg) were loaded for reduced SDS-PAGE analysis (Fig. 3B). Cell media was not concentrated and was loaded at 16 ul per lane in Fig. 3B.
Aqueous humor samples
All experimental animals used in this study were treated in compliance with the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use of animals in research. Rabbit aqueous humor samples (135–190 μl) were taken from anesthetized New Zealand Albino (NZA) rabbits and mixed with protease inhibitor cocktail (Complete; Roche) to prevent myocilin degradation. Human aqueous humor samples were obtained as previously described . The equivalent of 4 μl AH was used per lane in Fig. 3A.
Cells were rinsed with PBS and solubilized in a commercial mammalian extraction buffer (M-Per™; Pierce, Rockford, IL) supplemented with a protease inhibitor cocktail (Complete; Roche) followed by centrifugation at 12,000 × g for 5 min. Protein concentration of the supernatant was determined with Coomassie Plus Protein Assay Reagent (Pierce). Cell extracts were stored at -20°C.
Aqueous humor, cell media, and cell extracts were analyzed using pre-cast Criterion 7.5% Tris-HCl polyacrylamide gels (Bio-Rad) and the Bio-Rad electrophoresis system (Bio-Rad). Proteins were electroblotted to Hybond-P PVDF membranes (Amersham Pharmacia Biotech), blocked with gelatin, and probed with affinity purified rabbit anti-human myocilin antibody 129 (AB129; generated to human myocilin amino acids 156–171) [12, 17] and an anti-rabbit IgG secondary antibody (Amersham). Immunoreactivity was detected with the ECL Plus detection system (Amersham). Blots were exposed to BioMax MR film (Eastman Kodak) and scanned with a HP ScanJet 7400 c scanner (Hewlett Packard).
Deglycosylation of aqueous humor myocilin
Aqueous humor from human, monkey and rabbit was subjected to deglycosylation with a mixture of N-linked and O-linked glycosidases (PNGase F, sialidase, β-galactosidase, glucosaminidase and O-glycosidase) using the Enzymatic CarboRelease Kit according to the manufacturers instructions (QA-Bio). AH samples in buffer were boiled for 5 min in the presence of denaturant followed by incubation for 3 hr at 37°C with the glycosidase mixture. NuPAGE sample buffer (Invitrogen) was added to stop the reaction, samples were heated for 10 min at 70°C and subjected to NuPAGE electrophoresis on 10% NuPAGE gels (Invitrogen). Samples were transferred to PVDF, immunoblotted versus AB129, and developed using ECL Plus.
This work was supported by NIH grant RO1-EY-10564 (ES) and Alcon Research, Ltd. ES is an Investigator of the Howard Hughes Medical Institute.
- Stone EM, Fingert JH, Alward WLM, Nguyen TD, Polansky JR, Sunden SLF, Nishimura D, Clark AF, Nystuen A, Nichols BE: Identification of a gene that causes primary open angle glaucoma [see comments]. Science. 1997, 275: 668-70. 10.1126/science.275.5300.668.View ArticlePubMedGoogle Scholar
- Fingert JH, Heon E, Liebmann JM, Yamamoto T, Craig JE, Rait J, Kawase K, Hoh ST, Buys YM, Dickinson J: Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum Mol Genet. 1999, 8: 899-905. 10.1093/hmg/8.5.899.View ArticlePubMedGoogle Scholar
- Adam MF, Belmouden A, Binisti P, Brezin AP, Valtot F, Bechetoille A, Dascotte JC, Copin B, Gomez L, Chaventre A: 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: 2091-7. 10.1093/hmg/6.12.2091.View ArticlePubMedGoogle Scholar
- Schwartz B, McCarty G, Rosner B: Increased plasma free cortisol in ocular hypertension and open angle glaucoma. Arch Ophthalmol. 1987, 105: 1060-5.View ArticlePubMedGoogle Scholar
- Rozsival P, Hampl R, Obenberger J, Starka L, Rehak S: Aqueous humour and plasma cortisol levels in glaucoma and cataract patients. Curr Eye Res. 1981, 1: 391-6.View ArticlePubMedGoogle Scholar
- Weinstein BI, Gordon GG, Southren AL: Potentiation of glucocorticoid activity by 5 beta-dihydrocortisol: its role in glaucoma. Science. 1983, 222: 172-3.View ArticlePubMedGoogle Scholar
- Weinstein BI, Iyer RB, Binstock JM, Hamby CV, Schwartz IS, Moy FH, Wandel T, Southren AL: Decreased 3 alpha-hydroxysteroid dehydrogenase activity in peripheral blood lymphocytes from patients with primary open angle glaucoma. Exp Eye Res. 1996, 62: 39-45. 10.1006/exer.1996.0005.View ArticlePubMedGoogle Scholar
- Kitazawa Y, Horie T: The prognosis of corticosteroid-responsive individuals. Arch Ophthalmol. 1981, 99: 819-23.View ArticlePubMedGoogle Scholar
- Lewis JM, Priddy T, Judd J, Gordon MO, Kass MA, Kolker AE, Becker B: Intraocular pressure response to topical dexamethasone as a predictor for the development of primary open-angle glaucoma. Am J Ophthalmol. 1988, 106: 607-12.View ArticlePubMedGoogle Scholar
- 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: 6341-50. 10.1074/jbc.273.11.6341.View ArticlePubMedGoogle Scholar
- Shepard AR, Jacobson N, Fingert JH, Stone EM, Sheffield VC, Clark AF: Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2001, 42: 3173-81.PubMedGoogle Scholar
- Jacobson N, Andrews M, Shepard AR, Nishimura D, Searby C, Fingert JH, Hageman G, Mullins R, Davidson BL, Kwon YH: Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor. Hum Mol Genet. 2001, 10: 117-25. 10.1093/hmg/10.2.117.View ArticlePubMedGoogle Scholar
- Lutjen-Drecoll E, May CA, Polansky JR, Johnson DH, Bloemendal H, Nguyen TD: Localization of the stress proteins alpha B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci. 1998, 39: 517-25.PubMedGoogle Scholar
- Cheng LE, Ueda J, Wentz-Hunter K, Yue BY: Age independent expression of myocilin in the human trabecular meshwork. Int J Mol Med. 2002, 10: 33-40.PubMedGoogle Scholar
- Fingert JH, Clark AF, Craig JE, Alward WL, Snibson GR, McLaughlin M, Tuttle L, Mackey DA, Sheffield VC, Stone EM: Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension. Invest Ophthalmol Vis Sci. 2001, 42: 145-52.PubMedGoogle Scholar
- Zhou Z, Vollrath D: A cellular assay distinguishes normal and mutant TIGR/myocilin protein. Hum Mol Genet. 1999, 8: 2221-8. 10.1093/hmg/8.12.2221.View ArticlePubMedGoogle Scholar
- Clark AF, Steely HT, Dickerson JE, English-Wright S, Stropki K, McCartney MD, Jacobson N, Shepard AR, Clark JI, Matsushima H: Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest Ophthalmol Vis Sci. 2001, 42: 1769-80.PubMedGoogle Scholar
- Polansky JR, Fauss DJ, Chen P, Chen H, Lutjen-Drecoll E, Johnson D, Kurtz RM, Ma ZD, Bloom E, Nguyen TD: Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica. 1997, 211: 126-39.View ArticlePubMedGoogle Scholar
- Zimmerman CC, Lingappa VR, Richards JE, Rozsa FW, Lichter PR, Polansky JR: A trabecular meshwork glucocorticoid response (TIGR) gene mutation affects translocational processing. Mol Vis. 1999, 5: 19-PubMedGoogle Scholar
- Caballero M, Rowlette LL, Borras T: Altered secretion of a TIGR/MYOC mutant lacking the olfactomedin domain. Biochim Biophys Acta. 2000, 1502: 447-60. 10.1016/S0925-4439(00)00068-5.View ArticlePubMedGoogle Scholar
- Caballero M, Borras T: Inefficient processing of an olfactomedin-deficient myocilin mutant: potential physiological relevance to glaucoma. Biochem Biophys Res Commun. 2001, 282: 662-70. 10.1006/bbrc.2001.4624.View ArticlePubMedGoogle Scholar
- Huang W, Jaroszewski J, Ortego J, Escribano J, Coca-Prados M: Expression of the TIGR gene in the iris, ciliary body, and trabecular meshwork of the human eye. Ophthalmic Genet. 2000, 21: 155-69. 10.1076/1381-6810(200009)21:3;1-Z;FT155.View ArticlePubMedGoogle Scholar
- Lorenzetti OJ: Effects of corticosteroids on ocular dynamics in rabbits. J Pharmacol Exp Ther. 1970, 175: 763-72.PubMedGoogle Scholar
- Knepper PA, Breen M, Weinstein HG, Blacik JL: Intraocular pressure and glycosaminoglycan distribution in the rabbit eye: effect of age and dexamethasone. Exp Eye Res. 1978, 27: 567-75.View ArticlePubMedGoogle Scholar
