Preparation of photoreceptor membrane

All preparations were made under dim-red light. Bovine ROS was prepared as described by Smith et al., (1975). Disk membrane was isolated by Ficoll floatation methods (Smith et al., 1975). In order to obtain sealed intact disks, 3 ml of disks suspension containing 15 mg of rhodopsin suspended in TBS (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2 mM MgCl₂) with 1 mM CaCl₂ and MnCl₂ was mixed with an equal volume of ConA-sepharose (Pharmacia) so that broken and inside-out disks bind to the resin. The mixture was gently agitated overnight at 4°C and centrifuged to separate unbound disks. The supernatant (termed sealed intact disk) was stored at 0°C until use. A portion of sealed intact disks was frozen-thawed for 5 times between liquid nitrogen and room temperatures to obtain unsealed disks (Balkema and Draeger, 1985). Porcine ROS was prepared by the same procedure as bovine. Photoreceptor membranes from octopus and crayfish retina were prepared according to Shichida et al. (1978) and Hamacher and Stieve (1984), respectively. Rhodopsin content in each membrane preparation was estimated from a difference absorbance before and after bleaching in the presence of hydroxylamine. Molecular weights and the extinction coefficients of the above animal rhodopsins are assumed to have similar values to bovine (MW=41399, ε500 nm = 42,000) (Shichi, 1983).

Monoclonal Antibody Binding Assay

Rhodopsin-specific mAb, Rh29, was purified from ascites fluid by protein A column chromatography (Bio-Rad). Enzyme-linked immunosorbent assay (ELISA) was employed for the detection of the antigenic fragment and for the competitive binding study. Bleached ROS was used as an immobilized antigen for the competitive binding assay after frozen-thawing five times to expose the antigenic determinant for Rh29. Microtiter plate wells (Nunc) were coated by means of incubating 50 μl of ROS (3 μg rhodopsin/ml) for 2 hr at 37°C and subsequently 100 μl of blocking solution (TBS containing 3% BSA (bovine serum albmin), 1% normal goat serum) for 30 min at 37°C. A competitor serially diluted with TBS was mixed with Rh29 (final concentration is 65 ng/ml which gave 50–60% of full binding) and preincubated for 1 hr at 37°C. Each mixture of competitor and antibody (50 μl) was placed in wells and incubated for 1 hr at 37°C. After extensive wash with Tw-TBS (TBS containing 0.05% of Tween20) wells were treated with HRPO-conjugated second antibodies (DAKO) for 1 hr at 37°C, rinsed with extensive Tw-TBS and reacted with o-phenylenediamine for 30 min. The reation was stopped by adding 100 μl of 2 N H₂SO₄ and the absorbance at 492 nm was measured.

When disks solubilized with ammonyx-LO or digitonin (2%) were used as competitors, detergent concentration was decreased to 0.05% in a competitor-antibody mixture to avoid the decrease in binding ability of the antibody. When column fractions were tested for their antigenicity, each 20 μl of elution was dried in microtiter plate well (Sumitomo Bakelite) at 50°C and treated with Rh29 (1–2 μg/ml) and second antibodies, as mentioned above.

ConA-Binding Assay

ConA has affinity for mannose or glucose and also for sugar moiety of rhodopsin or HRPO (horse radish peroxidase). Advantage that ConA forms tetramer at neutral pH was taken for the detection of the oligosaccharides of rhodopsin or its glycopeptide. The assay (termed as ConA-binding assay, hereafter) includes essentially the same steps in ELISA described above. Blocking solution used in ConA-binding assay was free from ConA-reactive fractions by passing through ConA-sepharose column. Instead of mAb and second antibodies in ELISA, the wells were treated with ConA and HRPO dissolved in TBS containing 1 mM CaCl₂ and MnCl₂.

Immunoblotting and ConA-blotting

SDS-PAGE was carried out by the discontinuous slab gels (Laemmli, 1970). Routinely, 2–10 μl of disks or photoreceptor membranes (1 μg rhodopsin/μl) was loaded on 15% gel for proteolysis study or 10% gel for experiments of cross-reaction. Proteins were stained by Coomassie blue (CB), or by amido black after transferred to nitrocellulose filter. Protein blots were also sequentially treated with mAb and second antibodies (immunoblotting) or with ConA and HRPO (ConA-blotting) like mAb- or ConA-binding assay, respectively. Both blots were developed by diaminobenzidine.

