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Preparation and characterization of bovine retinal pigment epithelial cell plasma membrane

University of British Columbia

A 7-9 fold enriched preparation of bovine retinal pigment epithelial cell plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight retinal pigment epithelial cellular adhesion to the rod photoreceptor cells. The contaminating rhodopsin was partially removed by anti-rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or silver. Monoclonal antibodies raised against the retinal pigment epithelial plasma membrane preparation cross reacted with rod outer segment preparations. A monoclonal antibody, designated Rho-5A3, was classified as an IgG₃ kappa light chain immunoglobulin. It was shown to be specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment membrane proteins electrophoretically transferred to CNBr-activated paper. Limited proteolytic digestion of rhodopsin followed by electrophoretic transfer to CNBr- activated paper localized the binding site of this antibody to the N-terminal two-thirds of the rhodopsin molecule. Competition assays with rhodopsin polypeptides further defined the antigenic site to be within the 17-39 amino-acid segment of rhodopsin. The Rho-5A3 antibody did not bind to sealed ROS discs or frozen-thawed ROS discs but did bind to Triton X-100 solubilised discs indicating a detergent solubilisation dependence for antigenic site accessibility. Cultures of bovine retinal pigment epithelial cells were started by initial enzymatic isolation followed by recovery in RPMI-1640 culture medium. The retinal pigment epithelial cells established a doubling time of 52 hours until the cells reached culture confluency . The cells also maintained many of their in vivo characteristics such as a high degree of pigmentation and an abundance of microvilli. Cell surface glycoproteins labelled with FITC-Con A, FITC-WGA, and FITC-RCA showed dense and random surface labelling patterns. Fluorescent labels were induced to redistribute to central spots and clear from the cell surface by incubating the labelled cells in buffer for 60 minutes at 37°C. Treatment by the appropriate saccharide inhibitors indicated that the labelled sites had undergone endocytosis by the cell. Continuous labelling experiments indicated that redistribution and internalization is constantly occurring so that previously unlabeled receptors become accessible for labelling. As a result a dense pattern of label on the cell surface was maintained. The protein actin, with apparent Mr =46,000, was detected with rabbit anti-actin antisera labelling of the RPE plasma membrane preparation proteins electrophoretically transferred to nitrocellulose paper. Immunofluorescent labelling using the rabbit anti-actin antisera confirmed biochemical studies that actin was a major component of the bovine RPE cell. The activation of actin filaments may play an important role in the phagocytosis of bovine rod outer segments by retinal pigment epithelial cells in tissue culture. Scanning electron microscopy and transmission electron microscopy have shown that 2 week old bovine retinal pigment epithelial cells in vitro can be induced to recognize, attach, and engulf dark adapted sealed rod outer segments. In summary a monoclonal antibody was raised against a bovine retinal pigment epithelial cell plasma membrane preparation, however, the antibody raised proved to be specific for rhodopsin, a contaminating ROS protein found in the preparation. The monoclonal antibody, designated Rho-5A3, was fully characterized and its antigenic site determined. Finally, bovine retinal pigment epithelial cells grown in tissue culture acted as a model system for studying cell surface components and rod outer segment phagocytosis at the levels of fluorescence, scanning electron microscopy, and transmission electron microscopy.

Text

2010-05-19

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http://hdl.handle.net/2429/24835

Biochemistry and Molecular Biology

University of British Columbia

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