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Characterization of myocilin-myocilin interactions
1Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA. fautsch@mayo.edu
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.