Mass spectrometric analysis of rhodopsin from light damaged rats

[摘要] Purpose: It is well established that the retina is damaged byintense visible light. Rhodopsin has been proposed to be involved inthis process. We therefore undertook to examine whether rhodopsinisolated from light damaged animals is structurally altered at themolecular level.Methods: Dark reared and dim cyclic light reared 8 week oldSprague-Dawley rats were exposed to intense visible light and sacrificedimmediately or 24 h after exposure together with unexposed controlanimals reared under the same conditions. Rod outer segments wereisolated by sucrose gradient ultracentrifugation, their membranestreated with urea, then washed with Tris buffer. The rhodopsinpreparations were then reduced, pyridylethylated, delipidated, andcleaved with CNBr. Reversed phase HPLC was used to separate thefragments, and the effluent was analyzed online with a Finnigan LCQ iontrap mass spectrometer. C-terminal phosphorylation was investigatedfollowing Asp-N cleavage. MALDI-TOF mass spectrometry was used for theidentification of glycosylation.Results: The rat rhodopsin protein was mapped with the exception oftwo single amino acid fragments. The reported sequence was confirmedwith the exception of the controversial T/S320 residue, which was foundto be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms ofrhodopsin were found in the light damaged animals. Three sites ofphosphorylation were confirmed with MS/MS (tandem mass spectral) data.Single or double phosphorylations were found among these three sites, invarious combinations. Dark adaptation completely reversed thephosphorylation in all light damaged animals. Other posttranslationalmodifications were as previously reported.Conclusions: Our results indicate that intense visible lightexposure of rats does not lead to oxidative or other primary structuralalterations in the rhodopsin protein of rod outer segments. We alsoreport that the mutated rhodopsin (P23H) is present in rat rod outersegments from heterozygous animals and that residue 320 in both normaland mutated rhodopsins is threonine, not serine.

[学科分类] 生物化学/生物物理