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

Adamus, Grażyna; Zam, Z. Suzanne; Arendt, Anatol; Palczewski, Krzysztof; McDowell, J. Hugh; Hargrave, Paul A.

doi:10.1016/0042-6989(91)90069-h

Cited by 221 publications

(120 citation statements)

References 38 publications

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“…The purified protein was used to immunize mice as described previously (16). Purified RPE65 was emulsified with the adjuvant system (Sigma) according to the manufacturer's protocol, and the antigen (50 g/body) was injected intraperitoneally into 4-week-old BALB/c mice.…”

Section: Methodsmentioning

confidence: 99%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

Journal of Biological Chemistry

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Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A 2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.In vertebrates, the perception of light is preceded by a quantum event in which a photon of light hits the 11-cis-retinylidene chromophore of rhodopsin in the retina. Photoisomerization of this chromophore to its all-trans isomer results in activation of rhodopsin triggering a cascade of signal transduction events (summarized in Ref. 1). This process simultaneously leads to loss of photoreceptor light sensitivity making continuous regeneration of 11-cis-retinal a requirement to sustain vision. The visual (retinoid) cycle is the fundamental series of reactions responsible for regeneration of the visual chromophore that occurs in photoreceptors and the retinal pigment epithelium (RPE) 4 (summarized in Ref.2). The key component of this enzymatic cycle is the RPE-specific microsomal membrane protein known as RPE65 (3-5). The importance of RPE65 in the retinoid cycle is underscored by the metabolic dysfunction found in Rpe65 Ϫ/Ϫ mice, which is characterized by complete lack of 11-cis-retinoid production accompanied by accumulation of the all-tr...

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Section: Methodsmentioning

confidence: 99%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

Journal of Biological Chemistry

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“…Dot blots and data analysis were performed as described previously, using X. laevis eye extracts, monoclonal antibody (mAb) B630N (Adamus et al, 1991) and mAb 1D4 (MacKenzie et al, 1984) (Chemicon, Temecula, CA) primary antibodies, IR-dye800-conjugated secondary antibodies, and a LICOR Odyssey imaging system . Each blot included standards containing 100% X. laevis rhodopsin (from wild-type retinas) and 100% transgenic rhodopsin (from cell culture extracts), allowing the percentage of transgenic rhodopsin expression levels to be determined from the ratio of mAb B630N (recognizes total rhodopsin) to mAb 1D4 label (recognizes only transgenic rhodopsins).…”

Section: Methodsmentioning

confidence: 99%

“…After 36 h, cells were solubilized in 1% N-dodecyl maltoside (Anatrace, Maumee, OH) with 0.1 mg/ml PMSF in PBS for 1 h on ice, followed by centrifugation at 36,000 ϫ g for 30 min. Western blot analysis was performed as described previously , using mAb B630N or mAb A5-3 (Adamus et al, 1991) at 1:10 and mAb 1D4 at 1:750.…”

Section: Methodsmentioning

confidence: 99%

Dark Rearing Rescues P23H Rhodopsin-Induced Retinal Degeneration in a TransgenicXenopus laevisModel of Retinitis Pigmentosa: A Chromophore-Dependent Mechanism Characterized by Production of N-Terminally Truncated Mutant Rhodopsin

Tam¹,

Moritz²

2007

J. Neurosci.

100

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To elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic ϳ27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

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“…For reconstructing the topographic distribution of disease, 1-m retinal plastic sections, extending from the optic disk to the periphery (ora serrata), were evaluated from continuous overlapping 110-m fields (25). For immunocytochemical studies, sections from DGD-embedded retinas were labeled with mAb K16-107C, directed at the C-terminal domain of opsin (26).…”

Section: Optical Coherence Tomography (Oct)mentioning

confidence: 99%

Naturally occurring rhodopsin mutation in the dog causes retinal dysfunction and degeneration mimicking human dominant retinitis pigmentosa

Kijas

Cideciyan

Alemán

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

150

109

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Rhodopsin is the G protein-coupled receptor that is activated by light and initiates the transduction cascade leading to night (rod) vision. Naturally occurring pathogenic rhodopsin (RHO) mutations have been previously identified only in humans and are a common cause of dominantly inherited blindness from retinal degeneration. We identified English Mastiff dogs with a naturally occurring dominant retinal degeneration and determined the cause to be a point mutation in the RHO gene (Thr4Arg). Dogs with this mutant allele manifest a retinal phenotype that closely mimics that in humans with RHO mutations. The phenotypic features shared by dog and man include a dramatically slowed time course of recovery of rod photoreceptor function after light exposure and a distinctive topographic pattern to the retinal degeneration. The canine disease offers opportunities to explore the basis of prolonged photoreceptor recovery after light in RHO mutations and determine whether there are links between the dysfunction and apoptotic retinal cell death. The RHO mutant dog also becomes the large animal needed for preclinical trials of therapies for a major subset of human retinopathies. R hodopsin, the visual pigment of rod photoreceptors, is one of many G protein-coupled receptors involved in human disease (1-3). Numerous rhodopsin gene (RHO) mutations (mostly point mutations) cause retinitis pigmentosa (RP), a blinding human retinal degeneration (see RetNet, http:͞͞ www.sph.uth.tmc.edu͞Retnet͞; refs. 4 and 5). RP caused by RHO mutations exhibits two different phenotypes (4, 5). One is an early-onset disorder with rapid loss of rods uniformly across the retina. The second has a protracted natural history of vision loss and puzzling features: rod vision can be normal early in life, and degeneration slowly spreads from a disease focus in one retinal region. Furthermore, and independent of disease stage, there is abnormally slow recovery of rod vision after bright light exposure. Variation in severity of this second phenotype may indicate that epigenetic factors play an important role in its progression (5-7) and suggests that it may be particularly amenable to preventive or ameliorative therapies.Other than in humans, naturally occurring disease-causing RHO mutations have not been identified previously in mammals. Genetically engineered animals and mutagenized flies have been the mainstay for in vivo research and treatment attempts in the past decade (reviewed in ref. 8).Naturally occurring hereditary retinal degenerations in dogs, termed progressive retinal atrophies (PRAs), are widespread and have provided several models of autosomal recessive and X-linked RP (9-13). We have now identified an autosomal dominant form of PRA, one that closely resembles the second human phenotype described above. This model should advance understanding of the pathophysiology of the disease and therapies for this major subset of dominant RP. Materials and MethodsRhodopsin Gene Sequence Analysis. Mutation analysis by comparative sequencing of the five c...

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

Cited by 221 publications

References 38 publications

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Dark Rearing Rescues P23H Rhodopsin-Induced Retinal Degeneration in a TransgenicXenopus laevisModel of Retinitis Pigmentosa: A Chromophore-Dependent Mechanism Characterized by Production of N-Terminally Truncated Mutant Rhodopsin

Naturally occurring rhodopsin mutation in the dog causes retinal dysfunction and degeneration mimicking human dominant retinitis pigmentosa

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