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Vasanth Rao, Rasiah Pratheepa kumari, Nikolai Skiba; Characterization of Lens Fiber Cell Membrane Microdomains by Proteomics Analysis of Lipid Rafts. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
To better understand the role of scaffolding proteins in membrane organization of cell adhesive, cytoskeletal and signaling protein complexes in lens fibers, detergent-resistant low density membrane rafts were isolated from porcine lens fibers and characterized.
Detergent-resistant membrane rafts were isolated from freshly enucleated porcine lens cortical fibers using non-ionic detergent (Triton X-100) and sucrose gradient ultracentrifugation. The solvent (chloroform/methanol) extracted protein fraction of detergent-resistant low density membrane rafts was separated on gradient polyacrylamide gel, then protein bands were in-gel-digested with trypsin and identified by mass spectrometry using Synapt G2 and immunoblot analysis.
Two independent analyses of lens fiber cell lipid rafts derived from adult porcine lenses yielded consistent results with respect to protein composition based on mass spectrometry. The lipid rafts isolated from the porcine lens were confirmed to be enriched in phosphatidylinositol 4, 5-bisphosphate (PIP2) and flotillin. Proteins from various structural and functional subfamilies including cytoskeletal, cell adhesive, signaling, scaffolding, transport and enzymes were identified in the lens lipid raft fraction. Importantly, some of the major proteins present in the lipid rafts include Ankyrin-B, periaxin, actin, spectrin, N-cadherin, various catenins, neuronal cell adhesion molecules, filensin, phakinin, grifin, flotillin, ezrin, radixin, Band 4.1, profilin, cofilin, vimentin, aquaporin-0, LIM2, brain acid soluble protein-1 (BASP1), paralemmin, Rho, Rac1, Rap1 , Ras and Rab GTPases, zeta-crystallin, NADH-cytochrome b5 reductase, FABP5, PEA15, various 14-3-3 proteins, crystallins, voltage gated anion channels, ZO-1, gelsolin, heat shock proteins, ponsin, annexins and S100 family proteins.
To the best of our knowledge, this is the first study to describe the proteomics analysis of lipid rafts isolated from the lens fibers and reveals that periaxin, Ankyrin-B, paralemmin, BASP1, N-cadherin, catenins, filensin, phakinin, actin, spectrin, aquaporin-0 and small GTPases are some of the prominent components of lens lipid rafts. Studies are underway to quantify and profile lens fiber cell lipid raft proteins in a comprehensive manner.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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