Abstract
Purpose: :
We and others have recently established RPE65 as the visual cycle retinol isomerase. As the rate of pigment regeneration in rodents is slower than in humans or other mammals tested, we ask whether this is due to lowered isomerase activity of rodent RPE65 compared to other species. Also, as the methionine (Met) variant of mouse RPE65 residue 450 (normally Leu) is associated with lowered light sensitivity and with resistance to light damage in C57Bl/6 mice, consistent with lowered activity, we ask if this would also occur in other RPE65s.
Methods: :
Site–directed mutagenesis was used to change residues in mouse and dog RPE65. Mutants were tested for isomerase activity in a transient transfection cell culture model of the visual cycle (Redmond et al., Proc. Natl. Acad. Sci. USA 102: 13658–63, 2005) and compared with wildtype mouse and dog RPE65 activity.
Results: :
The relative isomerase activity of mouse and dog RPE65 was similar. Replacing RPE65 L450 with Met reduced relative activity of dog RPE65 to 65% of wildtype and to 25% of wildtype in mouse RPE65. As residues R358 and V459 in other mammalian RPE65s are both Met in rodent RPE65s, we wished to see if these differences affected RPE65 activity. Replacing either R358 or V459 in dog RPE65 with Met had a very modest effect on 11–cis retinol production giving about 90% of wildtype dog RPE65 activity. Conversely, changing M358 and M459 in mouse RPE65 to arginine and valine, respectively, had greater (though still moderate) effect: M358R was 80% and M459V was 60% of wildtype activity.
Conclusions: :
Wildtype mouse and dog RPE65s are equal in relative isomerase activity, so the slower regeneration rate in mouse is probably not due to the RPE65/isomerase step per se. The L450M variant showed reduced production of 11–cis retinol by both dog and mouse RPE65s, while variation at residues 358 (R or M) and 459 (V or M) had little effect on activity of dog RPE65 and only moderate effect on that of mouse RPE65. Overall, "tuning" of mouse RPE65 isomerase activity is not likely to be a mechanism for the lower rhodopsin regeneration rate in mouse. "Tuning" could occur but its effects may be more subtly expressed.
Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • regeneration