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Protocols for photoreceptor outer segment (POS) isolation that can be used in phagocytosis assays of retinal pigment epithelium (RPE) cells have routinely used a large number of cow or pig eyes. However, when working with large animal models (e.g., dog, cats, transgenic pigs) of inherited retinal degenerative diseases, access to retinal tissues may be limited. An optimized protocol is presented in this paper to isolate sufficient POS from a single canine retina for use in RPE phagocytosis assays.

Purpose: In experimental eye research, zebrafish has become a powerful model for human retina disorders. The purpose of the present study is the characterization of antibodies commonly employed in zebrafish models for rod photoreceptor degeneration., Methods: The 1D4 monoclonal antibody, developed against bovine rhodopsin, has been widely used in studies addressing structural and functional features of rhodopsin and was reported as an informative marker to stain rod outer segments in both mice and zebrafish. We have used transgenic reporter lines and histologic analysis to determine the photoreceptor types identified by 1D4 and other antibodies in zebrafish., Results: We demonstrate that 1D4, in contrast to what has been reported previously, does not recognize rod outer segments in zebrafish, but instead labels long double cone outer segments consistent with sequence conservation of the respective epitope. As an alternative marker for zebrafish rods, we characterized the monoclonal antibody zpr-3, which was found to stain outer segments of both rods, as well as double cones., Conclusions: Our findings highlight the importance to confirm specificity of antibodies in cross-species experiments for correct interpretation of experimental data. Our findings clarify conflicting published information arising from studies using 1D4 and zpr-3 antibodies in zebrafish.

We had previously reported that transduction of the channelrhodopsin-2 (ChR2) gene into retinal ganglion cells restores visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats. In this study, we attempted to reveal the safety and influence of exogenous ChR2 gene expression. Adeno-associated virus (AAV) type 2 encoding ChR2 fused to Venus (rAAV-ChR2V) was administered by intra-vitreous injection to dystrophic RCS rats. However, rAAV-ChR2 gene expression was detected in non-target organs (intestine, lung and heart) in some cases. ChR2 function, monitored by recording visually evoked potentials, was stable across the observation period (64 weeks). No change in retinal histology and no inflammatory marker of leucocyte adhesion in the retinal vasculature were observed. Although antibodies to rAAV (0.01-12.21 μg ml(-1)) and ChR2 (0-4.77 μg ml(-1)) were detected, their levels were too low for rejection. T-lymphocyte analysis revealed recognition by T cells and a transient inflammation-like immune reaction only until 1 month after the rAAV-ChR2V injection. In conclusion, ChR2, which originates from Chlamydomonas reinhardtii, can be expressed without immunologically harmful reactions in vivo. These findings will help studies of ChR2 gene transfer to restore vision in progressed retinitis pigmentosa.

Staining by antibodies to rhodopsin (Rh) and fluorescence of N-retinylopsin (RO) have shown that digitonin (DIG)- , dodecyl-β-D-maltoside (DM)- , and sodium dodecyl sulfate (SDS)-solubilized frog Rh after BN- and HRCN-PAGE is situated in the gradient gel in the state of dimer with a slight content of higher oligomers (trimer, tetramer, etc.). With increasing detergent harshness (DIG < DM < SDS), the proportion of higher oligomers in extracts becomes more prominent. Formation of RO in rod outer segments (ROS) in the presence of 0.7 M NaBH(3)CN at pH 5.0 occurs only when Rh is simultaneously photolyzed during reduction. Dithiothreitol at the concentration of 0.005 M failed to induce RO production. Formation of a stable C-N bond between all-trans-retinal and opsin in RO is accompanied by decrease in the dimer share and increase in the share of the higher oligomers due to secondary dissociation-aggregation of solubilized opsin. The position of the Rh dimer in relation to the anode during both native electrophoreses is determined not only by its molecular mass, but probably also depends on unfolding degree (or form): the harsher the detergent, the closer to the anode the dimer is located. Treatment of ROS by agents modifying the cholesterol component of lipid membrane (MβCD, filipin III, nystatin, saponin) did not change the character of Rh oligomerization, thus showing that integrity of the cholesterol component of photoreceptor membrane is not a crucial factor for oligomerization of opsin. It is supposed that the dimer-oligomer "portrait" of frog Rh, which has been found by two methods of native electrophoresis in three detergents with different degree of harshness, corresponds to a physiological state of this protein in native photoreceptor membrane.

