N-linked deglycosylated melanopsin retains its responsiveness to light.
Melanopsin is an opsin expressed in the plasma membrane of retinal ganglion cells that mainly project to the circadian clock and thus is important for nonvisual responses to light. Rat melanopsin contains two potential sites (Asn31 and Asn35) for N-linked glycosylation in the N-terminal extracellular part. To investigate if melanopsin is N-linked glycosylated and whether N-bound glycans influence the response of melanopsin to light as evidenced by Fos mRNA induction, we transfected PC12 cells to stably express rat wild-type melanopsin or mutant melanopsin lacking both N-linked glycosylation sites. Immunoblotting for membrane-bound melanopsin from the PC12 cells transfected to express wild-type melanopsin disclosed two immunoreactive bands of 62 and 49 kDa. Removal of N-linked glycosylation by tunicamycin or PNGase F changed the 62 kDa band to a 55 kDa band, while the 49 kDa band corresponding to the core melanopsin protein was unaffected. Likewise, mutation of the two extracellular N-linked glycosylation sites gave a melanopsin size comparable to that of PNGase F or tunicamycin treatment (55 kDa). Further in vitro O-linked deglycosylation of wild-type or mutant melanopsin with O-glycosidase and neuraminidase converted the 55 kDa band to a 49 kDa band. Finally, neither in vivo N-linked deglycosylation nor mutations of the two N-linked glycosylation sites significantly affected melanopsin function measured by Fos induction after light stimulation. In conclusion, we have shown that heterologously expressed rat melanopsin is both N-linked and O-linked glycosylated and that N-linked glycosylation is not crucial for the melanopsin response to light.
Department of Clinical Biochemistry, Bispebjerg Hospital, Faculty of Health Sciences, University of Copenhagen, DK-2400 Copenhagen NV, Denmark. jf01@bbh.regionh.dk
Jan
J
Fahrenkrug
Birgitte
B
Falktoft
Birgitte
B
Georg
Lene
L
Rask
D000818:Animals; D002478:Cells, Cultured; D006031:Glycosylation; D008027:Light; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D000099210:Melanopsin
D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D017299:Rod Opsins; D014415:Tunicamycin; D000099210:Melanopsin