Mass spectrometric analysis of rhodopsin from light damaged rats

PURPOSE: It is well established that the retina is damaged by intense visib le light. Rhodopsin has been proposed to be involved in this process. We th erefore undertook to examine whether rhodopsin isolated from light damaged animals is structurally altered at the molecular level. METHODS: Dark reared and dim cyclic light reared 8 week old Sprague-Dawley rats were exposed to intense visible light and sacrificed immediately or 24 h after exposure together with unexposed control animals reared under the same conditions. Rod outer segments were isolated by sucrose gradient ultra centrifugation, their membranes treated with urea, then washed with Tris bu ffer. The rhodopsin preparations were then reduced, pyridylethylated, delip idated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion tr ap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identificatio n of glycosylation. RESULTS: The rat rhodopsin protein was mapped with the exception of two sin gle amino acid fragments. The reported sequence was confirmed with the exce ption of the controversial T/S320 residue, which was found to be a threonin e. Mono-, di-, tri-, and tetraphosphorylated forms of rhodopsin were found in the light damaged animals. Three sites of phosphorylation were confirmed with MS/MS (tandem mass spectral) data. Single or double phosphorylations were found among these three sites, in various combinations. Dark adaptatio n completely reversed the phosphorylation in all light damaged animals. Oth er posttranslational modifications were as previously reported. CONCLUSIONS: Our results indicate that intense visible light exposure of ra ts does not lead to oxidative or other primary structural alterations in th e rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous ani mals and that residue 320 in both normal and mutated rhodopsins is threonin e, not serine.