Protein interactions with myocilin.
OBJECTIVE
To identify factors that interact in vivo with myocilin, a glaucoma gene product.


METHODS
The yeast two-hybrid system with myocilin as the bait and a human skeletal muscle cDNA library as the prey was used to identify potential factors that interact with myocilin. Interactions were also examined in bovine trabecular meshwork (TM) cells through a mammalian two-hybrid system. Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro translated proteins was also used to confirm results obtained from yeast analysis.


RESULTS
Twenty positive clones isolated through yeast two-hybrid screening were deemed potential myocilin partners. Sequence analysis determined that two of them encoded for myocilin from amino acids 64 to 268. Myocilin was also found to interact with a component of the myosin motor protein, myosin regulatory light chain (RLC). The myocilin-myocilin and myocilin-RLC interactions revealed by the yeast system were further confirmed and demonstrated in cultured TM cells, by means of a mammalian two-hybrid system, and through biochemical coimmunoprecipitation, subcellular fractionation, immunofluorescence, and immunogold double labeling.


CONCLUSIONS
These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain. Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin-RLC interaction. The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM.
Wentz-Hunter
Kelly
K
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Ueda
Jun
J
Yue
Beatrice Y J T
BY
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D016350:Leucine Zippers / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae; D014129:Trabecular Meshwork / Q000378:metabolism* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D017931:DNA Primers; D005136:Eye Proteins; D006023:Glycoproteins; D018994:Myosin Light Chains; D000099179:Myocilin