[show abstract][hide abstract] ABSTRACT: 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.
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.
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 MYOC(WT)/MYOC(mutant) 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.
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.
[show abstract][hide abstract] ABSTRACT: We determined the effects of a low dose of the actin-disrupting agent latrunculin (LAT)-A on dexamethasone (DEX)-induced changes in actin organization, focal adhesions, and production of extracellular matrix proteins in cultured human trabecular meshwork (HTM) cells. HTM cells were cultured to a highly confluent stage with stable endothelium-like morphology and incubated with 0.1 or 0.2 microM DEX and/or 0.1 microM LAT-A. Changes in the actin cytoskeleton and vinculin-containing focal contacts were evaluated by immunofluorescence microscopy. Expression of thrombospondin-1 (TSP1) and fibronectin (FN) in HTM cells was evaluated by Western blot analysis. The results showed that DEX induced morphological changes and actin reorganization in HTM cells. The cells partly recovered after DEX withdrawal, but the addition of low dose LAT-A hastened the recovery. In addition, DEX failed to induce changes when co-incubated with LAT-A for at least 4 weeks, and for at least 2 weeks when cells were pre-treated with LAT-A for 2 weeks. HTM cells treated with 0.1 microM LAT-A only for 5 days showed mild disorganization of the actin cytoskeleton and focal adhesions, which persisted during the 4 weeks of treatment. DEX stimulated production of FN in HTM cells independent of LAT-A treatment. LAT-A and, to a lesser extent, DEX inhibited production of TSP1 by HTM cells. Although LAT-A is not a DEX receptor antagonist, it is able to prevent the effects of DEX on the actin cytoskeleton in cultured HTM cells at a dose subthreshold for increasing outflow facility in monkeys. This suggests that LAT-A at low doses may be useful in treating steroid and other glaucomas. TSP1 may be an important target of LAT-A in HTM cells and modulation of TSP may influence the actin cytoskeleton of the trabecular meshwork (TM), and consequently, intraocular pressure.
Experimental Eye Research 09/2003; 77(2):181-8. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: A major variant of myocilin (MYOC) [TIGR/MYOC mt.1 (-1000 C/G)], present in the gene's promoter, is found to be associated with more rapid progression of the glaucoma disease state. Time-to-event analyses using the Cox proportional hazards model produced substantial statistical evidence that this TIGR/MYOC mt.1(+) variant accelerates worsening for both optic disc and visual field measures of disease progression. These analyses were based on evaluations of 147 patients with primary open-angle glaucoma (POAG) over 35 years of age with an average follow-up of approximately 15 years. Our analyses showed that there are independent effects of the variant on disease progression, taking into account other relevant disease-related baseline risk factors, including age, family history, initial drug treatment, initial surgical treatment, diabetes, gender, myopia, and initial disease severity. The finding that a TIGR/MYOC mt.1(+) determination provided a strong marker for glaucoma progression, above and beyond the other baseline risk factors, suggests a clinical utility in testing for this promoter genotype.
[show abstract][hide abstract] ABSTRACT: To compare promoter usage in primary differentiated and SV40 TAg transformed human trabecular meshwork cells (HTM and TM1 cells).
Cultured HTM and TM1 cells were transfected with vectors expressing MYOC/TIGR from the CMV-IE, IE4/5 (HSV immediate early 4/5), ICP6 (early gene ICP6 of HSV), EF-1 alpha (human elongation factor 1 alpha-subunit), or the UB6 (human ubiquitin) promoters, respectively. Immunoblotting was used to measure MYOC/TIGR protein expression. MYOC/TIGR expression at the RNA level was detected by Northern blotting.
In primary HTM cells, CMV was the only promoter displaying substantial activity. In TM1 cells, several promoters were functional with the order in decreasing activity being EF-1 alpha > or = CMV > or = UB6 > IE4/5.
The difference between the normal and transformed HTM cells suggests that the latter cell type has alterations that influence cellular promoter function. The type of cell used is likely to be a crucial factor in evaluating the functions of promoter elements for genes expressed in the trabecular meshwork and in screening promoters for use in gene delivery studies, especially for evaluations of the MYOC/TIGR gene in relation to glaucoma mechanisms.
Current Eye Research 01/2003; 25(6):347-53. · 1.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: To determine whether trabecular meshwork-inducible glucocorticoid response/myocilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells.
The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/MYOC. Solid phase binding assays using 125I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain.
TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cDNA. In solid phase binding assays, 125I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin.
TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the TM and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
[show abstract][hide abstract] ABSTRACT: Primary open-angle glaucoma (POAG) is a highly prevalent optic neuropathy and a major cause of irreversible blindness, with elevation of intraocular pressure (IOP) being a primary risk factor. The trabecular meshwork-inducible glucocorticoid response (TIGR)/MYOCILIN (MYOC) gene coding region is mutated in 3-4% of POAG patients. Here, in a retrospective study of 142 POAG patients, we evaluated the influence on glaucoma phenotype of a novel biallelic polymorphism (-1000C/G) located in the upstream region of the MYOC gene. Allele frequencies were similar among patients and controls. However, the G allele (frequency 17.6%), also designated as MYOC.mt1, was associated with an increased IOP (+4.9 mmHg, p=0.0004) and a more damaged visual field (p=0.02). Both effects were predominant in females. Moreover, whereas IOP in MYOC.mt1 noncarriers decreased very markedly to the normal range between diagnosis and inclusion in the study (p=3 x 10(-5) in both males and females), reflecting successful therapy, it decreased less noticeably in MYOC.mt1+ male patients (p=0.005) and not at all in MYOC.mt1+ female patients. MYOC.mt1 appears therefore to be an indicator of poor IOP control and greater visual field damage in diagnosed POAG patients, potentially due to a lack of response to therapeutic intervention. Its typing might help in the selection of treatment paradigms for the management of POAG patients.
[show abstract][hide abstract] ABSTRACT: Mutations in the trabecular meshwork inducible glucocorticoid response (TIGR) gene, also known as myocilin, have recently been linked to some forms of glaucoma. Recent studies have shown that TIGR protein also is expressed in the ciliary muscle. Because uveoscleral outflow, which traverses the ciliary muscle, is increased by prostaglandins (PGs), the present study assessed whether topical PGs alter the amount of TIGR protein within the ciliary muscle.
Vehicle was topically applied to one eye, and 2 microg PGF(2alpha)-isopropyl ester (PGF(2alpha)-IE) was applied to the other eye of cynomolgus monkeys twice daily for 5 days. Pressure reductions of 5 mm Hg in the PGF(2alpha)-IE-treated eyes were confirmed. The eyes were then fixed and paraffin sections were cut from each eye. The distribution of TIGR protein in the ciliary muscle was determined by confocal scanning laser microscopy. Additional sections were immunostained with a polyclonal antibody to recombinant TIGR protein or with a polyclonal antibody to a synthetic peptide corresponding to the leucine zipper region within the TIGR protein. Staining intensity in the ciliary muscle was assessed by measuring optical density (OD) along two line segments overlying the ciliary muscle, by using a high-resolution imaging densitometer.
TIGR protein immunoreactivity was observed in ciliary muscle fibers throughout the ciliary muscle. Extracellular TIGR immunoreactivity colocalized with collagen type IV immunoreactivity. Intracellular staining also was present. Immunoreactivity was less intense in the sections from the PGF(2alpha)-IE-treated eyes compared with the vehicle-treated eyes. This was reflected in the reduction of mean OD scores in each monkey. Overall, the reduction of mean OD scores in the treated eyes was 42.1% +/- 9.9% (P < 0.005) with the anti-recombinant TIGR antibody and 27.3% +/- 10.4% with the anti-TIGR peptide antibody (P < 0.005).
TIGR protein immunoreactivity was present both intracellularly and extracellularly in the ciliary muscle of the cynomolgus monkey. This suggests that extracellular TIGR protein is in contact with aqueous humor in the uveoscleral outflow pathway. Moreover, IOP-lowering topical PGF(2alpha)-IE treatment decreases the amount of TIGR protein in the ciliary muscle.
[show abstract][hide abstract] ABSTRACT: To determine the effect of the serine-threonine kinase inhibitor H-7, which blocks actomyosin contractility and increases outflow facility in live monkeys, on morphology, cytoskeleton, and cellular adhesions of human trabecular meshwork (HTM) cells in culture.
Cultured HTM cells were videographically recorded and evaluated before and after exposure to H-7 at different concentrations. The subcellular distribution of the actin-based cytoskeleton and associated anchor proteins including vinculin, paxillin, and beta-catenin, as well as phosphotyrosine-containing proteins were evaluated by fluorescence immunocytochemistry and digital fluorescence microscopy.
H-7 induced pronounced but reversible HTM cell thickening toward the cell center and deterioration of the actin cytoskeletal network. Cell-extracellular matrix (ECM) and cell-cell adhesions were also affected, but the beta-catenin-rich, vinculin-containing adherens junctions were clearly more resistant than focal contacts. Phosphotyrosine labeling in focal contacts was highly sensitive to H-7.
H-7 induces alterations in cell shape, actin cytoskeleton, and associated focal adhesions in cultured HTM cells, which may be responsible for the effects of H-7 on outflow facility in live monkey eyes.
[show abstract][hide abstract] ABSTRACT: Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.
