title: In vitro and in vivo characterization of disulfide bond use in myocilin complex formation.
abstract: OBJECTIVE
  Myocilin forms large complexes in aqueous humor. Part of this complex formation is due to myocilin-myocilin protein non-covalent interactions within the leucine zipper. However, additional covalent interactions also exist. We investigated the role of these covalent interactions in disulfide bond formation within myocilin.
  
  
  METHODS
  Human aqueous humor was separated by denatured/non-reduced SDS-PAGE followed by Western blot analysis with myocilin specific antibodies. In part two of the study, site-directed mutagenesis was used to selectively mutate one, two, three, four, and all five cysteine residues in the mature myocilin protein expressed in an in vitro system. Products were immunoprecipitated with a hemagglutinin polyclonal antibody following in vitro transcription/translation and analyzed by SDS-PAGE. In part three of the study, glaucoma associated myocilin mutations Arg82Cys and Cys433Arg were created and complex formation analyzed in trabecular meshwork cells.
  
  
  RESULTS
  Human aqueous humor showed myocilin in several distinct large complexes in non-reduced SDS-PAGE gels, indicating disulfide bonds occur. Similarly, in vitro expressed myocilin also produced large complexes. Mutation of all five cysteines (within the mature myocilin protein) eliminated this large complex formation. A combination of cysteine to alanine substitutions at amino acids 185, 245, and 433 had the most influence on myocilin complex formation under non-reducing conditions, however individual substitutions at each of the five cysteine amino acids had little influence on myocilin complexes. In trabecular meshwork cells, Arg82Cys was secreted but formed different sized complexes than wild type myocilin. Cys433Arg was not secreted and remained intracellular in a pattern that differed from wild type myocilin and Arg82Cys.
  
  
  CONCLUSIONS
  Myocilin complexes present in human aqueous humor are in part due to disulfide bond formation between cysteine amino acids. Glaucoma associated mutations that affect the number of cysteine residues may alter covalent interactions.
authors.affiliation: Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. fautsch@mayo.edu
authors.forename: Michael P
authors.initials: MP
authors.lastname: Fautsch
authors.forename: Anne M
authors.initials: AM
authors.lastname: Vrabel
authors.forename: Stefanie L
authors.initials: SL
authors.lastname: Peterson
authors.forename: Douglas H
authors.initials: DH
authors.lastname: Johnson
mesh_terms: D001082:Aqueous Humor / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005901:Glaucoma / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014129:Trabecular Meshwork / Q000378:metabolism; D000099179:Myocilin
chemical_list: D003545:Cysteine; D003598:Cytoskeletal Proteins; D004220:Disulfides; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin