J. Hugh McDowell

Anti-rhodopsin monoclonal antibodies of defined specificity: Characterization and application

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 rhodopsins cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsins 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 rhodopsins 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.

A humanized model of experimental autoimmune uveitis in HLA class II transgenic mice

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. Here we report the development of a humanized EAU model in HLA transgenic (TG) mice. HLA-DR3, -DR4, -DQ6, and -DQ8 TG mice were susceptible to IRBP-induced EAU. Importantly, HLA-DR3 TG mice developed severe EAU with S-Ag, to which wild-type mice are highly resistant. Lymphocyte proliferation was blocked by anti-HLA antibodies, confirming that antigen is functionally presented by the human MHC molecules. Disease could be transferred by immune cells with a Th1-like cytokine profile. Antigen-specific T cell repertoire, as manifested by responses to overlapping peptides derived from S-Ag or IRBP, differed from that of wild-type mice. Interestingly, DR3 TG mice, but not wild-type mice, recognized an immunodominant S-Ag epitope between residues 291 and 310 that overlaps with a region of S-Ag recognized by uveitis patients. Thus, EAU in HLA TG mice offers a new model of uveitis that should represent human disease more faithfully than currently existing models.

Arrestin migrates in photoreceptors in response to light: a study of arrestin localization using an arrestin-GFP fusion protein in transgenic frogs.

Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45-75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25-2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results.

Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin.

A synthetic heptaphosphopeptide comprising the fully phosphorylated carboxyl terminal phosphorylation region of bovine rhodopsin, residues 330–348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding phosphorylated rhodopsin or heparin. Unlike heparin, the phosphopeptide also induced light‐activated binding of arrestin to both unphosphorylated rhodopsin in disk membranes as well as to endoproteinase Asp‐N‐treated rhodopsin (des 330–348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin.

A high-throughput screening method for small-molecule pharmacologic chaperones of misfolded rhodopsin.

PURPOSE Many mutations in rhodopsin, including P23H, result in misfolding and mislocalization of the protein. It has been demonstrated that pharmacologic chaperones are effective in assisting the proper folding and targeting of P23H opsin. This study was designed to investigate a high-throughput screening strategy for identification of pharmacologic chaperones by using a combination of in silico, cell-based, and in vitro METHODS methods. A library of 24,000 drug-like small molecules was screened by in silico molecular docking with DOCK5.1. The top hits were assayed in an in vitro competition assay. The selected compound was then assayed for pharmacologic chaperoning activity in stable cell lines expressing wild-type and P23H opsin. RESULTS Beta-ionone was easily identified by the high-throughput screen. It strongly inhibits rhodopsin formation and, when incubated in cells expressing P23H opsin, resulted in a 2.5-fold rescue of P23H opsin. The screen also identified compound NSC45012 [1-(3,5-dimethyl-1H-pyrazol-4-yl)ethanone], a weak inhibitor of opsin regeneration and resulted in a 40% rescue of the mutant opsin. The level of rescue correlated well with the extent of inhibition. CONCLUSIONS A combination of in silico and cell-based screening provides a useful tool for identifying pharmacologic chaperones for P23H opsin. This approach identified both potent and weak pharmacologic chaperones. Both types of molecules may be potential candidates for treatment of opsin-related RP.

Binding pattern of anti-rhodopsin monoclonal antibodies to photoreceptor cells: An immunocytochemical study

A panel of anti-rhodopsin monoclonal antibodies (MAbs) of defined epitope specificity has been evaluated by immunocytochemistry. Most of the IgG class MAbs (23/27) gave positive results, but only a few of the IgM class MAbs (2/21) were useful for this application. MAbs specific to the N-terminal region stained rod outer segments most strongly, with progressively less staining in the Golgi, perikarya, plasma membrane of the inner segment, and synaptic region. Phagosomes located basally in the pigment epithelium were stained; cone cells were consistently negative. Antibodies to the C-terminus of rhodopsin labeled the same cell structures (except for phagosomes) but showed diversity in their binding pattern. Many of these MAbs bound to cone outer segments in addition to rods, and showed different patterns of binding to red/green and blue cones. Antibodies specific for rhodopsin sequence 340-348 labeled different types of cone cells, indicating differences in their binding sites. Two MAbs were found to label hydrophilic loop sequences which connect rhodopsins transmembrane segment: MAb K42-41 which binds loop 5-6, and MAb A1-55 which binds loop 2-3. At least these two regions of the rhodopsin sequence in addition to the C- and N-termini, are available for antibody reaction in fixed retina.

