Biomedical subjects

F Gonzalez-Fernandez

Publications and source records attributed to F Gonzalez-Fernandez.

At least 19 recordsLinked to original sources

PubMed2002

Evolution of the visual cycle: the role of retinoid-binding proteins.

The trafficking of retinoids in the retina represents a model to study soluble hormone-binding proteins in a complex system subject to profound evolutionary adaptations. Although a remarkable illustration of convergent evolution, all visual systems detect light in the same way, that is through the photoisomerization of an 11-cis retinoid to a corresponding trans isomer. What is strikingly different between the systems, is the mechanism by which the 11-cis chromophore is reformed and visual pigment regenerated in a process known as the visual cycle. The variations of the cycle address a problem inherent to retinoids themselves. That is, the properties that make these molecules suited for light detection also account for their susceptibility to oxidative and isomeric degradation, and cellular toxicity. The cycle therefore provides an opportunity to examine the role of soluble hormone-binding proteins within an integrative and evolutionary context. The present review focuses on interphotoreceptor retinoid-binding protein (IRBP), a controversial glycolipoprotein that recruits a protein fold common to Cterminal-processing proteases and the crotonase family. This unorthodox retinoid-binding protein is entrapped in the subretinal compartment of those eyes that translocate visual cycle retinoids between the photoreceptors and the retinal pigment epithelium. Recent studies suggest that we should look beyond a strictly carrier function if we are to appreciate the role of IRBP in the visual cycle. Here we draw lessons from other soluble hormone-binding proteins to anticipate avenues of future research likely to provide insight into the structure and function of IRBP in vision.

Amino Acid Sequence↗

PubMed2001

The functional unit of interphotoreceptor retinoid-binding protein (IRBP)--purification, characterization and preliminary crystallographic analysis.

To define the relationship between the structure and function of interphotoreceptor retinoid-binding protein (IRBP) we seek to prepare crystals of IRBP suitable for X-ray crystallographic analysis. As recent studies suggest that each of IRBPs four homologous regions or modules possess ligand-binding activity, we here explore the feasibility of preparing crystals from an individual module. Xenopus laevis IRBP, which has a similar four-module structure as that of mammalian and avian IRBPs, was selected for these studies in view of the advantage of the Xenopus retina for cellular and transgenic approaches. In the present study we focused on the second module of Xenopus IRBP. This module was expressed as a thioredoxin/histidine-patch fusion protein to promote its soluble expression in Escherichia coli and subsequent purification. The ligand-binding properties of the fusion protein were determined by fluorescence spectroscopy. For the preparation of crystals, the module was enzymatically separated from the fusion tag. Crystals of the native and selenomethionine derivatized module were prepared by vapor diffusion in hanging drops. Module II of IRBP binds 1.57 +/- 0.041 and 1.49 +/- 0.15 equivalents of at all- trans retinol and 9-(9-anthroyloxy) stearic acid, respectively, with KDs in the 0.1 microM range. Crystals of this module had an elongated rectangular beam-like morphology. A complete dataset of a frozen selenomethionine crystal extending to 1.85 A resolution was collected. Focusing on the individual modules will likely provide an effective strategy to correlate biochemical and structural data to define the functional domains of IRBP. The quality and resolution of the data obtained suggests that it will be possible in the near future to solve the X-ray crystal structure of the IRBP modules.

Animals↗

PubMed2001

Orbital lipogranulomatous inflammation harboring Mycobacterium abscessus.

PURPOSE: A case of iatrogenic Mycobacterium abscessus orbital infection is presented to call attention to its distinct histopathologic appearance, mechanism of organism, virulence, and clinical management. METHODS: Clinicopathologic analysis of an orbital infection caused by M. abscessus is described. Detailed histologic analysis is performed to provide insight into the mechanism of infection and correlate the features of the orbital infection with that of atypical mycobacteriosis at other body sites. RESULTS: A 71-year-old woman had an orbital mass in the supranasal orbit after a blepharoplasty. The mass consisted of a dimorphic inflammatory reaction with a superficial purulent reaction and a deeper granulomatous process consisting of epithelioid tubercles, each centered about a lipid vacuole. Acid-fast bacilli were found in the lipid vacuoles but not elsewhere in the specimen. Each tubercle was surrounded by a zone of lymphocytes and a desmoplastic reaction. Microbiologic culture studies identified M. abscessus. A combination of surgical intervention and antibiotic therapy for 4 weeks eliminated the infection. CONCLUSIONS: Acid-fast stains should be performed on any orbital lesion showing an apparent lipogranulomatous reaction. Although clinical management of orbital atypical microbacteriosis is difficult, the combination of surgical and specific antimicrobial intervention is effective. Our study contributes to an evolving understanding of the mechanism of human infectivity of these low-virulence organisms by suggesting that the orbital fat is a source of lipid material that can harbor the organisms, allowing them to escape host immunosurveillance.

