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At least 289 records · Page 16Linked to original sources
PubMed1993

Role of anti-recoverin autoantibodies in cancer-associated retinopathy.

PURPOSE: To examine the retina and test the serum of a patient with cancer-associated retinopathy syndrome who was diagnosed with small cell carcinoma of the lung and experienced unexpected visual loss. METHODS: Proteins from normal human retina were extracted, separated by one- and two-dimensional gel electrophoresis, transferred to PVDF membrane, and used for immunostaining. Antibody specificity was determined by use of solid-phase peptides in a solid-phase immunoassay. RESULTS: Histologic examination of the retina showed loss of the photoreceptor cell layer. This finding correlated with the results of clinical (loss of vision) and electrophysiologic (abnormal electroretinograph [ERG]) tests. The patient's serum antibodies specifically recognized recoverin, a protein predominantly found in retinal photoreceptor cells. The patient's serum also labeled some higher molecular weight proteins present in normal lung and other normal tissues, as well as in lung cell carcinoma cell lines. The only other tissue in which immunoreactivity against p23 could be found was the optic nerve. Our data revealed a lack of cross-reactivity between specific anti-recoverin antibodies and lung proteins. The results indicate that the patient serum contains more than one type of antibody activity. The autoantibodies were tested for fine immunospecificity by use of solid-phase peptides in a solid-phase immunoassay. Patient's antibodies reacted with a major determinant located in the recoverin sequence 62-68 (PKAYAQH) and with several minor ones. CONCLUSION: Based on the fact that the recoverin appears to be distributed in several different cell types, we suggest that this protein may be present in cancer cells and may play a role in the pathogenesis of some cancer-associated retinopathies.

Amino Acid Sequence↗
PubMed1995

Rem-1, a putative direct target gene of the Myb-Ets fusion oncoprotein in haematopoietic progenitors, is a member of the recoverin family.

The Myb-Ets oncoprotein encoded by the E26 avian leukaemia virus represents a fusion of two transcription factors which cooperate in transforming multipotent haematopoietic progenitors (MEPs) in vitro and in vivo. Previous studies with a temperature sensitive mutant in ets (ts1.1 E26) have suggested that the Ets part of the Myb-Ets fusion protein blocks multilineage differentiation of transformed MEPs, by regulating specific target genes. Using this system in a differential screening approach we have now identified a new gene, called rem-1, as a target for the E26 virus. Following shift of ts1.1 mutant transformed cells to the nonpermissive temperature a decreased expression of rem-1 was observed which increased upon downshift. The finding that this reexpression did not require new protein synthesis suggests that the Ets component of the fusion protein directly regulates rem-1 transcription. Rem-1 is related to a family of EF-hand-containing calcium-binding proteins that are predominantly expressed in the brain and in retinal cells. This family includes recoverin and visinin, proteins that have been implicated in regulating photoreception. Rem-1 is likewise expressed in these tissues but in addition in haematopoietic cells and in the gut. Enforced expression of rem-1 in ts1.1-transformed MEP cells, using a retroviral vector, showed that this gene is not sufficient to block their differentiation, but that it may provide them with a growth advantage.

Amino Acid Sequence↗
PubMed1994

Genetic and physical mapping of human recoverin: a gene expressed in retinal photoreceptors.

PURPOSE: Recoverin is a calcium-binding protein that may be involved in phototransduction in mammalian retinal photoreceptors, and is considered to be a candidate gene for retinitis pigmentosa. This study was undertaken to develop the recoverin locus into a polymorphic marker for future linkage studies on retinitis pigmentosa families. METHODS: A human genomic cosmid clone was isolated and used to map the recoverin gene to a human chromosome through hybridization to a panel of somatic hybrid cell line DNAs, and to human metaphase chromosomes by fluorescence in situ hybridization. A dinucleotide repeat polymorphism located within the coding region of the recoverin gene was identified, and used to genetically map the recoverin gene relative to index markers. In addition, three restriction fragment length polymorphisms revealed by the cosmid clone were identified and characterized. RESULTS: Hybridization to the somatic hybrid cell line DNAs localized the recoverin gene to chromosome 17. Recoverin was further localized to 17p12-p13 by fluorescence in situ hybridization. The dinucleotide repeat polymorphism and restriction fragment length polymorphisms at the recoverin locus have a cumulative polymorphic information content = 0.71. CONCLUSIONS: These polymorphic markers and additional closely linked markers will be useful for linkage analysis of families with retinitis pigmentosa.

Base Sequence↗
PubMed1994

[Function of the calcium-binding protein p26 (recoverin) in bovine retinal rods].

The bovine retina rod cell protein with an apparent M(r) 26 kDa (p26 [1] or recoverin [2]) was suggested to be a calcium-sensitive regulator of photoreceptor guanylate cyclase. The data obtained show that highly purified p26 is not capable of restoring guanylate cyclase in washed ROS membranes up to the level of a ROS suspension. At the same time we found that a Ca(2+)-sensitive complex of p26 and a protein with apparent M(r) 67 kDa, presumably rhodopsin kinase, is present in the ROS extract. We suggest that rhodopsin kinase can be a functional target for p26 in retina rod cells.

Animals↗
PubMed1994

Expression of soluble phototransduction-associated proteins in ground squirrel retina.

PURPOSE: This study describes the expression and distribution of arrestin, phosducin, and recoverin in the cone-dominant retina of the ground squirrel Spermophilus tridecemlineatus. METHODS: mRNA expression was studied by blot hybridization of ground squirrel retinal RNA, with human and murine RNA as controls. The distribution of the gene products in the ground squirrel retina was investigated by immunocytochemistry using radial and consecutive tangential sections. RESULTS: Northern blot hybridization showed messages for arrestin (1.9 kb), phosducin (1.4 kb), and recoverin (1.2 kb) in ground squirrel retinal RNA. Both controls showed transcripts of the same or similar sizes. Rod-like cells and blue cones were stained by antibodies against arrestin and phosducin. The arrestin antiserum stained the whole cell bodies, most intensely in the myoid region, whereas phosducin immunoreactivity was confined to the outer and inner segments, which were stained with approximately equal intensity. The strongest immunoreaction was found in the photoreceptor plasma membrane. Recoverin antibodies recognized the entire soma of all photoreceptor cells. The myoid region and the synaptic pedicles were most heavily stained. No light-dependent migration was observed with either antiserum in any photoreceptor type. CONCLUSION: The presence of arrestin immunoreactivity in rod-like cells and blue cones is consistent with previous reports on other mammals. However, it has not been reported previously that phosducin immunoreactivity is distributed in the same way. The colocalization of arrestin and phosducin in rod-like cells and blue cones is yet another trait distinguishing blue cones from red and green cones.

Animals↗
PubMed1994

Recoverin is highly uveitogenic in Lewis rats.

PURPOSE: Recoverin, a calcium-binding protein that selectively localizes to the retina and pineal gland, has been identified as the target for the putative pathogenic autoimmune process of cancer-associated retinopathy (CAR). The present study was aimed at testing the capacity of recoverin to induce experimental autoimmune uveoretinitis and pinealitis in Lewis rats. METHODS: Lewis rats were immunized against recombinant myristoylated recoverin by a single footpad injection of the protein, at various doses, emulsified in complete Freund's adjuvant. Development of uveoretinitis was monitored by clinical and histologic examinations, whereas pinealitis was detected by histologic examination. RESULTS: Immunization with recoverin induced severe panuveitic changes that closely resemble those induced by S-antigen (arrestin). The effect was dose-dependent, with 10 micrograms/rat the lowest immunopathogenic dose. Rats immunized with recoverin also developed pineal inflammation. CONCLUSION: Recoverin is highly immunopathogenic in Lewis rats. Although the ocular changes induced in rats differ from those seen in CAR, the data recorded here are in line with the concept that recoverin can initiate pathogenic autoimmune processes in the eye.

Animals↗
PubMed1994

Heterogeneous N-acylation is a tissue- and species-specific posttranslational modification.

Heterogeneous N-terminal glycine acylation recently has been reported for two proteins involved in visual signal transduction. Similar N-acylations have typically involved only myristate; however, none of the previously examined proteins were isolated from retinas. To determine whether heterogeneous N-acylation is tissue-specific or protein sequence-specific, the N-terminal modifications of the catalytic subunit of cAMP-dependent protein kinase, partially purified from bovine retinas, heart, and brain tissues, were characterized. Using tandem mass spectrometry and liquid chromatography coupled directly to an electrospray mass spectrometer, we found only myristate at the N termini of catalytic subunits from brain and heart tissue, whereas the N termini of the retina-derived subunits were heterogeneously acylated in a manner similar to recoverin and transducin. Thus it appears that the nature of N-terminal glycine acylation is determined by the cell or tissue type in which it is located, and not by the sequence of the modified protein. We also examined the N-acylation of recoverin purified from human retinas, as well as transducin purified from frog retinas, to determine if heterogeneous acylation of retinal proteins is a uniquely bovine phenomenon. Interestingly, human recoverin was modified by the same family of fatty acids found on the bovine retinal proteins, while frog transducin was modified homogeneously not with myristate, but with a doubly unsaturated (C14:2) fatty acyl group.

