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A.M. Vrabel, M.P. Fautsch, S.L. Peterson, D.H. Johnson; Characterization of Disulfide Bonds in Myocilin Complex Formation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1150.
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Purpose: Myocilin forms large complexes in aqueous humor. Part of this complex formation is presumably due to myocilin-myocilin interactions within the leucine zipper. However, additional chemical interactions also exist. We investigated the role of the cysteine amino acids in disulfide bond formation within myocilin. Methods: Human aqueous humor and cultured trabecular meshwork cells (gift from Abe Clark, Alcon, Inc.) were separated by denatured/non-reduced SDS-PAGE followed by Western blot analysis with myocilin specific antibodies. Site-directed mutagenesis was used to selectively mutate one, two, three, four, and all five cysteine residues in the mature myocilin protein (does not contain signal peptide sequence). Products were immunoprecipitated following in vitro transcription/translation and analyzed by SDS-PAGE under denatured/non-reduced conditions. Myocilin containing Arg82Cys, a known glaucoma causing mutation, was constructed and also analyzed by this method. Results: Human aqueous humor and media collected from cultured human trabecular meshwork cells showed myocilin in several distinct large complexes. Mutation of all five cysteines (within the mature myocilin protein) eliminated large complex formation. Examination of products containing various cysteine mutations identified cysteines 185, 245, and 433 as having the most influence on myocilin complex formation under denatured/non-reducing conditions. Arg82Cys disrupted some myocilin interactions resulting in smaller than normal complexes. Conclusions: Myocilin is present in complexes in human aqueous humor that in part may be due to disulfide bond formation between cysteine amino acids.
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