Aged↗

PubMed2001

Trilateral retinoblastoma variant indicative of the relevance of the retinoblastoma tumor-suppressor pathway to medulloblastomas in humans.

Results of recent studies have led investigators to suggest that the retinoblastoma tumor-suppressor (rb) gene plays an underappreciated role in the genesis of brain tumors. Such tumors cause significant rates of mortality in children suffering from hereditary retinoblastoma. It has been assumed that the pineal gland, which is ontogenetically related to the retina, accounts for the intracranial origin of these trilateral neoplasms. To address this issue, the authors describe an unusual trilateral retinoblastoma variant. The authors provide a detailed clinicopathological correlation by describing the case of a child with bilateral retinoblastoma who died of a medulloblastoma. The intraocular and intracranial neoplasms were characterized by performing detailed imaging, histopathological, and postmortem studies. Karyotype analysis and fluorescence in situ hybridization were used to define the chromosomal defect carried by the patient and members of her family. An insertion of the q12.3q21.3 segment of chromosome 13 into chromosome 18 at band q23 was identified in members of the patient's family. This translocation was unbalanced in the proband. The intraocular and cerebellar neoplasms were found to be separate primary neoplasms. Furthermore, the pineal gland was normal and the cerebellar neoplasm arose within the vermis as a medulloblastoma. Finally, the two neoplasms had different and characteristically identifiable cytolological and immunohistochemical profiles. The findings of the present study, taken together with those of recent molecular and transgenic studies, support the emerging concept that rb inactivation is not restricted to central nervous system regions of photoreceptor lineage and that inactivation of this tumor suppressor pathway may be relevant to the determination of etiological factors leading to medulloblastoma in humans.

Cerebellar Neoplasms↗

PubMed2000

Xenopus IRBP, a phylogenetically remote protein, is uveitogenic in Lewis rats.

Mammalian interphotoreceptor retinoid-binding proteins (IRBPs) are highly uveitogenic in Lewis rats. Xenopus laevis IRBP resembles mammalian IRBP in its four-fold structure, and has approximately 70% amino acid sequence identity with the bovine protein. This study investigated the uveitogenicity of recombinant Xenopus IRBP and two of its derived peptides in Lewis rats. Rats immunized with Xenopus IRBP developed uveoretinitis as well as pineal inflammation. The Xenopus molecule was, however, less immunopathogenic than the bovine IRBP. Of the two Xenopus IRBP peptides tested, 1180-1191 was remarkably uveitogenic, whereas sequence 521-540 exhibited low activity. It is assumed, therefore, that as with bovine IRBP, peptide 1180-1191 is the major uveitogenic sequence in Xenopus IRBP. The role individual residues of these peptides play in the immunopathogenic process is discussed. Our data thus demonstrate that despite its being phylogenetically remote, Xenopus IRBP is uveitogenic in Lewis rats

Animals↗

PubMed2000

Coordination between production and turnover of interphotoreceptor retinoid-binding protein in zebrafish.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), which is secreted by the photoreceptors of most vertebrates, is the major soluble protein component of the interphotoreceptor matrix (IPM). Recent studies suggest that IRBP is short lived in the IPM (half-life, approximately 11 hours). The mechanisms coordinating the production and removal of IRBP are not known. Zebrafish provide a useful system to study the regulation of these two processes, because its IRBP mRNA levels are under circadian regulation. In the present study, the relationship between the quantity of IRBP, the rate of its turnover, and the expression of its mRNA in the zebrafish retina were examined. METHODS: Full-length zebrafish IRBP was expressed in Escherichia coli and an antiserum generated against purified recombinant IRBP. Western and protein dot blot analyses and indirect immunofluorescence were used to define the temporal and spatial patterns of IRBP expression in the adult zebrafish. In vivo and in vitro metabolic labeling experiments were used to examine the regulation of IRBP turnover by both environmental light and the light-dark cycle. RESULTS: Despite the known rhythmicity in IRBP mRNA expression, neither the amount of IRBP nor its localization changes significantly during the light-dark cycle. IRBP is rapidly removed from the zebrafish eye (half life, approximately 7 hours). This rapid turnover is independent of environmental lighting conditions during subjective day and is more rapid during the day than at night. CONCLUSIONS: Because the amount of IRBP remains constant throughout the day, the enhanced daytime IRBP mRNA expression may function to compensate for an increased turnover of the protein during the day. These findings suggest that the processes of IRBP production and removal are coordinately regulated.