Acylation↗
PubMed1993

Recoverin has S-modulin activity in frog rods.

In vertebrate photoreceptors, light induces hydrolysis of cGMP by activating cGMP phosphodiesterase (PDE), which results in closure of the cGMP-activated cation channel. During light adaptation, the cytoplasmic Ca2+ concentration decreases, and this decrease is one of the underlying mechanisms of light adaptation. Sensitivity-modulating protein (S-modulin) is a Ca(2+)-binding protein involved in light adaptation in frog rods; it regulates both the light sensitivity of PDE and the lifetime of activated PDE by controlling rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin has been reported as a Ca(2+)-dependent regulator of guanylate cyclase in bovine rods (Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A. Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Here, we show that recoverin has similar activity as S-modulin, and the amino acid sequences of both proteins are similar. The results strongly suggest that recoverin is bovine S-modulin and regulates PDE activation.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Recoverin, but not visinin, is an autoantigen in the human retina identified with a cancer-associated retinopathy.

PURPOSE: We investigated the hypothesis that visinin, a cone-specific protein first characterized in chicken retina, is a cone homologue of recoverin and may be the cancer-associated retinopathy (CAR) autoantigen in human cone cells. METHODS: Visinin was purified from chicken retinas and tested for binding by CAR antisera. In addition, antibodies specific to visinin were used immunocytochemically and for Western analysis to determine whether visinin is present in human or bovine retinas. Anti-peptide antibodies against recoverin were used immunocytochemically to localize recoverin to mammalian cone cells. RESULTS: CAR antisera recognized recoverin but not visinin. Furthermore, visinin could not be detected in mammalian retinas by immunocytochemical methods or by attempts to purify the protein. In contrast to visinin, antibodies specific for different regions of the recoverin molecule stained both rod and cone cells in the human retina. CONCLUSIONS: Visinin is not the CAR autoantigen in human cone cells. Differences between recoverin and visinin probably reflect species differences rather than rod-cone differences. Recoverin, or a nearly identical molecule, is present in mammalian cones and likely is the cone cell CAR autoantigen.

Amino Acid Sequence↗
PubMed1996

[Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E. coli cells and comparison of their functional activity].

A recombinant plasmid was constructed for expressing a gene for bovine recoverin under the control of the lac promoter. Coexpression of the recoverin and N-myristoyl transferase genes was performed to prepare recombinant myristoylated recoverin. The obtained systems provide high levels of biosynthesis of the recombinant recoverins in the E. coli cells. Using a reconstructed system, containing urea-washed rod outer segment membranes, purified rhodopsin kinase (RK), and a recoverin, it was shown that the three recoverin forms (natural, recombinant nonmyristoylated, and recombinant myristoylated ones) perform the calcium-dependent regulation of the activity of RK with half a maximum effect at a free calcium concentration of 2 microM. Interestingly, the N-terminal myristoylation of recoverin increased substantially its functional activity.

Acyltransferases↗
PubMed1997

Apoptotic retinal cell death induced by antirecoverin autoantibodies of cancer-associated retinopathy.

