Effect of Leu/Met variation at residue 450 on isomerase activityand protein expression of RPE65 and its modulation by variation at otherresidues

摘要

Purpose: RPE65 is the visual cycle retinol isomerase and missensemutations in its gene cause severe retinal dystrophies in man, due tolack of chromophore. While the rate of opsin regeneration in mouse isslower than in man, the methionine (M) variant of mouse RPE65 residue450 (normally L) is associated with additionally lowered lightsensitivity and with resistance to light damage in C57Bl/6 mice,consistent with lowered total activity. We wished to determine how thisvariant affects RPE65 and if it is modulated by other rodent-specificvariations.Methods: Site-directed mutagenesis was used to make variantconstructs in mouse and dog RPE65, which were tested for isomeraseactivity by transient transfection in 293-F cells.Results: The isomerase activity of dog RPE65 is slightly higher thanmouse. Replacing L at aa450 with M reduces total activity of dog toapproximately 70% and mouse to approximately 45% of respective wild typeRPE65, and also reduces protein levels of both variants. Replacing K ataa446 in mouse with R, as in other species, reduces total activity inmouse RPE65, whereas the converse case, changing dog aa446 from R to K,increases activity. Exchanges of residues at aa457 and 459 had littleoverall effect. Human variants at two of these positions, L450R andT457N, had disparate effects, abolishing and augmenting activity,respectively.Conclusions: Wildtype dog RPE65 is more active than wildtype mouseRPE65, perhaps partially explaining the slower regeneration rate in themouse. The effect of Met at aa450 is more severe in mouse RPE65 than indog. The effects of variation at residues 446 (K or R) modulatevariation at aa450. The sensitivity of aa450 to change is underscored bythe abolition of activity in the pathogenic human L450R mutation. Theseresults suggest that subtle species-specific residue changes may beinvolved in "tuning" of RPE65 activity to required evolutionarycriteria.