In vitro and in vivo characterization of disulfide bond use inmyocilin complex formation

[摘要] Purpose: Myocilin forms large complexes in aqueous humor. Part ofthis complex formation is due to myocilin-myocilin protein non-covalentinteractions within the leucine zipper. However, additional covalentinteractions also exist. We investigated the role of these covalentinteractions in disulfide bond formation within myocilin.Methods: Human aqueous humor was separated by denatured/non-reducedSDS-PAGE followed by Western blot analysis with myocilin specificantibodies. In part two of the study, site-directed mutagenesis was usedto selectively mutate one, two, three, four, and all five cysteineresidues in the mature myocilin protein expressed in an in vitro system.Products were immunoprecipitated with a hemagglutinin polyclonalantibody following in vitro transcription/translation and analyzed bySDS-PAGE. In part three of the study, glaucoma associated myocilinmutations Arg82Cys and Cys433Arg were created and complex formationanalyzed in trabecular meshwork cells.Results: Human aqueous humor showed myocilin in several distinctlarge complexes in non-reduced SDS-PAGE gels, indicating disulfide bondsoccur. Similarly, in vitro expressed myocilin also produced largecomplexes. Mutation of all five cysteines (within the mature myocilinprotein) eliminated this large complex formation. A combination ofcysteine to alanine substitutions at amino acids 185, 245, and 433 hadthe most influence on myocilin complex formation under non-reducingconditions, however individual substitutions at each of the fivecysteine amino acids had little influence on myocilin complexes. Intrabecular meshwork cells, Arg82Cys was secreted but formed differentsized complexes than wild type myocilin. Cys433Arg was not secreted andremained intracellular in a pattern that differed from wild typemyocilin and Arg82Cys.Conclusions: Myocilin complexes present in human aqueous humor arein part due to disulfide bond formation between cysteine amino acids.Glaucoma associated mutations that affect the number of cysteineresidues may alter covalent interactions.

[学科分类] 生物化学/生物物理