- Ticho U, Lahav M, Berkowitz S, Yoffe P: Ocular changes in rabbits with corticosteroid-induced ocular hypertension. Br J Ophthalmol. 1979, 63: 646-50.PubMed CentralView ArticlePubMedGoogle Scholar
- Hester DE, Trites PN, Peiffer RL, Petrow V: Steroid-induced ocular hypertension in the rabbit: a model using subconjunctival injections. J Ocul Pharmacol. 1987, 3: 185-9.View ArticlePubMedGoogle Scholar
- Bonomi L, Perfetti S, Noya E, Bellucci R, Tomazzoli L: Experimental corticosteroid ocular hypertension in the rabbit. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1978, 209: 73-82.View ArticlePubMedGoogle Scholar
- Kubota R, Noda S, Wang Y, Minoshima S, Asakawa S, Kudoh J, Mashima Y, Oguchi Y, Shimizu N: A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping. Genomics. 1997, 41: 360-9. 10.1006/geno.1997.4682.View ArticlePubMedGoogle Scholar
- Fingert JH, Ying L, Swiderski RE, Nystuen AM, Arbour NC, Alward WL, Sheffield VC, Stone EM: Characterization and comparison of the human and mouse GLC1A glaucoma genes. Genome Res. 1998, 8: 377-84.PubMed CentralPubMedGoogle Scholar
- Tomarev SI, Tamm ER, Chang B: Characterization of the mouse Myoc/Tigr gene. Biochem Biophys Res Commun. 1998, 245: 887-93. 10.1006/bbrc.1998.8541.View ArticlePubMedGoogle Scholar
- Taniguchi F, Suzuki Y, Kurihara H, Kurihara Y, Kasai H, Shirato S, Araie M: Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells [In Process Citation]. Invest Ophthalmol Vis Sci. 2000, 41: 2070-5.PubMedGoogle Scholar
- Taguchi M, Kanno H, Kubota R, Miwa S, Shishiba Y, Ozawa Y: Molecular cloning and expression profile of rat myocilin [In Process Citation]. Mol Genet Metab. 2000, 70: 75-80. 10.1006/mgme.2000.2986.View ArticlePubMedGoogle Scholar
- Nielsen H, Engelbrecht J, Brunak S, von Heijne G: Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997, 10: 1-6. 10.1093/protein/10.1.1.View ArticlePubMedGoogle Scholar
- Fautsch MP, Johnson DH: Characterization of myocilin-myocilin interactions. Invest Ophthalmol Vis Sci. 2001, 42: 2324-31.PubMedGoogle Scholar
- Wentz-Hunter K, Ueda J, Yue BY: Protein interactions with myocilin. Invest Ophthalmol Vis Sci. 2002, 43: 176-82.PubMedGoogle Scholar
- Borras T, Rowlette LL, Tamm ER, Gottanka J, Epstein DL: Effects of elevated intraocular pressure on outflow facility and TIGR/MYOC expression in perfused human anterior segments. Invest Ophthalmol Vis Sci. 2002, 43: 33-40.PubMedGoogle Scholar
- 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: 983-6.PubMedGoogle Scholar
- Rao PV, Allingham RR, Epstein DL: TIGR/myocilin in human aqueous humor. Exp Eye Res. 2000, 71: 637-41. 10.1006/exer.2000.0920.View ArticlePubMedGoogle Scholar
- Shimizu S, Lichter PR, Johnson AT, Zhou Z, Higashi M, Gottfredsdottir M, Othman M, Moroi SE, Rozsa FW, Schertzer RM: Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma. Am J Ophthalmol. 2000, 130: 165-77. 10.1016/S0002-9394(00)00536-5.View ArticlePubMedGoogle Scholar
- Imperiali B, O'Connor SE: Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol. 1999, 3: 643-9. 10.1016/S1367-5931(99)00021-6.View ArticlePubMedGoogle Scholar
- Bernard BA, Yamada KM, Olden K: Carbohydrates selectively protect a specific domain of fibronectin against proteases. J Biol Chem. 1982, 257: 8549-54.PubMedGoogle Scholar
- Kim BS, Savinova OV, Reedy MV, Martin J, Lun Y, Gan L, Smith RS, Tomarev SI, John SW, Johnson RL: Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function. Mol Cell Biol. 2001, 21: 7707-13. 10.1128/MCB.21.22.7707-7713.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Wiggs JL, Vollrath D: Molecular and clinical evaluation of a patient hemizygous for TIGR/MYOC. Arch Ophthalmol. 2001, 119: 1674-8.View ArticlePubMedGoogle Scholar
- Lam DS, Leung YF, Chua JK, Baum L, Fan DS, Choy KW, Pang CP: Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2000, 41: 1386-91.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.