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the rod cells of the retina and detects light/dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

Unlike most other tissues, the optimal fixative for preserving eye morphology is considered to be Davidson's fixative or modified Davidson's rather than formalin. However, the methodology for antibodies to be used in tissues fixed this way is not normally outlined in current antibody datasheets. Additionally, where eyes have been stored in Davidson's fixative, the efficacy of retrospective analysis of eye morphology by immunohistochemistry is largely unknown. The aim of this study was to compare a panel of six antibodies in both Davidson's-fixed and formalin-fixed pigmented and non-pigmented rat eyes, in order to provide optimal methods for future retinal immunohistochemical evaluation with image analysis. The antibodies evaluated were raised against rhodopsin, synaptophysin, glutamine synthetase, glial fibrillary acidic protein (GFAP), cleaved caspase-3 and phospho-histone H3 (PH3). Overall, the staining quality of these antibodies was found to be optimal in Davidson's compared to formalin-fixed tissues after a time period of up to 4 days in fixative. The methods outlined thus provide a platform for future detailed analysis of retinal pathology in Davidson's-fixed eyes.

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg(alpha1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the alpha1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and alpha1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln238 and Glu239, in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.

To investigate retinal involvement in chronic Chagas' disease, we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency, under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors, all related to the same subfamily of G-protein-coupled receptors.

Aim: To develop a conceptual model of using catalytic autoantibodies as diagnostic and monitoring tools in organ-specific autoimmune disorders., Material and Methods: A total of 99 patients (56 males and 43 females aged 21-52 years) with autoimmune myocarditis (AM) and 198 patients (77 males and 121 females aged 8-79 years) with autoimmune uveitis (A U) participated in the study. AM patients were examined for anticardiomyosin and anti-DNA autoantibodies (ACM, ADNAab), AU patients - for autoantibodies to S-antigen, IRBP, redopsin, phosphocine, autoDNA., Results: AM patients had double level of DNA-binding autoantibodies. In 1/3 of them there was hydrolysing DNA and cytotoxic activity. In AU patients maximal titers were in Behcet's disease, sympathic ophthalmia, generalized uveitis and viral uveitis., Conclusion: Autoantibodies with different specificity and function including DNA-abzymes can be additional diagnostic and prognostic markers.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.

Chameleons (Order, Reptilia: Family, Lacertilia) are unique among vertebrates in being able to make independent eye movements. The organisation of their retina, however, closely ressembles that of other diurnal lizards; based on morphological studies, it is typically described as containing only cone photoreceptors. We show here that a subpopulation of the photoreceptors are immunolabelled by an antibody directed against rhodopsin, suggesting the presence of rods. We conclude that in the nonmammalian retina, rods and cones cannot be exclusively distinguished on purely morphological grounds.

Metastatic melanoma still has a very poor prognosis since it withstands conventional therapies like surgery or chemotherapy. A paraneoplastic autoimmune manifestation of this disease is melanoma-associated retinopathy (MAR). MAR has been associated with prolonged survival and may be an early marker of tumor progression. By screening a retina and a melanoma cDNA phage library by SEREX using sera of patients suffering from melanoma and, in some cases, clinical symptoms of MAR, we identified 20 new antigens (HD-MM-28-47), of which 14 clones had high homology to well-known genes. Six of these genes had previously been associated with retina: rhodopsin, visual arrestin, MEK1, SRPX, BBS1 and galectin-3. Individual clones were recognized by up to 43% of patients' sera, while sera of healthy volunteers were negative except in 2 cases. The expression profile of the antigens identified on the basis of homologous EST database entries in healthy tissues was ubiquitous to differential. Using RT-PCR, we found frequent expression of preselected antigens in melanoma cell lines. For rhodopsin, this could be quantified by quantitative PCR. Retinal proteins were recognized by serum antibodies of melanoma patients but not healthy controls. The role of these antigens in MAR awaits further investigation. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.)

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained "inside out" and "right side out" membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the beta-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands.

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21-25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh 29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.

[alpha-(15)N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside-phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20 degrees C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [(13)C]Ser-338/[(15)N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330-Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.

