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Figure 1. N termini of red/green cone opsins. A, N termini of human visual opsins. Red indicates Ser/Thr residues that potentially could be O-glycosylated. Green indicates N-glycosylated Asn residues. The epitope of the 7G8 mAB is boxed. B, NetOGlyc 4.0 score and ISOGlyP (beta2.1) O-glycosylation EVP results for human green cone opsin (NetOGly score values >0.5 and ISOGlyP EVP values >1.0 suggest possible glycosylation. See Table. S1 for the full ISOGlyP output.). Residues in red belong to the epitope of 7G8 mAB. The input in both cases was the full hOPSG sequence. C, conserved Ser/Thr residues at the N termini of vertebrate red/green cone opsins (highlighted in blue). The epitope of 7G8 mAB is in a black box. Human residues Ser22, Thr23, Ser25, and Ser26 are marked with asterisks. N-Glycosylated Asn residues are highlighted in green.
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Figure 2. O-Glycosylation of red/green cone opsins in retina of Euarchontoglires. 7G8 mAB immunoblots of retinal detergent extracts treated with different glycosidases. The retina tissues were obtained from macaque (A), human (B) and 13LGS eyes (C). The lanes labeled with letters correspond to the enzymatic treatment of the samples: untreated (Un), treated with O-glycosidase (O), PNGase F (P), and/or neuraminidase (N).
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Figure 3. O-Deglycosylation of human red/green cone opsins on PVDF membrane. hOPSR/hOPSG from a human retinal detergent extract was immunoprecipitated with nanobody Nb-E9. The sample was treated with different glycosidases, subjected to SDS-PAGE, and transferred simultaneously to two PVDF membranes. A, top immunoblot of red/green cone opsins incubated with 7G8 mAB. The lanes labeled with letters correspond to the enzymatic treatment of the samples: untreated (Un), treated with O-glycosidase (O), and/or PNGase F (P). B, membrane from A was washed, incubated with O-glycosidase, re-incubated with 7G8 mAB, and developed again. C, immunoblot of bottom membrane incubated with anti-C terminus polyclonal antibody AB5405.
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Figure 4. Jacalin binds to OPSR/OPSG of different vertebrates in immunoblots. A–C, hOPSR/hOPSG purified from human retina was treated with different glycosidases, subjected to SDS-PAGE, and transferred simultaneously to three PVDF membranes. A, top membrane, incubated with jacalin; B, middle membrane, incubated with anti-C-terminal antibody AB5405; C, bottom membrane, incubated with 7G8 mAB. D–G, Jacalin blots and AB5405 immunoblots of chicken COS/ROS samples (D), and of retinal extracts from X. laevis (E), 13LGS (F), and pig (G). The lanes labeled with letters correspond to the enzymatic treatment of the samples: untreated (Un), treated with PNGase F (P), neuraminidase (N), β(1–3)-galactosidase (B) and/or O-glycosidase (O).
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Figure 5. Purification of bOPSR by cation-exchange chromatography. An LMNG-solubilized bovine COS/ROS sample was loaded onto a column equilibrated with 10 mm HEPES, pH 7. 4, 10 mm NaCl, 0.2 mm LMNG, and it was eluted with a NaCl gradient (0.01–2 m) in 12 fractions, followed by further elution at 2 m NaCl. The samples and fractions were subjected to SDS-PAGE; the proteins in the gel were transferred simultaneously to three PVDF membranes, and the membranes were incubated for blot analysis with anti-hOPSR/hOPSG polyclonal antibody AB5405, jacalin, or anti-rhodopsin 1D4 mAB. FT, flow-through; En, elution fraction n.
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Figure 6. Identification of O-glycosylated peptide by MS/MS analysis. Fragmentation of a precursor ion (m/z 1124. 50603+) using high-energy collisional dissociation during MS/MS analysis yielded a series of b and y ions that accurately identified the peptide as 10LAGGQPQANFEESTQGSIFTYTNSNSTR37, in which the 13th residue (Ser22) was O-glycosylated and the 25th residue (Asn34) was deamidated. Matched ions are shown in red; internal fragmentation ions are not labeled in the spectrum.
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Figure 7. Regeneration with 11-cis-retinal of hOPSG deletion constructs expressed in Sf9 cells. In all constructs, intracellular loop 3 was replaced by T4L, and the 1D4 mAB epitope was fused at the C terminus. A, N-terminal sequence of the four constructs used in this figure. B, Coomassie-stained SDS-PAGE of the four immunopurified OPSG/T4L constructs: full-length OPSG/T4L (FL), Δ16N OPSG/T4L (Δ16), Δ27N OPSG/T4L (Δ27), and Δ43N OPSG/T4L (Δ43). The relative band intensities (normalized to that of Δ16N) were 0.94, 1, 0.62, and 0.66 for FL, Δ16N, Δ27N, and Δ43N OPSG/T4L, respectively. The three samples labeled with an asterisk had been incubated with PNGase F. C, absorbance spectra of the four purified OPSG/T4L constructs. The absorbance was normalized to account for the molecular mass of the constructs and their respective electrophoretic band intensity in B. The discontinuous line marks the A530 nm.
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Figure 8. Human green cone opsin expressed in mammalian and insect cells is partially N- and O-glycosylated. A–C, immunoblots of purified hOPSG expressed in N-glycosylation–deficient HEK293SGnTI− cells, incubated with anti-N terminus 7G8 mAB (A), anti-C terminus 1D4 mAB (B), or jacalin (C). D–F, immunoblots of purified hOPSG expressed in Sf9 cells, incubated with anti-N terminus 7G8 mAB (D), anti-C terminus 1D4 mAB (E), or jacalin (F). The bands marked with an asterisk are not detected by 7G8 mAB. The samples were untreated (Un) or treated with PNGase F (P).
