Increasing the Stability of Recombinant Human Green Cone Pigment

Abstract

Three types of cone cells exist in the human retina, each containing a different pigment responsible for the initial step of phototransduction. These pigments are distinguished by their specific absorbance maxima: 425 nm (blue), 530 nm (green), and 560 nm (red). Each pigment contains a common chromophore, 11-cis-retinal covalently bound to an opsin protein via a Schiff base. The 11-cis-retinal protonated Schiff base has an absorbance maxima at 440 nm in methanol. Unfortunately, the chemistry that allows the same chromophore to interact with different opsin proteins to tune the absorbance of the resulting pigments to distinct λmax values is poorly understood. Rhodopsin is the only pigment with a native structure determined at high resolution. Homology models for cone pigments have been generated, but experimentally determined structures are needed for a precise understanding of spectral tuning. The principal obstacle to solving the structures of cone pigments has been their innate instability in recombinant constructs. By inserting five different thermostabilizing proteins (BRIL, T4L, PGS, RUB, and FLAV) into the recombinant green opsin sequence, constructs were created that were up to 9-fold more stable than WT. Using cellular retinaldehyde-binding protein (CRALBP), we developed a quick means of assessing the stability of the green pigment. CRALBP testing also confirmed an additional 48-fold increase in pigment stability when varying the detergent used. These results suggest an efficient protocol for routine purification and stabilization of cone pigments that could be used for high-resolution determination of their structures, as well as for other studies.

Human red and green cone opsins are O-glycosylated at an N-terminal Ser/Thr–rich domain conserved in vertebrates

“…Protein database searching of MS data unambiguously identified Ser 22 as the O-glycosylated residue and Asn 34 as the N-glycosylation site (Fig. 6).…”

“…However, due to lack of specific fragment ions, the actual site cannot be unambiguously determined. In addition to Ser 22 , we suspect that Thr 23 and/or Ser 26 may represent additional O-glycosylation sites in bOPSR.…”

“…6). MS/MS analysis of a parent ion (m/z 1124.5060 3ϩ ) yielded a series of b and y ions accurately identifying the peptide as 10 LAGGQPQANFEESTQGSIFTYT-NSNSTR 37 , in which the 13th residue (Ser 22 ) was modified with a Hex-HexNAc O-glycan and the 25th residue (Asn 34 ) was deamidated, representing an O-glycosylated peptide with N-glycan removed by PNGase F cleavage (Fig. 6 and Table S2).…”

“…Next, we confirmed the presence of O-glycans on OPSR and OPSG from several vertebrate species, including mammals, birds, and amphibians. Finally, the analysis of bovine OPSR by MS identified an O-glycan on Ser 22 , a residue that is semi-conserved (Ser or Thr) among vertebrate OPSR and OPSG. These results suggest that O-glycosylation is a fundamental feature of red and green cone opsins, which may be relevant to their function or to cone cell development, and that differences in this post-translational modification also could contribute to the different morphologies of rod and cone photoreceptors.…”

“…In this investigation, we demonstrate that OPSR and OPSG in the retina of humans and several other vertebrates are O-glycosylated in a relatively homogeneous manner and with a full occupancy. Mass spectrometry (MS) analysis of bovine OPSR identified Ser 22 as the O-glycosylation site, a residue that is semi-conserved (Ser or Thr) among vertebrates, suggesting an important role of O-glycosylation, perhaps in the unique features of green and red cone photoreceptors. In addition, our findings show that OPSG heterologously expressed in mammalian and insect cells is O-glycosylated but has a glycan pattern and/or occupancy that is different from that observed in retina, emphasizing that heterologous expression systems are often unable to mimic the PTMs observed in native tissues.…”