The identification of myocilin-associated proteins in the human trabecular meshwork.
Myocilin forms high molecular weight complexes in vivo presumably due to interaction with itself and other myocilin binding proteins. To identify myocilin interacting proteins, yeast 2-hybrid analysis was performed on >1x10(6) human trabecular meshwork cDNA clones. Coimmunoprecipitation and Far Western analysis were also performed on cell lysates obtained from fresh human trabecular meshworks or cultured human monolayer trabecular cell lines. Among the different methods, 46 candidate myocilin-associated proteins were identified, including molecules associated with the extracellular matrix, cytoskeleton, signaling, and metabolism. The most consistent interaction was myocilin-myocilin binding. Yeast-2 hybrid and Far Western analysis also found an association between myocilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). None of the other candidate myocilin interacting proteins were identified in more than one method. Characterization of these potential interacting proteins may help to better understand the function of myocilin in the trabecular meshwork and aqueous outflow pathway.
Fautsch
Michael P
MP
Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. fautsch.michael@mayo.edu
Vrabel
Anne M
AM
Johnson
Douglas H
DH
D000368:Aged; D026481:Blotting, Far-Western / Q000379:methods; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005109:Extracellular Matrix / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005136:Eye Proteins / Q000032:analysis* / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D050505:Mutant Proteins / Q000032:analysis; D009154:Mutation; D015398:Signal Transduction / Q000502:physiology; D014129:Trabecular Meshwork / Q000737:chemistry* / Q000166:cytology; D020798:Two-Hybrid System Techniques; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006023:Glycoproteins; D050505:Mutant Proteins; D000099179:Myocilin