Radiation Research 12/2000; 154(5):477-84. · 2.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro.
HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis.
HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells.
HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.
[show abstract][hide abstract] ABSTRACT: Determine the effects of the actin cytoskeleton disrupting compound latrunculin-A (LAT-A) on morphology, cytoskeleton, and cellular adhesions of cultured human trabecular meshwork (HTM) cells.
HTM cells were cultured to high confluence with endothelial-like morphology and treated with LAT-A at different doses and duration. Topography of living cells was evaluated by videomicroscopy. Distribution and organization of the actin-based cytoskeleton, vinculin- and paxillin-containing focal contacts, and beta-catenin-rich intercellular adhesions were determined by immunofluorescence and digital microscopy.
LAT-A induced pronounced but highly reversible rounding of HTM cells, intercellular separation, and disruption of actin filaments. beta-catenin-rich intercellular adherens junctions were particularly sensitive to LAT-A. Vinculin- and paxillin-containing focal contacts were only partially affected and appeared to be more resistant to the drug than the intercellular interactions.
The increase in outflow facility in the living primate eye induced by LAT-A may be due to the disorganization and disruption of the actin cytoskeleton and its associated cellular adhesions in the trabecular meshwork.
[show abstract][hide abstract] ABSTRACT: A number of genetic loci have been implicated in the pathogenesis of primary open angle glaucoma (POAG). The aim of this study was to identify the genetic cause of POAG in a large Scottish family and, if possible, offer genetic screening and advice to family members.
Family members were examined to determine their disease status. Base excision sequence scanning was carried out in order to test for the presence of a POAG causing mutation at known genetic loci. Direct DNA sequencing was performed in order to determine the mutation sequence.
All family members of known affected disease status and two family members of unknown disease status were found to have a mutation in the TIGR gene. The mutation resulted in the substitution of a glycine residue with an arginine residue at codon 252 (Gly252Arg). No other sequence variations were present in any members of the family.
The Gly252Arg mutation in the TIGR gene results in the development of POAG in this family. It was possible to identify younger, currently unaffected, members of the family who carry the mutation and who are therefore at a very high risk of developing POAG themselves. This is the first demonstration that Gly252Arg can be a disease causing mutation rather than a benign polymorphism. The possible pathogenic mechanisms and wider implications of the mutation are considered.
British Journal of Ophthalmology 08/2000; 84(7):722-6. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glucocorticoid (GC) treatment of human trabecular meshwork (HTM) cells produces delayed, progressive cellular and extracellular protein/glycoprotein inductions with characteristics matching those for intraocular pressure elevation with corticosteroid eyedrops. The cloning of the Trabecular Meshwork Inducible Glucocorticoid Response (TIGR) gene from this system has suggested possible environmental and genetic influences in relation to glaucoma mechanisms. As reported here, the major GC-induced increase of TIGR expression in HTM cells is reduced approximately 4-fold by basic fibroblast growth factor (bFGF, 100-1000 pM), with a somewhat smaller inhibition noted with the thyroid hormone triiodothyronine (T3, 100 nM). Such endogenous 'protective' factors could help balance stimulatory effects on TIGR gene expression from 'stress' and/or mechanical perturbations in the trabecular meshwork. TIGR coding region mutations affecting the gene's olfactomedin (OLF) homology domain may also perturb biosynthetic pathways and cellular homeostatic functions. Our recent studies have shown the OLF domain corresponds to a major translocational 'pause', an area where critical processes for normal TIGR biogenesis are expected to take place. Observations that Glu323Lys (and other mutations early in the OLF domain) altered the pattern of paused protein intermediates provide possible clues to previously unexplained pathogenetic mechanisms. HTM cell transfection studies using TIGR-green fluorescent protein (GFP) fusions showed increased and altered distribution of the expressed protein with constructs missing the OLF domain, an effect also found with the Pro370 Leu mutation for early-onset glaucoma. The data suggest an activation of stress/apoptotic pathways in HTM cells as a potential mechanism for environmental/genetic interactions in glaucoma pathogenesis.
[show abstract][hide abstract] ABSTRACT: Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 10(9) light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7)-10(9) light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene products. Degraded dendrimers and high molecular weight PEI exhibited the best combination of high activity and low toxicity in RPE cell transfection.
Journal of Drug Targeting 01/2000; 7(6):413-21. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: To examine possible effects of the E323K mutation in the trabecular meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYOC]), using assays of translocational processing through the endoplasmic reticulum (ER). The E323K mutation was of particular interest, since the mutation shows a strong association with early onset open-angle glaucoma, but has a minimal predicted effect on protein structure.
Normal and mutant TIGR cDNA constructs were used to generate protein products in the presence of endoplasmic reticulum (ER) membranes, using an assay previously developed to detect alterations in the ER translocation function. "Paused" regions for potential protein modifications were defined by proteinase K (PK) sensitivity in the presence of ER membranes, with the ability to restart translocation when treated with EDTA. The effects of the E323K mutation were evaluated, as well as mutations located on either side of E323K (G246R, G364V, P370L) as the other mutations had substantial predicted structural changes in addition to clear disease associations.
The native TIGR molecule was observed to have a paused region that corresponds to the region of highest olfactomedin (OLF) homology. The E323K mutation, located near the beginning of this region, dramatically altered the normal pattern of nascent proteins observed in the translocational pausing assay. A prominent band appeared with the E323K mutation, which could represent a new product or a marked enhancement of a faint band normally seen, approximately 3 kDa higher than the major paused band. The other TIGR mutants examined did not show this effect.
The major translocational pause that starts near the beginning of the region of high OLF homology may help to explain the high frequency of glaucoma-associated mutations in this area. The observed effect of the E323K mutation on the products of translocational processing suggests a delay in the normal pausing process of TIGR biogenesis. This delay points to a potentially distinct pathogenic mechanism for E323K as compared with the other TIGR mutations so far evaluated.
[show abstract][hide abstract] ABSTRACT: Cyclic AMP production in the presence or absence of various adrenergic agonists and antagonists was determined in cultured human trabecular cells using a gammaflow automated radioimmunoassay that allowed multiple simultaneous experiments and reproducible results. Adrenergic agonists and antagonists showed activation and inhibition constants consistent with the presence of beta2-receptors: Ka of isoproterenol < epinephrine < norepinephrine < phenylephrine; Ki of timolol < betaxolol < celiprolol < atenolol. Selective ICI antagonists showed beta2-specificity.
Ophthalmic Research 02/1999; 31(1):53-8. · 1.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to screen affected members of glaucoma families for mutations in the Trabecular Meshwork Inducible Glucocorticoid Response (TIGR) gene also known by the name myocilin (MYOC) or by combined names such as TIGR/MYOC. Our primary objectives were (1) to identify mutations responsible for glaucoma in members of three families for which we have shown linkage between chromosome 1 GLC1A-region markers and the primary open angle glaucoma (POAG) phenotype, and (2) to determine the relationship of these and other mutations to key points of predicted function and structure of the TIGR/MYOC protein.
DNA sequence determination was used to identify sequence changes in sections of the TIGR/MYOC gene that were PCR-amplified from genomic DNA from the probands of three previously-reported GLC1A juvenile-onset POAG families, UM:JG1, UM:JG3, UM:GL57, and unmapped family UM:JG5. Allele-specific oligonucleotide hybridization was used to screen for the identified mutations in PCR-amplified DNA from individual members of each pedigree and from a panel of 11 additional juvenile glaucoma family probands, 42 adult POAG family probands, and 43 normal individuals. Computerized algorithms were used to identify functional motifs and predict structures of normal and mutant forms of the protein.
Sequence changes were found that alter amino acids in the olfactomedin-like domain near the carboxy terminal end of the TIGR protein in affected members of families UM:JG1 (Pro370Leu), UM:JG3 (Val426Phe), UM:GL57 (Glu323Lys) and UM:JG5 (Gly252Arg). Co-segregation of glaucoma and Pro370Leu, Val426Phe, and Gly252Arg in known GLC1A families suggests that these are mutations. Although the Gly252Arg substitution observed in UM:JG5 is non-conservative, it was not possible to distinguish whether it is a mutation or a polymorphism. None of the sequence changes described in these families were observed in other juvenile glaucoma cases in this study, nor in any of the POAG or phenotypically normal individuals tested here. Analysis of amino acid sequence changes resulting from mutations described in this and other works demonstrate localization of many mutations in the vicinity of predicted functional motifs in the olfactomedin-like domain. Identification of rat latrophilin (LPH1/CIRL) as a new member of the olfactomedin-like protein family to which TIGR/MYOC belongs suggests that the region of olfactomedin homology is a protein domain that can occur in different protein contexts.
Location of mutations described in this and previous work suggests that some specific predicted protein motifs in the olfactomedin-like domain may be important to TIGR/MYOC function. In some cases, the role of TIGR/MYOC in the etiology of glaucoma may result from alteration of the sequences recognized by modifying enzymes such as casein kinase II. In other cases altered protein folding may affect access of enzymes to their target sequences on TIGR/MYOC. Although modifications and structures discussed here are predicted rather than proven, they provide a useful theoretical framework for design of subsequent experiments. Alterations to protein folding and predicted modification motifs cannot explain the pathogenic mechanisms of all of the known TIGR/MYOC glaucoma mutations.