Purpose. To determine whether myocilin (MYOC; also referred to as TIGR) is present as a complex in human aqueous humor, whether part of the complex formation may be due to MYOC-MYOC interactions and to characterize the sites of interaction. Methods. Human aqueous humor was analyzed by using a gel filtration column for the identification of MYOC complexes. MYOC-MYOC interactions were studied with a yeast two-hybrid system. Expression of full-length and truncated MYOC proteins in AH109 yeast was analyzed for growth and color on minimal medium. Site-directed mutagenesis was used to selectively mutate eight leucine residues within the leucine zipper motif. In vitro transcription and translation was used to verify yeast two-hybrid analysis. Results. MYOC was found to be present in human aqueous humor as a complex ranging from 120 to 180 kDa. Expression of full-length MYOC in yeast as well as in vitro binding studies revealed that MYOC can interact with itself. MYOC-MYOC interactions occurred mainly within amino acids 117-166, a region containing a leucine zipper domain. Glycine substitution for selective leucine residues confirmed that MYOC-MYOC interactions occurred mainly within the leucine zipper domain. Conclusions. MYOC is present in human aqueous humor, not as a monomer but as a complex. Part of this complex may form due to MYOC-MYOC interactions that take place mainly within the leucine zipper domain.
|Original language||English (US)|
|Number of pages||8|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Sep 26 2001|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience