Evaluation of tyrosinase and tyrosinase related proteins in the rpe/choroid of vitiligo (mi ^vit/mi ^vit) mice

Purpose: The basic-helix-loop-helix zipper protein, microphthalmia (mi), regulates the transcription of the gene encoding tyrosinase, the rate-limiting enzyme in melanin biosynthesis, by binding the DNA sequence: CATGTG. This binding site is present also in the genes encoding two tyrosinase related proteins, TRP1 and TRP2. The purpose of this study was to determine whether there was a difference in the levels of these enzymes in the RPE/choroid of the vitiligo (mt ^vit/mt ^vit) mouse, in which a mutation of the m gene alters RPE pigmentation and function leading to slow progressive loss of photoreceptor cells. Methods: RPE/choroid was dissected from eyes of vitiligo and C57B1/6 control mice at postnatal ages 2, 4, 7, 10, 14, 21, & 42 days. Extracts of pooled tissues (4 eyes/assay) were subjected to electrophoresis and immunoblotting. The levels of tyrosinase, TRP1 and TRP2 were determined densitometrically following immunodetection with rabbit antipeptide antisera. In addition, the tyrosine hydroxylase activity of tyrosine was assayed radiometrically. Results: Levels of TRP1 were 3 -7 fold greater in control RPE/choroid compared with mutants. This marked difference in protein level was observed at the earliest age examined (P2) and persisted throughout the first two weeks. Tyrosinase levels in mutants were similar to controls at P2 and P4, but were reduced at P10 and beyond. Tyrosinase activity was also diminished in mutants by P10. Levels of TRP2 were similar between mutants and controls, although the typical decrease seen in controls after P14 was attenuated in the mutant mice. Condusigns: There is a significant reduction in the level of TRP1 in the RPE/choroid of the m ^vit/mt ^vit mouse. The data suggest that transcription of the TRPt gene is extremely dependent upon functional microphthalmia and provides evidence that m can regulate the transcription of this gene as well as tyrosinase. Supported by NIH (EY09682, EY10223) & Research to Prevent Blindness.