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Expression and characterization of retinal disease linked ABCR mutants

University of British Columbia

The retinal A B C transporter, ABCR, is a 220 kDa glycoprotein localized in the outer segments of the photoreceptor cells. As a member of the A B C superfamily, A B C R is organized into two tandem halves each containing a transmembrane domain and a nucleotide binding domain. Studies have suggested that A B C R may either act as an alltrans- retinal extruder or a retinylidene-phosphatidylethanolamine flippase. Eighty-nine mutations in ABCR gene have been identified and associated with a number of retinal degenerative diseases including Stargardt's disease. In this study the effects of Stargardt's disease causing mutations (D846H, T1526M, R2038W, R2077W and R2106C) on the structure and function of A B C R were investigated. As a first step toward this goal, the five mutant constructs were expressed in the mammalian COS cell expression system. When the expression levels of the proteins were compared, it was found that the R2038W and R2077W mutants were expressed at 57-59% of the wild type while R2106C and T1526M expressed at 71% and 81% of the wild type protein, respectively. The expression level of the D846H mutant varied between 31-68% of the wild type, depending on the transfection method used. The recombinant proteins were purified from CHAPS solubilized COS cell by immunoaffinity chromatography. Immunofluorescence microscopy of the transfected COS cells revealed that the expressed wild type A B C R was localized in the vesicles, whereas almost 100% of both D846H and T1526M, 77% of R2038W, 80% of R2077W and 34% of R2106C showed an endoplasmic reticulum/Golgi labeling pattern. Calnexin interaction studies showed that all of the mutants examined co-purified with calnexin to a greater extent than the wild type ABCR. The ATPase activity of purified proteins reconstituted in liposomes rich in phosphatidylethanolamine was measured. In the presence of 50 uM all-frvms-retinal, the basal activity of the wild type A B C R was increased by 1.6 fold; however, no stimulation was seen in T1526M and R2038W variants. The ATPase activities of both D846H and R2077W were impaired, whereas the R2106C mutant showed a retinal-stimulated activity similar to the wild type. The azido-ATP labeling study showed that the wild type, T1526M and R2106C bound ATP, whereas the D846H and R2077W did not.

Thesis/Dissertation

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2009-08-06

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http://hdl.handle.net/2429/11833

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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