Human autoimmune uveitides are diverse and complex. Animal models have been developed for studying the pathogenesis of uveitis because of the difficulties in obtaining tissues from a patient's inflamed eye for experiments. There are animal models for experimental uveitis that provoke inflammation of different tissues of the eye and represent different forms of uveitis. Since inflammatory cells can infiltrate any part of the uvea and spill over to nonuveal tissues, such as retina, various antigens have been used to induce uveitis. Most of those models that represent autoimmune forms of uveitis are induced with proteins specific for photoreceptor cells (S-antigen, IRBP, rhodopsin, recoverin, phosducin). Nonretinal antigens, including melanin-associated proteins and myelin basic protein, are also good inducers of uveitis in animals.

The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.

Visual pigments in the regressed eye and pineal of the depigmented neotenic urodele, the blind cave salamander (Proteus anguinus anguinus), were studied by immunocytochemistry with anti-opsin antibodies. The study included light- and electron-microscopic investigations of both the eye and the pineal organ. A comparison was made with the black pigmented subspecies Proteus anguinus parkelj (black proteus), which has a normal eye structure. In the retina of the black proteus, we found principal rods, red-sensitive cones and a third photoreceptor type, which might represent a blue- or UV-sensitive cone. Photoreceptors in the regressed eye of the blind cave salamanders from the Planina cave contained degenerate outer segments, consisting of a few whorled discs and irregular clumps of membranes. The great majority of these outer segments showed immunolabelling for the red-sensitive cone opsin and only a few of them were found to be positive for rhodopsin. An even more pronounced degeneration was observed in the photoreceptors of the animals derived from the Otovec doline, which are completely devoid of an outer segment, most of them not even possessing an inner segment. Even in some of these highly degenerate cells, the presence of rhodopsin could be detected in the plasma membrane; however, immunoreactions with antibodies recognizing cone visual pigment were negative. In the pineals of all studied animals, the degenerate photoreceptor outer segments were recognized exclusively by the antibody against the red-sensitive cone opsin. The presence of immunopositive visual pigments indicates the possibility of a retained light sensitivity in the blind cave salamander photoreceptors.

A monoclonal anti-rhodopsin antibody (B6-30N), characterized by Hargrave and coworkers [Adamus, G., Zam, Z. S., Arendt, A., Palczewski, K., McDowell, J. M. & Hargrave, P. (1991) Vision Res. 31, 17-31] as recognizing a short peptide sequence at the N terminus, failed to bind to rhodopsin when the latter was solubilized in dodecylmaltoside (DM). Of the detergents tested thus far, DM affords maximum stability to rhodopsin. Solubilization of rhodopsin in cholate allowed binding of the antibody, but the binding caused destabilization as evidenced by the accelerated loss of absorbance at 500 nm. The result provides support for the earlier conclusion that the N-terminal segment is an integral part of a tertiary structure in the intradiscal domain of native rhodopsin coupled to a tertiary structure in the transmembrane domain. Additional comparative studies on the stability of rhodopsin in different detergents were carried out after direct solubilization from rod outer segments and after extensive treatments to remove the endogenous phospholipids. Purification of rhodopsin in DM resulted in essentially quantitative removal of endogenous phospholipids. When rhodopsin thus purified was treated with the above antibody in DM and in cholate, enhanced destabilization (5-fold) was observed in the latter detergent.

Eleven single-cysteine substitution mutants have been prepared in the sequence 325-340 of rhodopsin, corresponding to the C-terminal domain. Each of the cysteine mutants was modified with a selective nitroxide reagent to introduce a spin-labeled side chain. The electron paramagnetic resonance spectra of the labeled proteins were analyzed in terms of side chain dynamics. At all sites, the spectra reflected the presence of two populations of different mobility, although one was always dominant. The mobility of the dominant population increased in a regular fashion from the palmitoylation sites at 322C and 323C to the C-terminus, where the spectra resembled those of an unfolded protein. This apparent mobility gradient is only slightly affected in mutants lacking the palmitoyl groups, suggesting that they are not responsible for physically anchoring the C-terminal peptide at one end. Binding of a monoclonal antibody to its epitope at the C-terminus dramatically reduces the mobility of nearby residues, creating a local mobility gradient opposite that in the absence of the antibody. These results indicate that the C-terminal domain of rhodopsin, beyond the palmitoylation sites, is highly disordered and dynamic, resembling an unfolded peptide tethered at one end.

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell-autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild-type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild-type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival-promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival-promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival-promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice.

