Abstract
Abstract: :
Purpose: To identify myocilin interacting proteins in human trabecular meshwork (TM). Identification of myocilin binding partners will give insights into the physiologic role of myocilin. Methods: Three techniques were performed to identify myocilin interacting proteins. For yeast 2–hybrid analysis, a myocilin expressing yeast cell line was mated with >1 x 106 human smooth muscle cDNA clones. Positive interacting colonies were selected for growth on minimal media, isolated and identified. For coimmunoprecipitation studies, a protein lysate from 51 human TMs was incubated with either anti–myocilin antibodies attached to sepharose beads or control sepharose beads (no antibody). Captured myocilin protein complexes were washed, eluted from antibody, and separated by SDS–PAGE. Proteins present after myocilin coimmunoprecipitation that were not present in control were excised, digested with trypsin, and identified by LC–MS/MS. For Far Western analysis, 12 human TMs were lysed, separated by 1 or 2–dimensional (D) gel electrophoresis, transferred to PVDF membrane, and probed with purified recombinant myocilin (a. a. 1–504 fused to a C–terminal tag that contains a histidine and V5 epitope). Western blot procedures were performed to identify specific myocilin interactions. Proteins in silver–stained gels that matched Far Western blot features were identified. Results: We have identified 23 candidate myocilin interacting proteins by yeast 2–hybrid, coimmunoprecipitation, and Far Western analysis. These include molecules with cellular functions associated with the cytoskeleton, signaling, and metabolism. Two proteins were identified in more than one technique. Myocilin was found to interact with itself in yeast 2–hybrid and by coimmunoprecipitation. Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) was found to interact with myocilin in yeast 2–hybrid and Far Western (both 1 and 2–D analysis). Conclusions: We have identified candidate–binding partners for myocilin. In addition to myocilin forming a complex with itself, GAPDH appears to be a potential binding partner for myocilin. Originally regarded as a protein involved in the glycolytic pathway, GAPDH is now known to function in endo– and exocytosis, membrane fusion, and apoptosis. Its association with key proteins implicated in Alzheimer’s and Huntington’s disease suggests it may also have a role in age–related neurodegenerative disorders. Further analysis of GAPDH and the other myocilin binding proteins in normal and glaucomatous TMs is warranted.
Keywords: trabecular meshwork • proteomics • anterior segment