Protein interactions with myocilin

Purpose: 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.