Abstract
Purpose :
Human vision relies on four visual opsins: blue, green, and red cone opsins (OPSB, OPSG and OPSR) for color vision, and rhodopsin for low-light conditions. All visual opsins of vertebrates are N-glycosylated at one or two N-terminal Asn residues. N-glycosylation of rhodopsin is necessary for its incorporation into the rod outer segments (OS), for the morphogenesis and maintenance of the OS, and for proper biochemical function. In addition to a single N-glycosylation site, our laboratory has previously demonstrated that OPSG and OPSR are also O-glycosylated at their N-terminus with 100% occupancy in tetrapod homologs, from amphibians to humans. The long-term goal of this work is to investigate the role of O-glycans of OPSG and OPSR in cone photoreceptor formation, maintenance, and function.
Methods :
We generated knock-in mice with point mutations on three Ser/Thr residues that could potentially be O-glycosylated in mOPSG (also known as M-opsin). M-opsin expression was quantified by immunoblot, and cone cells were visualized by immunocytochemistry. Electroretinogram (ERG) recordings measured differences in response to light stimuli. O-glycosylated opsin was identified by lectin- and immuno-blots, while mass spectrometry identified the O-glycosylation sites on w.t. M-opsin. Purification of M-opsin was achieved by cation exchange chromatography followed by lectin chromatography.
Results :
Quantification by immunoblot revealed a progressive loss of M-opsin in knock-in mice, with an expression level of 28.7% (±6.5%) compared to w.t. mice at 6.3 months of age, with a concomitant reduction in the number of M-cones. However, photopic and scotopic ERG showed no significant difference between 6-mo-old w.t. and knock-in mice. Mass spectrometry identified two O-glycosylation sites on w.t. M-opsin at Ser17 and Thr18. We also confirmed by lectin- and immuno-blots that green/red cone opsins of fish are O-glycosylated.
Conclusions :
The results of this work reinforce the idea that N- and O-glycosylation on visual opsins is necessary for maintaining the viability of photoreceptors in vertebrates. However, the precise mechanism of action of N- and O- glycans remains unknown. One possibility is that N-terminal glycans of visual opsins interact to produce and maintain the flatness of rod and cone OS discs. The O-glycosylation site of OPSG/OPSR being strictly conserved in all vertebrates suggests an important role in vision.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.