Purpose: The presence of anti-rhodopsin antibodies in patients with normal pressure glaucoma (NPG) has been previously demonstrated with western blot analysis and enzyme-linked immunosorbent assay. To learn more about the characteristics, origin, and possible significance of these antibodies, the epitopic specificity of the anti-rhodopsin antibodies was examined in four NPG patients., Methods: Antibodies in patient sera were assayed by western blot analysis against purified bovine rhodopsin. Peptides derived from particular segments of the rhodopsin sequence were tested for activity in competing for rhodopsin-antibody binding., Results: Of a series of nine peptides that constitute most of the hydrophilic regions of rhodopsin, only one, consisting of the C-terminal 25 amino acids, prevented binding of the patient antibodies to rhodopsin. Higher resolution mapping using a set of dodecamers of overlapping sequences from the C-terminal region demonstrated that antibody binding is completely dependent on the last two amino acids. Removing the C-terminal alanine alone, or amidating the C terminus carboxyl group, also eliminated antibody binding., Conclusions: Because four of four patient antibodies examined exhibited the identical epitopic specificity, it is likely that a common mechanism underlies their generation. This may indicate that molecular mimicry has occurred, because several pathogens contain similar C-terminal sequences. Although they may serve as diagnostic markers, and provide evidence that there is an autoimmune component in some patients with glaucoma, the role, if any, that these antibodies play in the pathogenesis of the disease remains unknown.

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11-cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

Protein extracts of optic cushion tissue from the asteroid Asterias forbesi and arm tissue from the ophiuroid Ophioderma brevispinum were subjected to Western blot analysis. Both tissues contain a membrane-associated protein that reacts with two monoclonal antibodies raised against bovine rhodopsin. This protein migrates slightly behind bovine rhodopsin during sodium-dodecyl-sulfate polyacrylamide gel electrophoresis, suggesting that its molecular weight is slightly larger. Immunohistochemical examination of the optic cushions of A. forbesi revealed a substance that reacts with both monoclonal antibodies; moreover, this substance is more abundant in dark-adapted animals than in light-adapted animals. The arms and central disk of O. brevispinum were also examined immunohistochemically. The tips of the arm spines contain a substance that reacts with both monoclonal antibodies, and at higher magnification this immunoreactive material is localized to small regions within the stroma of the ossicles. Taken together, the biochemical and immunochemical evidence suggests that the cross-reacting protein is homologous to other known rhodopsins and is serving as a visual pigment in echinoderms.

IgG Ab titers elicited to bovine rhodopsin in CFA differ 8- to 10-fold between H2s identical inbred strains A.SW/snJ (high responder) and SJL/snJ (low responder). This variation in IgG Ab titer resulted from a dramatic difference in the rise in Ab titer occurring during the maturation of the T-dependent humoral immune response. To determine the positions of non-MHC genes controlling this quantitative variation in T-dependent humoral immune responsiveness, 206 reciprocal (A.SW/snJ x SJL/snJ)F2 female progeny were immunized and assayed for anti-rhodopsin responsiveness. The genomes of these progeny were screened with 115 polymorphic simple sequence repeat markers covering >90% of the mouse genome. interval mapping analysis localized the positions of these non-MHC immune response genes to genomic intervals on chromosomes 1, 5, and 13. Interestingly, these three intervals coincide exactly with three intervals recently shown to contain genes contributing to susceptibility to systemic lupus erythematosus and/or the production of autoimmune anti-dsDNA Abs. These results suggest that some genes affecting levels of humoral immune responsiveness to exogenous Ag may also play a role in genetic susceptibility to humoral autoimmune diseases. Analyses of the modes of inheritance demonstrated that high responder alleles were inherited from both parental genomes, indicative of epistatic interactions among genes influencing humoral immune responsiveness.

Purpose: To explore further the potential role autoimmunity may play in the pathogenesis of normal-pressure glaucoma (NPG) in some patients, the authors examined the sera of patients with NPG for the presence of antibodies directed toward retinal antigens., Methods: Using patient sera, immunoblotting was performed on subcellular fractions of retina, purified bovine rhodopsin, and immunoaffinity-purified recombinant human rhodopsin. A chemiluminescence-enzyme-linked immunosorbent assay (ELISA) to detect anti-rhodopsin antibodies was developed and used., Results: A patient with NPG was found to have a high titer of immunoglobulin M-lambda antibody against a 40-kd retina-specific glycoprotein antigen subsequently identified as rhodopsin. ELISA analysis conducted on sera from 28 patients with NPG and 26 patients with primary open-angle glaucoma (POAG) revealed highly significant differences in anti-rhodopsin antibody activity between these groups (P < 0.0002, Mann-Whitney test). For example, the majority of patients with NPG (19/28; 68%) had anti-rhodopsin antibody activity higher than the highest value obtained from among 26 age-matched patients with POAG., Conclusions: An elevated anti-rhodopsin antibody count is related to NPG. This may indicate that there is an autoimmune component in the optic neuropathy in these patients. The specific role of these autoantibodies, if any, in the pathogenesis of the disease remains to be determined.

