A trabecular meshwork glucocorticoid response (TIGR) gene mutation affectstranslocational processing

PURPOSE: To examine possible effects of the E323K mutation in the trabecula r meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYO C]), using assays of translocational processing through the endoplasmic ret iculum (ER). The E323K mutation was of particular interest, since the mutat ion shows a strong association with early onset open-angle glaucoma, but ha s a minimal predicted effect on protein structure. METHODS: Normal and mutant TIGR cDNA constructs were used to generate prote in 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 define d by proteinase K (PK) sensitivity in the presence of ER membranes, with th e ability to restart translocation when treated with EDTA. The effects of t he E323K mutation were evaluated, as well as mutations located on either si de of E323K (G246R, G364V, P370L) as the other mutations had substantial pr edicted structural changes in addition to clear disease associations. RESULTS: The native TIGR molecule was observed to have a paused region that corresponds to the region of highest olfactomedin (OLF) homology. The E323 K mutation, located near the beginning of this region, dramatically altered the normal pattern of nascent proteins observed in the translocational pau sing assay. A prominent band appeared with the E323K mutation, which could represent a new product or a marked enhancement of a faint band normally se en, approximately 3 kDa higher than the major paused band. The other TIGR m utants examined did not show this effect. CONCLUSIONS: 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 E 323K mutation on the products of translocational processing suggests a dela y in the normal pausing process of TIGR biogenesis. This delay points to a potentially distinct pathogenic mechanism for E323K as compared with the ot her TIGR mutations so far evaluated.