Dynamics of Arrestin-Rhodopsin Interactions LOOP MOVEMENT IS INVOLVED IN ARRESTIN ACTIVATION AND RECEPTOR BINDING

In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.

Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin

Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated phosphorylated rhodopsin. This study describes the combined use of insertional mutagenesis and immunochemical approaches to probe the structural determinants of arrestin function. Recombinant arrestins with insertions of a 10-amino acid c-Myc tag (EQKLISEEDL) were expressed in yeast and characterized. When the tag was placed on the C terminus after amino acid 399, between amino acids 99 and 100 or between residues 162 and 163, binding to rhodopsin was found to be very similar to that of wild-type arrestin. Two stable mutants with Myc insertions in the 68–78 loop were also generated. Binding to rhodopsin was markedly decreased for one (72myc73) and completely abolished for the other (77myc78). Limited proteolysis assays using trypsin in the absence or presence of heparin were performed on all mutants and confirmed their overall conformational integrity. Rhodopsin binding to either 162myc163 or 72myc73 arrestins in solution was completely inhibited in the presence of less than a 2-fold molar excess of anti-Myc antibody relative to arrestin. In contrast, the antibody did not block the interaction of the 399myc or 99myc100 arrestins with rhodopsin. These results indicate that an interactive surface for rhodopsin is located on or near the concave region of the N-domain of arrestin.

Molecular, enzymatic and functional properties of rhodopsin kinase from rat pineal gland

Rhodopsin kinase activity from rat pineal gland and from rat retina are indistinguishable, based upon determination of a variety of enzymatic and molecular properties. Both activities are independent of calcium, cyclic nucleotides, and calmodulin. Both are activated by spermine and inhibited by adenosine and some rhodopsin kinase specific adenosine derivatives such as sangivamycin. The Kms for rhodopsin, ATP, and GTP are indistinguishable for the protein kinase in extracts from the retina and from the pineal gland. The apparent molecular weight of the kinase from both sources, as determined by gel filtration and autoradiography of the 32P-labeled autophosphorylated kinase, is about 70 kDa. Rhodopsin kinase activity from pineal binds in a light-dependent manner to rhodopsin in rod outer segments as does the enzyme from retina. Monoclonal antibodies against bovine rhodopsin were used in an immunochemical study that identified a rhodopsin-immunoreactive protein in rat pineal gland and retina. Using an ELISA we demonstrated the presence of a rhodopsin-immunoreactive protein in rat pineal gland equivalent to 0.075 pmol rhodopsin per gland. Frog pineal organ (Rana catesbiana) contains 33 times more of this rhodopsin-like protein than does rat pineal gland.

Uveitis-Associated Epitopes of Retinal Antigens Are Pathogenic in the Humanized Mouse Model of Uveitis and Identify Autoaggressive T Cells

Noninfectious uveitis is a leading cause of blindness and thought to involve autoimmune T cell responses to retinal proteins (e.g., retinal arrestin [soluble-Ag (S-Ag)]). There are no known biomarkers for the disease. Susceptibility is associated with HLA, but little is known about susceptible class II alleles or the potentially pathogenic epitopes that they present. Using a humanized HLA-transgenic mouse model of S-Ag–induced autoimmune uveitis, we identified several susceptible and resistant alleles of HLA-DR and -DQ genes and defined pathogenic epitopes of S-Ag presented by the susceptible alleles. The sequences of these epitopes overlap with some previously identified peptides of S-Ag (“M” and “N”), known to elicit memory responses in lymphocytes of uveitis patients. HLA-DR–restricted, S-Ag–specific CD4+ T cells could be detected in blood and draining lymph nodes of uveitic mice with HLA class II tetramers and transferred the disease to healthy mice. Importantly, tetramer-positive cells were detected in peripheral blood of a uveitis patient. To our knowledge, these findings provide the first tangible evidence that an autoimmune response to retina is causally involved in pathogenesis of human uveitis, demonstrating the feasibility of identifying and isolating retinal Ag-specific T cells from uveitis patients and may facilitate their development as biomarkers for the disease.