A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation.

The spectral sensitivity of the dark-adapted eye of the snail Helix lucorum L. was investigated in semi-intact "optic cupoptic nerve-cerebral ganglion" preparations. The data of the recording of the electroretinogram in response to monochromatic radiation of varied intensities were used to plot the spectral sensitivity functions. The averaged spectral sensitivity function is well described by a Dartnall nomogram for the photopigment rhodopsin with a sensitivity peak at 496 nm.

Rhodopsin, a membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) in Lewis rats. Synthetic peptides derived from rhodopsin sequences that cover hydrophilic, exposed regions of the protein were tested for their capacity of eliciting in vitro T cell proliferation and their ability for inducing EAU in Lewis rats. Rats were injected with rhodopsin's peptides mixed in complete Freund's adjuvant containing M. tuberculosis H37Ra (5 mg/ml) three days after pretreatment with cyclophosphamide (20 mg/kg). ELISA results indicate that all peptides induce antibody responses; however antibody titers differ among sera tested. Immunization with four peptides--the amino-terminus (2-32), loop I-II (61-75), loop V-VI (230-251), and the carboxyl-terminus (324-348 and 331-342) induced both antibody and T cell responses. In all cases, the proliferative responses of cells derived from peptide-injected rats were stronger against the immunizing peptide than against native protein. Three distinct uveitogenic epitopes were identified on rhodopsin's cytoplasmic surface--within the rhodopsin carboxyl-terminus (324-348), loop I-II (61-75), and loop V-VI (230-250). Histopathologically, at the immunized doses, total destruction of the photoreceptor cell layer was observed as compared to the control group. Loop V-VI caused severe inflammation of the retina while the other pathogenic peptides produced less severe destruction with few inflammatory cells present. Our study indicates that the major immunodominant T cell epitope (331-342) is also involved in EAU induction but is not the primary uveitogenic site.

A mu opioid binding protein (OBP), previously purified to homogeneity from bovine striatal membranes, was examined by immunoblotting with six antisera against bovine rhodopsin. An antibody against the carboxyl-terminal tail of rhodopsin and one against membrane-associated rhodopsin gave strong signals at the appropriate molecular mass (65 kDa). An antibody directed against the first cytoplasmic loop of rhodopsin was weakly reactive. Three other antibodies did not recognize OBP. This pattern of crossreactivity was identical to that previously seen with beta-adrenergic receptors. The existence of domains in the OBP, which are antigenically similar to those in two other guanine nucleotide regulatory protein-coupled receptors, supports the hypothesis that mu opioid receptors have the structure characteristic of this receptor family.

Inbred strains of mice of independent haplotype were immunized with bovine rhodopsin. All mice tested except SJL developed significant titers of specific antibodies 21 days after a single immunization. Anti-rhodopsin antibody level differed among conventional inbred strains. Comparison of the immune response to rhodopsin of congenic mice on two different genetic backgrounds showed that animals with an A background typically produced higher levels of specific antibody than mice with a B10 background. Titer of specific antibodies in antisera of mice of the same H-2 haplotype but different Igh haplotype differed; e.g. for H-2d haplotype, NZB (Ighn) generated the highest level of antibody with BALB/c (Igha), DBA/2 (Ighc), and B10.D2 (Ighb) strains giving successively lower responses. The location of immunodominant regions of bovine rhodopsin was investigated in primary sera among strains of mice. Sera were tested for their binding of anti-rhodopsin antibodies to synthetic peptides covering the entire primary structure of rhodopsin. From direct binding studies with hydrophilic rhodopsin peptides, the majority of the antigenic binding sites were localized in the sequence of the amino terminus, the II-III loop and the carboxyl terminus. Binding to these antigenic peptides was not strain restricted. Application of the overlapping synthetic peptide strategy of Geysen enabled refinement of these epitopes and determination of an additional major epitope in the hydrophobic sequence 304-310.