Proteolytic Digestion of Intact Disks or Solubilized Disks

Chymotrypsin and TPCK-trypsin were purchased from Sigma. Papain was purchased from Wako. Unbleached intact disks freshly prepared (1 mg rhodopsin/ml) were digested with 5% (w/w) chymotrypsin (37°C, 24 hr) or 5% (w/w) papain (37°C, 6 hr) in a buffer of 50 mM Tris-acetate (pH 8.0), 2 mM CaCl₂ or 50 mM sodium phosphate (pH 7.0), 5 mM cysteine, 2 mM EDTA, respectively. Digestion was stopped by washing the disks three times.

To cleave N-terminal segment of opsin, bleached disks were solubilized with 2% (w/v) digitonin (Wako). After detergent concentration was decreased to 0.5%, disks in TBS containing 1 mM CaCl₂ were treated with each 1% (w/w) of TPCK-trypsin or chymotrypsin for 6 hr at 37°C. Reaction was stopped by the addition of DFP. The obtained peptide mixture was subjected to the competitive binding assay. Protection and deprotection of arginine residues with 1,2-cyclohexanedione were according to the method of Patthy & Smith (1975). Protected, deprotected opsin and control sample, which was treated along the same procedure without the addition of 1,2-cyclohexanedione, were dialyzed against 0.1 M sodium borate (pH 8.0) and digested with TPCK-trypsin (1%, w/w).

Isolation of mAb-Specific Peptide

Antigenic peptides were separated from protease-digested and CNBr-cleaved rhodopsin according to Brett and Findlay (1983). ROS (20 mg of rhodopsin) was digested with 5% (w/w) thermolysin (Sigma) at 37°C for 24 hr. Washed material was delipidated, carboxymethylated and chromatographed on Sephadex LH-60. F1 (large fragment of thermolysin-cleaved rhodopsin) was dried and then cleaved with CNBr (300 folds excess of methionine) in 70% trifluoroacetic acid for 24 hr at room temperature. Reaction mixture was separated by Sephadex LH-60 and then G-50 column chromatography. Amino acid composition of each peptide was analyzed by Hitachi 835S Amino Acid Analyzer.

Rh29 detected light-induced structural change of rhodopsin.

The striking difference in the antigenicity for Rh29 was observed between bleached and unbleached disks (Fig. 1). About 100-folds amount of unbleached disks were necessary for the same degree of binding inhibition with bleached disks (A in Fig. 1). Frozen-thawed disks also showed such a large difference in antigenicity (not shown).

Fig. 1

Effect of photo-bleaching on the binding of Rh29 to membrane-bound rhodopsin and solubilized rhodopsin. Competitive binding assay was made to examine the binding character of intact disks (A), disks solubilized by digitonin (B) and by ammonyx-LO (C). Open and closed symbols represent bleached and unbleached state of rhodopsin, respectively. Each point is a mean value of 3–4 wells.

Solubilization with strong detergent increased antigenicity of rhodopsin for Rh29.

Under digitonin solubilized conditions bleached rhodopsin was 20-fold more antigenic than unbleached one (B in Fig. 1). Taking it into account that digitonin is relatively mild detergent which could retain some authentic activities of the membrane-associated rhodopsin such as spectroscopic character and ability of regeneration from opsin and retinal (Hubbard, 1952), the results on both intact and digitonin-solubilized disks indicate that the antigenic region should be exposed to surrounding medium during photo-bleaching. In contrast, opsin and rhodopsin solubilized by ammonyx-LO showed the same degree of inhibition for the binding of Rh29 (C in Fig. 1). This would be accounted for that amm-onyx-LO effectively removed lipids from membrane-associated rhodopsin and that the antigenic determinant would be naked to be easily bound by Rh29.

Which side of disks does the antibody bind to?

