Glycosylation and palmitoylation are not required for the formation of the X-linked cone opsin visual pigments.
OBJECTIVE
This study was designed to test whether palmitoylation and glycosylation are required for the formation of the green opsin visual pigment.


METHODS
Stable cell lines were established by transfecting EBNA-293 cells with a pMEP4ss recombinant plasmid containing wild-type bovine rhodopsin or wild-type or mutant (N32S) green opsin cDNA molecules that included a tag for the eight amino acid residues located at the C-terminus of rhodopsin. The opsins were induced by addition of CdCl2 into the medium and then reconstituted with 11-cis-retinal. The reconstituted opsins were purified by immunoaffinity chromatography, then analyzed by difference spectra, and by binding 35S-GTP in the presence of bovine transducin. Non-reconstituted opsins were analyzed by Western blotting and by pulse-labeling with 3H-palmitic acid followed by immunoprecipitation.


RESULTS
Elimination of glycosylation by mutagenesis of the N-linked glycosylation site did not impair the ability of the resulting cone opsin to absorb light at the appropriate wavelength nor to activate transducin. Furthermore, as judged by pulse-labeling with 3H-palmitic acid and immunoprecipitation and by gas chromatography-mass spectroscopy, the wild type green opsin differs from rhodopsin by not being palmitoylated.


CONCLUSIONS
Glycosylation and palmitoylation are not required for the formation of cone opsin visual pigments. For the previously described green opsin C203R mutation, disruption of folding and transport, rather than altered glycosylation is sufficient to explain the associated color vision deficiency.
Ostrer
H
H
Human Genetics Program, Department of Pediatrics, New York University Medical Center, New York, NY 10016, USA. ostreh01@mcrcr6.med.nyu.edu
Pullarkat
R K
RK
Kazmi
M A
MA
D015153:Blotting, Western; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D016296:Mutagenesis; D019308:Palmitic Acid / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin / Q000032:analysis / Q000378:metabolism; D017299:Rod Opsins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015293:Transducin / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; C095854:middle-wavelength opsin; D019308:Palmitic Acid; D012243:Rhodopsin; D015293:Transducin