Rhodopsin, an integral membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) when injected into Lewis rats. This disease is characterized by a mononuclear and polymorphonuclear cellular infiltrate of the retina resulting in destruction of the photoreceptor cells. In this study the B and T cell specificities of the response to bovine rhodopsin by Lewis rats were determined. Antibodies induced by injection of rhodopsin were directed almost exclusively to the IV-V loop (residues 174-202). Later in the response, antibody to the N-terminus was also detected. At the T cell level, most activity was directed to the C-terminus as measured by in vitro lymphocyte proliferation. Other minor T cell epitopes were found in the II-III (96-114) and IV-V (174-202) loops. Further dissection of the amino acid sequence responsible for the activity isolated to the C-terminus indicated that a 12-amino acid-long sequence (331-342) elicited the strongest proliferative response.

Experimental autoimmune retinitis induced by immunization with rhodopsin was investigated in the Lewis rat using transmission electron microscopy and light microscopy. The first signs of retinitis consisted of scattered infiltrations of lymphocytes and other mononuclear cells, predominantly in the inner nuclear layer and outer plexiform layer. Occasionally, some macrophages were detected in the photoreceptor cell layer. Eyes exhibiting a clinically moderate or severe inflammation contained areas of normal retina coexistent with mildly to severely inflamed foci. The central retina was more frequently affected than the peripheral area. In moderately inflamed foci, macrophages infiltrated the photoreceptor cell layer, damaging and eliminating its structures. Inflammatory cells penetrated the photoreceptor outer segment layer which remained unaltered so far in spite of a high serum anti-(rhod)opsin antibody titer. In stages of severe inflammation, massive infiltrations of macrophages and polymorphonuclear cells destroyed the photoreceptor cells focally, leaving the retinal pigment epithelium virtually unaffected. Adjacent to these foci the pigment epithelial cells sometimes exhibited increased numbers of phagosomes and swelling. The locations of the cell infiltrations and lesions in progressive stages of development suggest that the rod outer segments are the target for the autoimmune damage. The described patterns of inflammation were compared with those of previous studies using other animal species and inciting antigens. Especially in rhodopsin-induced retinitis, the blood-retina barrier at the level of the Bruch's membrane/pigment epithelium appears to be highly resistant to cytotoxic cells. The present observations are in agreement with the concept that the cellular immune response plays a major role in the pathogenesis of (rhod)opsin-induced retinitis.

Paramecium bursaria is photosensitive and accumulates in a lighted area. The cells can be deciliated by a brief suspension in dilute ethanol. Both intact and deciliated cells showed depolarization in response to light stimulation by a step-increase from dark to above 0.7 mW cm-2 (550 nm). On the other hand, after a step-increase to below 0.4 mW cm-1, intact cells showed hyperpolarization, while the deciliated cells showed no change in membrane potential. This difference in membrane potential response between ciliated and deciliated cells suggests that both somatic and ciliary structures are photosensitive. In our search for the photoreceptive molecules, a polyclonal antibody induced in rabbits against frog rhodopsin was found to cross-react with a 63x10(3) Mr protein of P. bursaria, by immunoelectrophoresis. Immunocytochemical studies showed that the antibody labeling was localized on both the ciliary and the somatic membranes. These results raise the possibility that P. bursaria may contain a rhodopsin-like protein as a photoreceptor molecule.

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbent assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 118-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 10(8)-10(10) M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similarly, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

We have studied the immunoreactivity of turtle photoreceptors to a monoclonal antibody (MAb 15-18) which binds to the external loop connecting bovine rhodopsin helices IV-V. Three chromatic types of cone photoreceptors were identified by the presence and color of oil droplets. MAb 15-18 intensely labeled the outer segments of both rods and green cones. In addition, a weak cross-reactivity was also found in the outer segments of red cones having a pale-green oil droplet, and of blue cones. Other morphological subtypes of red cones, cones with a red oil droplet and both members of double cones, showed no labeling. Our results indicate that rhodopsin and green cone opsin have a similar antigenic determinant, and that two different structural forms of red cone opsin may be present in the turtle retina.

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.