It was tested which side of disks the antibody bound to. Fig. 2 shows the binding ability of Rh29 for bleached disks with or without freeze-thawing. Frozen-thawed disks were found to compete with the immobilized antigen at 33-times lower concentration (at half maximal inhibition) than sealed intact disks. Since freeze-thawing makes disks unsealed (Clark and Molday, 1979), the result suggests that an antigenic determinant of Rh29 is present in the intradiscal space. The carefully prepared sealed intact disks, although, inhibited the binding of Rh29 to immobilized ROS. It was shown in the Fig. 2 that the much higher amount of opsin as compared to the antibody (65 ng/ml) was required for the inhibition. Thus, the small inhibition of binding with our “sealed intact disks” was probably due to a contaminant of broken disks in the material. Assuming that a freeze-thawing makes completely disrupted disks which can be bound by Rh29, our “sealed intact disks” would contain broken ones up to 3%. Instead of mAb, the competitive ConA-binding assay also resulted in the increase in accessibility of ConA to frozen-thawed disks (data not shown) in agreement with the previous report (Molday and MacKenzie, 1983). On the basis of the rhodopsin structural model (Hargrave et al., 1983; Ovchinnikov, 1982), three loops (connecting segments) and N-terminal segment are exposed toward intradiscal space.

Fig. 2

Competitive inhibition by sealed intact disks (circles) and frozen-thawed disks (squares) in the binding of Rh29 to immobilized antigen. The microtiter plate wells were coated with bleached ROS. Rh29 was preincubated with competitors; bleached sealed intact disks or bleached frozen thawed disks. The wells were sequentially treated with the preincubated mixture and second antibodies. The percentage of 0 or 100 was defined as values obtained along the same treatment without Rh29 or competitor, respectively. Each point represents a mean value of 4 wells.

The epitope of Rh29 is N-terminal region of rhodopsin molecule.

To survey the antigenic determinants, intact disks treated with chymotrypsin or papain were analyzed by immunoblotting (Fig. 3). A pattern of intact disks stained with CB indicated opsin being major protein which banded at 39kDa. This band was recognized by Rh29 (lane 4 in Fig. 3).

Fig. 3

Immunoblotting and ConA-blotting of proteolytic intact disks. Approximately 10 μg of rhodopsin undigested (lane 1, 4 and 7), digested with chymotrypsin (lane 2, 5 and 8) and those with papain (lane 3, 6 and 9) were run in a 15% polyacrylamide gel and were stained with Coomassie blue (CB), with Rh29 (1.3 μg/ml) and the second antibodies (2 μg/ml) (Rh29) or with ConA (10 μg/ml) and HRPO (20 μg/ml)(ConA).

When intact disks were digested with chymotrypsin, the resultant fragments migrated in the region of 12–30 kDa. In these fragments, a band of 21 kDa reacted with Rh29. Papain cleaved rhodopsin in intact disks into 9.5 k, 11 k, 13 k and 18 kDa fragments. The band of 13 kDa was faint in the CB stain. However, this and a band at 18 kDa were recognized by Rh29 (lane 6 in Fig. 3). Upon the ConA-blotting, the 21 kDa of chymotryptic and the 13 k and 18 kDa of papainic fragments were detected (lanes 7, 8 and 9 in Fig. 3). Thus, these antigenic peptides should contain rhodopsin N-terminal segment attached by oligosaccharides.

In the next stage, we obtained a Rh29-reactive CNBr-peptide of rhodopsin. Unbleached ROS was digested with thermolysin which produced major two peptide fragments, F1 (1–240) and F2 (241–327) (Pober and Stryer, 1975).

After delipidation and carboxymethylation, F1 and F2 were separated on Sephadex LH-60 column. Rh29 and ConA reacted with a fraction of F1 fragment. The pool containing F1 fragment was cleaved by CNBr and chromatographed on Sephadex LH-60 column.

After the chromatography, Rh29 and ConA also reacted with the same fractions. These fractions were finally rechromatographed on Sephadex G-50 (Fig. 4). Rh29-binding assay showed that the first peak appeared in the void volume and the second peak included the antigenic peptide (the middle trace in Fig. 4). ConA also reacted with the same fractions (the bottom trace in Fig. 4). The second peak was revealed to contain a single peptide corresponding to residues 2-39 by amino acid analysis (Table 1).

