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M. Jin, S. Li, W.N. Moghrabi, A.R. Philp, G.H. Travis; Mutational Analysis to Determine Key Residues Essential for Activity and Membrane Association of Rpe65 Isomerohydrolase . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2043.
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© ARVO (1962-2015); The Authors (2016-present)
We previously identified Rpe65 as the retinoid isomerase in RPE cell. Rpe65 was shown to undergo palmitoylation of residues C231, C329 and C330. Reportedly, these modifications are effected by LRAT and are responsible for the association of Rpe65 with membranes. One purpose of this study was to investigate the role of these palmitoylated residues on isomerase activity and membrane association of Rpe65. Another was to define the roles of the four conserved histidines that coordinate Fe2+ in Rpe65. The final purpose is to determine the effects of mutations in the RPE65 gene that cause Leber congenital amaurosis (LCA) on isomerase activity of Rpe65.
We performed site directed mutagenesis to generate cDNAs that express Rpe65 with the indicated amino–acid substitutions. These cDNAs were expressed in Sf9 cells, 293T cells, or 293T cells that also express LRAT and CRALBP (293T–LC cells). Membranes from the cells were isolated by sucrose gradient ultracentrifugation. Isomerase activity was measured by monitoring 11–cis retinol (11cROL) formation from added all–trans retinol (atROL) or all–trans retinyl palmitate (atRP) substrate.
We detected isomerase activity with atROL substrate in 293T–LC cells expressing Rpe65 with the single or double substitutions: C213S, C329S, C330S, C231/329S, and C231/330S. However, C329/330S–double and C231/329/330S–triple substituted Rpe65 possessed no isomerase activity. All seven Cys–substituted forms of Rpe65 were tightly associated with membranes, even when expressed without LRAT. Isomerase activity was also lost in H180R, H241R, H313R or H527R–substituted Rpe65. Membranes from Sf9 cells expressing wild–type Rpe65 possessed isomerase activity. However, no activity was detected in membranes from Sf9 cells expressing Rpe65 with the LCA–associated mutations: R91W, Y239D and L408P. We observed similar isomerase activities in 293T–LC cells expressing wild–type or L450M–substituted Rpe65 at the same protein level.
1) Rpe65 remains associated with membranes following substitution of the three cysteines reported to undergo S–palmitoylation. This membrane association does not depend on LRAT. 2) Substitution of any histidines thought to bind Fe2+ result in complete loss of catalytic activity. 3) All substitutions due to LCA–causing mutations resulted in loss of isomerase activity. 4) The slower rhodopsin regeneration seen in mice with the M450L substitution results from reduced levels of the Rpe65 protein rather than a reduction in Rpe65 catalytic activity.
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