Light-induced phosphorylation of rhodopsin in bovine rod outer segment disk membranes inhibits the binding of three carboxyl-terminal-specific anti-rhodopsin antibodies and the cleavage of the carboxyl-terminal region of rhodopsin by trypsin and Staphylococcus aureus V-8 protease. Two monoclonal antibodies, rho 3A6 and rho 1C5, which previously have been shown to preferentially bind to the 8'-12' and the 9'-18' carboxyl-terminal segments of rhodopsin, respectively, are both highly sensitive to phosphorylation. When an average of one phosphate is incorporated per rhodopsin, the binding reactivity of rhodopsin for these antibodies decreases to 30% that of nonphosphorylated rhodopsin as measured in radioimmune competition assays. Reactivity of the rho 1D4 antibody whose primary binding site is localized in the 1'-8' C-terminal segment of rhodopsin is unaffected at this level of phosphorylation but decreases to 30% when three phosphates on average are incorporated per rhodopsin. Direct binding studies using 125I-labeled antibodies indicate that phosphorylation of rhodopsin decreases the maximum extent of rho 3A6 and rho 1C5 binding to rhodopsin. For rho 1D4, the maximum extent of binding is unaffected by phosphorylation, but the dissociation constant is increased by 10-fold. Phosphorylation of rhodopsin also inhibits cleavage of the 1'-9' and 1'-7' carboxyl-terminal peptides by trypsin and S. aureus V-8 protease, respectively. When an average of one phosphate per rhodopsin is incorporated, cleavage decreases to 40% that of nonphosphorylated rhodopsin as measured by high-performance liquid chromatography. Phosphorylation of rhodopsin had no effect on S. aureus cleavage of rhodopsin into the F1 (Mr 25 000) and F2 (Mr 12 000) fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

In the tree shrew retina individual rod and cone photoreceptors can be readily identified and quantified because their perikarya are arranged in a single layer. This retina is therefore an ideal system for testing the specificity of photoreceptor-directed antibodies. Here we describe the staining properties of polyclonal antibodies against (rhod)opsin and retinal S-antigen in the tree shrew retina. The (rhod)opsin antibody exclusively and completely labelled the rod population. The antibody against S-antigen also labelled all rods and, in addition, a regularly arrayed subpopulation of cones, which we argue to be the blue-sensitive cones. In the context of our findings, the labelling of pinealocytes with these antibodies is discussed.

Systemic immunization with purified homologous rhodopsin from retinal outer segments induced blindness in primates (Macaca mulatta). Inflammation and characteristic retinal changes were the earliest clinical signs of the disease. Perivasculitis, subretinal exudations and bullous detachments of the retina were progressive and unrelenting pathological processes leading to rapid and irreversible visual deterioration. Electroretinographic responses (ERG) at this stage of the disorder became abolished. Antibodies and delayed hypersensitivity to rhodopsin were demonstrated only in the experimental diseased animals. Homologous visual purple appears to be organ and immunopathologically specific. Histological confirmation of these findings showed a pathological spectrum of destructive alterations confirmed specifically to the outer segments of the entire retina. The pathologic reaction was supported by a distinct and pronounced granulomatous inflammatory response.

We investigated rhodopsin immunoreactivity in five well-differentiated retinoblastomas using a panel of monoclonal antibodies directed against specific antigenic sites in the amino- and carboxyl-terminal portions of rhodopsin. All five monoclonal antibodies bound to the rod cell outer segment of nontumorous retina in all 10% formaldehyde solution-fixed, paraffin-embedded tissue sections. A characteristic "halo" cell surface staining pattern was observed in four (80%) of five tumors treated with two monoclonal antibodies, B6-30 (rhodopsin amino-terminal specific) and K16-107 (rhodopsin carboxyl-terminal specific). In each case, the staining pattern was limited to well-differentiated areas of the tumor containing Flexner-Wintersteiner rosettes or fleurettes. One tumor was not stained by any monoclonal antibody, whereas all monoclonal antibodies stained the rod cell outer segments of nontumorous retina. Our studies indicate that selected retinoblastomas may be differentiated along a rod photoreceptorlike cell lineage.

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

We have produced monoclonal antibodies directed against defined amino acid sequences of rhodopsin by using peptides for hybridoma selection, rather than for immunization. Mice were immunized with rhodopsin, and hybridoma culture supernatants were assayed by ELISA to detect anti-rhodopsin antibodies. These antibodies were further tested by competition ELISA using synthetic peptides from the rhodopsin sequence in order to select antibodies with the desired amino acid sequence specificity. Site-specific monoclonal antibodies selected in this way have affinities for the protein which are typically higher than that of antibodies which have been generated against peptide-carrier conjugates as antigens.