Characterization of myocilin-myocilin interactions

Michael P Fautsch, D. H. Johnson

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

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 languageEnglish (US)
Pages (from-to)2324-2331
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume42
Issue number10
StatePublished - 2001

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Leucine Zippers
Aqueous Humor
Yeasts
Leucine
Two-Hybrid System Techniques
Site-Directed Mutagenesis
Glycine
Gel Chromatography
Color
Amino Acids
trabecular meshwork-induced glucocorticoid response protein
Growth
In Vitro Techniques

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Characterization of myocilin-myocilin interactions. / Fautsch, Michael P; Johnson, D. H.

In: Investigative Ophthalmology and Visual Science, Vol. 42, No. 10, 2001, p. 2324-2331.

Research output: Contribution to journalArticle

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abstract = "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.",
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