title: Effect of Leu/Met variation at residue 450 on isomerase activity and protein expression of RPE65 and its modulation by variation at other residues.
abstract: OBJECTIVE
  RPE65 is the visual cycle retinol isomerase and missense mutations in its gene cause severe retinal dystrophies in man, due to lack of chromophore. While the rate of opsin regeneration in mouse is slower than in man, the methionine (M) variant of mouse RPE65 residue 450 (normally L) is associated with additionally lowered light sensitivity and with resistance to light damage in C57Bl/6 mice, consistent with lowered total activity. We wished to determine how this variant affects RPE65 and if it is modulated by other rodent-specific variations.
  
  
  METHODS
  Site-directed mutagenesis was used to make variant constructs in mouse and dog RPE65, which were tested for isomerase activity by transient transfection in 293-F cells.
  
  
  RESULTS
  The isomerase activity of dog RPE65 is slightly higher than mouse. Replacing L at aa450 with M reduces total activity of dog to approximately 70% and mouse to approximately 45% of respective wild type RPE65, and also reduces protein levels of both variants. Replacing K at aa446 in mouse with R, as in other species, reduces total activity in mouse RPE65, whereas the converse case, changing dog aa446 from R to K, increases activity. Exchanges of residues at aa457 and 459 had little overall effect. Human variants at two of these positions, L450R and T457N, had disparate effects, abolishing and augmenting activity, respectively.
  
  
  CONCLUSIONS
  Wildtype dog RPE65 is more active than wildtype mouse RPE65, perhaps partially explaining the slower regeneration rate in the mouse. The effect of Met at aa450 is more severe in mouse RPE65 than in dog. The effects of variation at residues 446 (K or R) modulate variation at aa450. The sensitivity of aa450 to change is underscored by the abolition of activity in the pathogenic human L450R mutation. These results suggest that subtle species-specific residue changes may be involved in "tuning" of RPE65 activity to required evolutionary criteria.
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, NIH, Bethesda, MD 20892-0706, USA. redmond@helix.nih.gov
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
authors.forename: Charles H
authors.initials: CH
authors.lastname: Weber
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.forename: Shirley
authors.initials: S
authors.lastname: Yu
authors.forename: Susan
authors.initials: S
authors.lastname: Gentleman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D007930:Leucine; D008239:Lysine; D008715:Methionine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology / Q000235:genetics; D014162:Transfection; D019745:cis-trans-Isomerases / Q000235:genetics* / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
chemical_list: D001120:Arginine; D002352:Carrier Proteins; D005136:Eye Proteins; D007930:Leucine; D008239:Lysine; D008715:Methionine; D010105:Oxygenases; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C524249:apocarotenal oxygenase, Synechocystis