Molecular architecture and modifications of full-length myocilin

• Type of Material: Article
• Authors: Mackenzie D Martin, Georgia Institute of Technology; Dustin JE Huard, Georgia Institute of Technology; Ricardo C Guerrero-Ferreira, Emory University; Ishani M Desai, Georgia Institute of Technology; Brett M Barlow, Georgia Institute of Technology; Raquel L Lieberman, Georgia Institute of Technology
• Language: English
• Date: 2021-10-01
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2021 Elsevier Ltd. All rights reserved.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.exer.2021.108729
• Title of Journal or Parent Work: Experimental Eye Research
• Volume: 211
• Start Page: 108729
• End Page: 108729
• Grant/Funding Information: NIH R01EY021205, R01EY021205-09S1, and S10RR025679.
• Keywords: Protein Interaction Domains and Motifs; Glycosylation; Electron microscopy; Glaucoma; Eye; Extracellular matrix protein; Microscopy, Electron, Transmission; Glycoproteins; Proteomics; Cytoskeletal Proteins; Protein Processing, Post-Translational; N-linked glycosylation; Transmission electron microscopy; Trabecular Meshwork; Trabecular meshwork; Proteolysis; Gene Expression; Oligomerization; Cell Line; Transfection; Protein misfolding; Humans; Animals; Protein Multimerization; Protein Folding; Myocilin; Eye Proteins; Blotting, Western

Abstract

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.