Glaucoma is a major cause of blindness and is characterized by progressive degeneration of the optic nerve and is usually associated with elevated intraocular pressure. Analyses of sequence tagged site (STS) content and haplotype sharing between families affected with chromosome 1q-linked open angle glaucoma (GLC1A) were used to prioritize candidate genes for mutation screening. A gene encoding a trabecular meshwork protein (TIGR) mapped to the narrowest disease interval by STS content and radiation hybrid mapping. Thirteen glaucoma patients were found to have one of three mutations in this gene (3.9 percent of the population studied). One of these mutations was also found in a control individual (0.2 percent). Identification of these mutations will aid in early diagnosis, which is essential for optimal application of existing therapies.

Expression of the trabecular meshwork inducible glucocorticoid response (TIGR) gene progressively increases from barely detectable levels to greater than 2% of total cellular mRNA over 10 days exposure of trabecular meshwork (TM) cells to dexamethasone. Cycloheximide blocked most of the TIGR mRNA induction, suggesting a requirement for ongoing protein synthesis. The genomic structure of TIGR (ϳ20 kilobases) consists of 3 exons, and a 5-kilobase promoter region that contains 13 predicted hormone response elements, including several glucocorticoid regulatory elements, and other potentially important regulatory motifs. TIGR cDNA encodes an olfactomedin-related glycoprotein of 504 amino acids with motifs for N-and O-linked glycosylation, glycosaminoglycan initiation, hyaluronic acid binding, and leucine zippers. Recombinant TIGR (rTIGR) showed oligomerization and specific binding to TM cells. Anti-rTIGR antibody detected multiple translational/post-translational forms of TIGR produced by the cells (including secreted 66 kDa/55 kDa glycoproteins/proteins in the media and 55 kDa cellular proteins), whereas Northern blot showed a single mRNA species. The findings suggest potential mechanisms by which TIGR could obstruct the aqueous humor fluid flow and participate in the pathogenesis of glaucoma.The trabecular meshwork inducible glucocorticoid response (TIGR) 1 protein, which has significant homology in its C-terminal domain with olfactomedins, was initially cloned in our laboratories as a candidate gene for glaucoma using differential library screening in a trabecular meshwork cell culture model (1, 2). Mutations were recently found in this gene that co-segregated with both juvenile and adult forms of the disease (3).Glaucoma is a major cause of blindness, with its most prevalent form thought to involve the specialized endothelial cells lining the outflow pathway of the eye, termed the trabecular meshwork (TM) (4, 5). The synthesis and/or degradation of a variety of extracellular molecules in the meshwork are thought to be regulated by the TM cells, and alterations in the type or amount of connective tissue elements have been postulated to explain the increased outflow resistance seen in glaucoma cases (6). However, an understanding of the biochemical changes that actually contribute to this process has remained elusive.Previously, we described a highly expressed protein and related glycoprotein (55 and 66 kDa, respectively) found in the media of TM cell culture, but not in other cell types examined, after a prolonged exposure to dexamethasone (DEX) (2). We used this observation to define a cell culture model for "steroidinduced glaucoma" and elevated intraocular pressure due to corticosteroids. The extracellular induced proteins appeared as reasonable candidates for being involved in steroid glaucoma since the time course and dose response of their induction mimicked the intraocular pressure elevation and increased outflow resistance seen in patients receiving glucocorticoid (GC) therapy (7,8).Coincident with our res...

Studies of the effects of glucocorticoid (GC) and oxidative stress stimuli in differentiated cultures of human trabecular meshwork (HTM) cells have provided the rationale for our studies of a major new gene termed TIGR (trabecular meshwork inducible GC response). The TIGR clone was isolated by differential library screening using selection criteria based on the induction pattern of a new protein/glycoprotein found in HTM cultures after prolonged but not brief exposure to GCs. This GC induction patter matched the time course and dose response required for intraocular pressure elevation in patients receiving corticosteroids. The very large, progressive induction of TIGR combined with specific structural features of its cDNA suggested that TIGR should be considered a candidate gene for outflow obstruction in glaucoma. Among the properties of TIGR cDNA were a signal sequence for secretion, several structural features for interactions with glycosaminoglycans and other glycoproteins and putative sites for cell surface interactions. In addition, the leucine zippers in the structure were related to TIGR-TIGR oligomerization that was shown to occur with native and recombinant TIGR protein. The verification that TIGR was a major stress response protein in HTM cells following hydrogen peroxide (or phorbol esters) exposure provided a potential link between GC and oxidative mechanisms thought to be involved in glaucoma pathogenesis. Pharmacological evaluation showed that basic fíbroblast growth factory and transforming growth factor β decreased the GC induction of TIGR, and certain nonsteroidal anti-inflammatory drugs protected against both GC- and oxidation-induced stress responses in HTM cells. Our recent studies of TIGR’s genomic structure have shown motifs in the promoter region that suggest a basis by which multiple hormonal/environmental stimuli can regulate TIGR production and by which putative genetic alterations could lead to an overexpression of the protein. Further application of cell biology/biochemistry, molecular biology, genetic and histological approaches will be helpful in understanding the role of TIGR in different glaucoma syndromes.

An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated as well. Here we show that information encoded in the hydrophobic domain of a signal sequence influences the timing and efficiency of at least two steps in maturation, namely N-linked glycosylation and signal sequence cleavage. We demonstrate that these consequences correlate with and likely stem from the nature of the initial association made between the signal sequence and the translocon during the initiation of translocation. We propose a model by which these maturational events are controlled by the signal sequence-translocon interaction. Our work demonstrates that the pathway taken by a nascent chain through post-translational maturation depends on information encoded in its signal sequence.N-terminal signal sequences enable nascent secretory and transmembrane proteins to be targeted to the endoplasmic reticulum (ER) 1 for translocation. The signal sequence, newly emerged from the translating ribosome, is recognized in the cytoplasm by the signal recognition particle (1). By virtue of an interaction with its ER-localized receptor, signal recognition particle brings the ribosome-nascent chain complex to the ER membrane (2). Once at the ER, the ribosome is thought to facilitate the assembly or stabilization of the translocation channel, composed of the heterotrimeric Sec61 complex (3, 4). The 35-kDa polytopic Sec61␣ subunit appears to form the channel walls, whereas the smaller bitopic ␤-and ␥-subunits play an as yet undetermined role, perhaps in facilitating the insertion of the nascent chain into the channel (5-7).An important advance in the understanding of the initiation of translocation was the discovery of a second step of signal sequence recognition. In addition to being recognized by the signal recognition particle, the signal sequence also interacts with the translocation channel itself (8,9). Although the mechanism is not yet clear, this event is thought to stimulate the initiation of translocation. For the simplest secretory proteins, when the signal sequence is recognized by the channel, the ribosome assumes a tight interaction with the channel that shields the protein from the cytoplasm (8, 10). Concomitantly, the channel opens toward the ER lumen, either via a conformational change in the channel or removal of a molecular plug covering the luminal aperture (11,12).Not all signal sequences initiate translocation in the same way. Beyond merely stimulating the initiation of translocation, signal sequences can regulate the association between the ribosome and translocon and the exposure of the nascent chain to the cytoplasm or ER lumen (13-16). In at least one case, that of the prion protein, the consequence of this regulatory step is the governance of the ...

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.