Title : N-linked deglycosylated
melanopsin retains its responsiveness to light
Abstract :
- 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