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Figure S-1: Determining the epitope of the 7G8 mAB. (A) Immunoblots of three deletion variants of hOPSG fused to T4L (OPSG/T4L), using 7G8 mAB (left blot) or 1D4 mAB (right blot) as antibodies. The three constructs, that had been purified from Sf9 insect cells and N-deglycosylated with PNGase-F, are full length OPSG/T4L (FL), Δ16N OPSG/T4L (Δ16), and Δ27N OPSG/T4L (Δ27). The N-terminal sequence of these variants is described in Fig. 7A. (B) Full length OPSG/T4L purified from Sf9 insect cells was treated with PNGase F, mixed with a MW marker, run in five lanes of an SDS-polyacrylamide gel and transferred to a PVDF membrane. The lanes were cut to fit into 24-well plates and then, each membrane fragment was incubated with 7G8 mAB pre-incubated with one of five competing nonapeptides (a-e). Only peptides b and c were able to prevent 7G8 mAB’s binding to OPSG/T4L. The immunoblots in panels A and B were developed with an alkaline phosphatase reaction. (C) The N-terminal sequence of red/green human cone opsins is aligned with the competing peptides used in panel B. 7G8 mAB’s epitope is boxed.
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Figure S-2: Presence of N-glycans on Asn34 hinders binding of 7G8 mAB to its epitope. A 13LGS retinal detergent extract was incubated with O-glycosidase alone (lanes O) or with a mixture of O-glycosidase plus PNGase F (lanes PO). Duplicates of both samples were subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane then was cut through the central MW lane, and both sides were incubated separately with 7G8-AP mAB. In addition to antibody, the membrane on the right side also was incubated with PNGase F. After 1 h, the membranes were washed and developed with AP reagents. The band intensity ratio between lane PO and lane O is 2.3 for the left panel and 1.4 for the right panel.
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Figure S-3: In-blot O-deglycosylation of OPSR/OPSG from 13LGS (A,B), pig (D,E) and cows (G,H).
(A) Immunoblot of 13LGS retinal extract from Fig. 2C. (B) The developed membrane from panel A was washed, incubated with O-glycosidase, re-incubated with 7G8 mAB and developed again with an alkaline phosphatase reaction. (C) Immunoblot of the same 13LGS retinal samples, incubated with anti-C-terminus polyclonal antibody AB5405.
(D) Immunoblot of porcine retinal extract. Negative control in lane labeled as X contains only O-glycosidase and PNGase F. (E) The developed membrane from panel D was washed, incubated with O-glycosidase, re-incubated with 7G8 mAB and developed again with an alkaline phosphatase reaction. (F) Immunoblot of the same porcine retinal samples, incubated with anti-C-terminus polyclonal antibody AB5405.
(G) Immunoblot of bovine OPSR, semi-purified with cation-exchange chromatography. (H) The developed membrane from panel G was washed, incubated with O-glycosidase plus neuraminidase, re-incubated with 7G8 mAB and developed again with an alkaline phosphatase reaction. (I) The developed membrane from panel H was washed, incubated with anti-C-terminus polyclonal antibody AB5405 and re-developed. The red boxes correspond to the bands detected in panel H.
The lanes are labeled with the enzymatic treatment to which each sample was subjected: untreated (Un), O-glycosidase (O), neuraminidase (N) and/or PNGase F (P). The black boxes in panels C, F and I correspond to PNGase F, which cross-reacts with antibody AB5405.
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Figure S-4: O-glycosylation of green cone opsin from mouse retina. OPSG from a mouse retina detergent extract was immunoprecipitated with nanobody Nb-E9. (A) Immunoblot of mouse OPSG incubated with anti-C-terminus polyclonal antibody AB5405. The lanes are labeled with letters corresponding to the enzymatic treatment of the samples: untreated (Un), treated with O-glycosidase (O), neuraminidase (N) and/or PNGase F (P). (B) Band intensity plot of the four lanes showing a small mobility shift of the OPSG bands corresponding to samples treated with O-glycosidase plus neuraminidase.
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Figure S-5: Purification of bovine OPSR with immobilized jacalin. Fractions (from Fig. 5) containing bOPSR were combined, dialyzed and purified with immobilized jacalin. (Left panel) Silver stained samples after cation exchange column (Load) contained mainly rhodopsin, whereas the eluted fraction (Elut) contained mainly bOPSR. FT: flow-through. (Right panel) Same samples loaded in the left panel were subjected to immunoblot incubated with anti-C-terminus antibody AB5405 to confirm the identity of bOPSR. The samples for the immunoblot were normalized for sample volume.
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Figure S-6: Human green opsin expressed in mammalian cells is heterogeneously glycosylated. (A) Immunoblot of purified hOPSG (C-terminally tagged with 1D4 mAB’s epitope) expressed in N-glycosylation-deficient HEK293SGnTI- cells (treated with different glycosidases), incubated with anti-C-terminus 1D4 mAB. The bands marked with an asterisk are not detected by 7G8 mAB (see Fig. 8). (B and C) Immunoblots of purified hOPSG expressed in HEK293 cells incubated with anti-N-terminus 7G8 mAB (B) or anti-C-terminus 1D4 mAB (C). The lanes are labeled with letters corresponding to the enzymatic treatment of the samples: untreated (Un), treated with O-glycosidase (O), PNGase F (P) and/or neuraminidase (N).
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