Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of myocilin structure and its still enigmatic biological function.
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| http://dx.doi.org/10.1016/j.exer.2021.108729 | DOI Listing |
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Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible.
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Article Synopsis
- Recombinant antibodies with specific epitopes are essential for accurate immunoassays in biomedical research, particularly for understanding myocilin's role in glaucoma and other diseases.* -
- Existing antibodies struggle to differentiate various forms of misfolded myocilin, limiting research into its structure and function; new antibodies 2A4 and 1G12 were developed through protein engineering to target specific myocilin domains.* -
- The refined antibody 2H2 shows promise for use in human samples by successfully binding to and visualizing myocilin in cells, indicating its potential utility in glaucoma research and related studies across different organisms.*
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