Mass spectrometric analysis of rhodopsin from light damaged rats.
OBJECTIVE
It is well established that the retina is damaged by intense visible light. Rhodopsin has been proposed to be involved in this process. We therefore 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 ultracentrifugation, their membranes treated with urea, then washed with Tris buffer. The rhodopsin preparations were then reduced, pyridylethylated, delipidated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion trap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identification of glycosylation.


RESULTS
The rat rhodopsin protein was mapped with the exception of two single amino acid fragments. The reported sequence was confirmed with the exception of the controversial T/S320 residue, which was found to be a threonine. 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 adaptation completely reversed the phosphorylation in all light damaged animals. Other posttranslational modifications were as previously reported.


CONCLUSIONS
Our results indicate that intense visible light exposure of rats does not lead to oxidative or other primary structural alterations in the rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous animals and that residue 320 in both normal and mutated rhodopsins is threonine, not serine.
Ablonczy
Z
Z
Medical University of South Carolina, Charleston, SC 29425, USA. ablonczy@musc.edu
Knapp
D R
DR
Darrow
R
R
Organisciak
D T
DT
Crouch
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D008027:Light; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D011922:Rats, Mutant Strains; D017207:Rats, Sprague-Dawley; D012162:Retinal Degeneration / Q000503:physiopathology*; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D012243:Rhodopsin