PURPOSE: Recoverin has been identified as a target autoantigen for antirecoverin antibodies found in the sera of some patients with cancer-associated retinopathy. The aim of this study was to investigate the role of antirecoverin antibodies in cancer-associated retinopathy. METHODS: Human, rat, and rabbit antirecoverin antibodies were purified using a recoverin-affinity column. Purified biotinylated antibodies were cultured with recoverin-positive rat retinal cells E1A.NR3. Antibody uptake by retinal cells in vitro was analyzed by immunocytochemistry. Cytotoxic effect of antibodies on retinal cells was measured by the MTT colorimetric method. Apoptosis was shown by the ladder DNA fragmentation method and by fluorescent dye chromatin fragmentation analysis. RESULTS: Antirecoverin antibodies obtained either from sera from five cancer-associated retinopathy patients or from sera of immunized animals were internalized by E1A.NR3 cells. Only specific, antirecoverin antibodies produced destruction of the cells in a dose- and time-dependent manner. Normal immunoglobulin G did not have such effects on retinal cells. No additional cell destruction was observed in the presence of complement as compared with cultures incubated with antirecoverin antibodies alone. Internucleosomal DNA fragmentation and presence of apoptotic cells was observed throughout the culture treated with recoverin specific antibodies but not with normal antibodies. Cells not expressing recoverin (Y79, PC12, and GH3) were not susceptible to cell destruction because of antirecoverin antibody action. CONCLUSIONS: These studies showed that antibodies specific to recoverin are able to enter and cause death of cells expressing recoverin. In humans, autoantibodies originally elicited against recoverin expressed in tumor cells may damage retinal photoreceptors and play a role in the pathogenesis of cancer-associated retinopathy. Results suggest that autoantibody to recoverin, when given access to recoverin in the retina through the blood-retina barrier, could initiate photoreceptor degeneration leading to blindness. Such mechanism may be common for other paraneoplastic disorders or autoimmune diseases where antibodies interfere with the normal cell physiology.

Animals↗
PubMed1997

Evaluation of the human gene encoding recoverin in patients with retinitis pigmentosa or an allied disease.

PURPOSE: To determine whether defects in the human recoverin gene cause retinitis pigmentosa (RP) or an allied disease such as Usher syndrome, Leber congenital amaurosis, or the Bardet-Biedl syndrome. METHODS: Single-strand conformation polymorphism analysis and direct genomic sequencing techniques were used to screen 596 unrelated patients, comprising 167 patients with dominant RP, 168 with recessive RP, and 261 with an allied disease. RESULTS: Four sequence variants were discovered. The first was a missense change (Ala200Thr) found in one family with autosomal dominant RP and in one family with autosomal recessive RP; it did not segregate with disease. The second was a silent, single-base variation affecting codon Ser24 with a minor allele frequency of approximately 0.5%. The third was a silent, single-base variation affecting codon Va1122. The fourth was a single-nucleotide substitution in intron 2, 11 bp upstream of exon 3. CONCLUSIONS: The authors found no evidence that mutations in the recoverin gene are a cause of RP or another of the hereditary retinal diseases studied. The human phenotype associated with mutations of the recoverin gene remains unknown.

Calcium-Binding Proteins↗
PubMed1997

[Paraneoplastic retinopathy in 2 patients with breast carcinoma].

Paraneoplastic retinopathies are rare paraneoplastic phenomena resulting in retinal degeneration. They occur in association with different tumor types, yet most frequently encountered in small cell carcinoma of the lung. Clinical symptoms may be present before the diagnosis of the underlying malignancy. They are characterized clinically by progressive visual loss with ring scotomas, photopsia and night-blindness. An autoimmune disorder is suggested. In the sera of patients antiretinal antibodies may be detected that are sometimes reactive with the 23 kD retinal antigen recoverin, a photoreceptorprotein. We report on two patients with breast cancer who developed paraneoplastic retinopathy during the course of disease. Immunologic tests showed antiretinal antibodies that were not reactive with the 23 kD retinal antigen recoverin.

Autoantibodies↗
PubMed1997

Developmental expression pattern of phototransduction components in mammalian pineal implies a light-sensing function.

Whereas the pineal organs of lower vertebrates have been shown to be photosensitive, photic regulation of pineal function in adult mammals is thought be mediated entirely by retinal photoreceptors. Extraretinal regulation of pineal function has been reported in neonatal rodents, although both the site and molecular basis of extraretinal photoreception have remained obscure. In this study we examine the developmental expression pattern of all of the principal components of retinal phototransduction in rat pineal via cRNA in situ hybridization. All of the components needed to reconstitute a functional phototransduction pathway are expressed in the majority of neonatal pinealocytes, although the expression levels of many of these genes decline dramatically during development. These findings strongly support the theory that the neonatal rat pineal itself is photosensitive. In addition, we observe in neonatal pinealocytes the expression of both rod-specific and cone-specific phototransduction components, implying the existence of functionally different subtypes of pinealocytes that express varying combinations of phototransduction enzymes.

Animals↗