Fig. 4

Isolation of Rh29-reactive CNBr-fragment by Sephadex G-50 column chromatography. A large thermolitic fragment of opsin (F1; 1–249) was cleaved with CNBr and developed on Sephadex LH-60 column. Rh29-reacted fractions were collected and then rechromatographed on Sephadex G-50 column (upper trace). Portions of fractions were dried in the microtiter plate wells and reacted with Rh29 (middle trace) or with ConA (lower trace).

Table 1

Amino acid composition of Rh29- and ConA-bound pep-tide

The first peak did not contain any peptide expected from the digestion of rhodopsin with CNBr and seemed to be an aggregate of partially cleaved materials.

Based on the structural model, N-terminal residues up to 36 are thought to be localized in the intradiscal space (Hargrave et al., 1983). This agreed with our results that Rh29 recognized amino acids residues 2–39 and that unsealed disks became more antigenic than sealed intact disks.

In order to elicit the essential amino acid residues for the binding of Rh29, we examined the effect of proteolytic digestion of the solubilized disks on the binding to the antibody. Digitonin-solubilized disks were treated with trypsin or chymotrypsin which cleaves N-terminal region of opsin at several sites (Ovchinnikov, 1982; Pober and Stryer, 1975). The resultant fragment mixture was subjected to competitive binding assay. Each tryptic or chymotryptic fragment was found not to be an effective competitors for binding to Rh29 (Fig. 5A). In both cases digested disks required for 50% inhibition were over thousand times higher than undigested one.

Fig. 5

(A): Effect of proteolytic digestion of solubilized opsin on the binding of Rh29. Bleached disks were solubilized by digitonin and incubated with TPCK-trypsin (triangles) or chymotrypsin (circles). The reaction was stopped by DFP and then the antigenicity of the peptide mixture was examined by competitive binding assay. Closed symbols represent controls incubated with the each enzyme and DFP. DFP-treated trypsin and chymotrypsin did not affect the binding of Rh29 to antigen. (B): Effect of modification of arginine residue. Digitonin-solubilized bleached disks (1 mg opsin; a and b) were treated with 1,2-cyclohexanedione (0.15 M; c and d) and then with hydroxylamine for deprotection of modified arginine (e and f). Each samples were then incubated with 1% TPCK-trypsin (b, d and f) or with TPCK-trypsin and DFP (a, c and e).

The carboxylic side of Lys16 or Arg21 was reported to be attacked by trypsin (Ovchinnikov, 1982). The chemical modification of arginine with 1,2-cyclohexanedione prevent tryptic cleavage and the cleavage sites can be restricted within lysine (Patthy and Smith, 1975). We, thus, modified the arginine residues of solubilized disks and digested with trypsin. The arginine-protected opsin could bound Rh29 regardless of trypsin treatment, while unprotected and deprotected opsin apparently lost their anitigenicity with trypsin digestion (Fig. 5B). Therefore, the cleavage between Arg21 and Ser22 should be responsible for the loss of antigenicity.

The epitope for Rh29 was conserved among wide species.

Finally, we tested the cross-reactivities of Rh29 to rhodopsins of several animals (Fig. 6). Rh29 recognized opsins (molecular weight 39–41 kDa) and its oligomer of bovine and porcine ROS, and octopus microvilli. Analysis of ConA-blots also elicited that these opsins had ConA-specific oligosaccharides. Residual ConA-specific proteins were banded in higher molecular weight region. In crayfish rab-dome, no bands were detected with Rh29, although applied membrane samples contained as much opsin as the other samples (>3 μg determined by visible absorbance measurements). In addition, ConA did not bind to the protein smaller than 50 kDa in crayfish, suggesting that the primary structure around N-terminal segment and the sugar structure might be altered in arthropoda.

Fig. 6

Immunoblotting and ConA-blotting of photoreceptor membranes of several animals. SDS-PAGE using 10% polyacrylamide gel was made for bovine, porcine ROS, octopus microvilli and crayfish rhabdome. Proteins were transferred on nitrocellulose paper and stained with amido black (1, 4, 7 and 10), or treated with Rh29 (2, 5, 8 and 11) or ConA (3, 6, 9 and 12). The conditions for the reaction were same as in Fig. 3.