Animals↗

PubMed1999

11-cis retinol dehydrogenase mutations as a major cause of the congenital night-blindness disorder known as fundus albipunctatus.

PURPOSE: Patients with fundus albipunctatus uniformly experience difficulty with vision at night. Their retinas are spotted with characteristic light yellow flecks of unknown composition that typically spare the macula. A defect in the transport or utilization of visual cycle retinoids is thought to underlie this recessive disorder with variable clinical expression. To elucidate the molecular defect we considered the genes for interphotoreceptor retinoid-binding protein (RBP3) and 11-cis retinol dehydrogenase (RDH5) as candidates for this disease. METHODS: We examined two unrelated families with fundus albipunctatus. The diagnosis was determined clinically and RBP3 and RDH5 were analyzed by molecular screening methods and direct genomic sequencing. RESULTS: Each family had two affected members with typical fundus albipunctatus. The affected members were siblings born to unaffected parents who were seventh cousins in the first family and unrelated in the second family. The probands from both families were clinically similar except for the fundus dots that were more extensive in the second family to the point of involving the parafoveal region. In the initial phase of genetic screening RBP3 defects were ruled-out as the cause of the disease in both families. In contrast, RDH5 mutations were found in the affected siblings in both families. The proband in one had a homozygotic Gly238Trp missense mutation (GGG -> TGG) involving exon 4 and in the other carried compound heterozygotic changes Arg280His (CGC -> CAC) and Ala294Pro (GCC -> CCC) in exon 5. The disease phenotype was only manifested in family members with two abnormal RDH5 alleles consistent with autosomal recessive inheritance in both pedigrees. CONCLUSIONS: These findings strongly implicate defects of RDH5 as the cause of fundus albipunctatus and point to a heterogeneity of RDH5 mutations in this form of congenital stationary night blindness with variable expressivity.

Adult↗

PubMed1998

Arginine to glutamine substitutions in the fourth module of Xenopus interphotoreceptor retinoid-binding protein.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of approximately 300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions. METHODS: To study the arginines in an individual module without the interference of ligand-binding activity elsewhere in the protein, we expressed in E. coli the fourth module of Xenopus IRBP by itself as a soluble thioredoxin fusion protein (X4IRBP). Arginines 1005, 1041, 1073 and 1122 were separately replaced by glutamine using PCR overlap extension mutagenesis. The glutamine substitutions were confirmed by liquid chromatography-tandem mass spectrometry. The binding of all-trans retinol and 9-(9-anthroyloxy)stearic acid (9-AS) to X4IRBP and each of the mutants was evaluated by fluorescence spectroscopy. Binding was followed by monitoring the enhancement of ligand fluorescence and the quenching of protein endogenous fluorescence. The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring absorbance at 325 nm over time. RESULTS: The substitution of Gln for Arg1005 about doubled the amount of ligand necessary to attain saturation and about doubled the level of fluorescence enhancement obtained at saturation for both all-trans retinol and 9-AS. Although there was not a significant change in the Kd, the substitution increased the calculated number of binding sites (N) from approximately 2 to approximately 4 per polypeptide. The other Arg->Gln mutants did not significantly change the Kd or N. None of the mutations compromised the ability of the module to protect all-trans retinol from degradation. CONCLUSIONS: Our data suggest that the function of the conserved arginines in IRBP is fundamentally different from that of other retinoid-binding proteins. These residues do not appear to play a role in defining the specificity of the ligand-binding domain. Rather, Arg1005 appears to play a role in defining the capacity of the domain. Our data suggest that the binding site consists of a single hydrophobic cavity promiscuous for fatty acids and all-trans retinol.

Amino Acid Substitution↗

PubMed1998

Novel expression pattern of interphotoreceptor retinoid-binding protein (IRBP) in the adult and developing zebrafish retina and RPE.

PURPOSE: Interactions between the neural retina and retinal pigment epithelium (RPE) are mediated by the interphotoreceptor matrix (IPM). The transport of retinoids across the IPM is mediated by interphotoreceptor retinoid-binding protein (IRBP). To explore the possibility that IRBP is important during retinal development, we examined its spatiotemporal expression pattern in embryonic zebrafish. METHODS: IRBP mRNA expression was examined using RT-PCR and in situ hybridization. IRBP was localized using antiserum against recombinant zebrafish IRBP. IRBP synthesis and secretion were studied by in vitro metabolic labeling of retinas and RPE-eyecups. RESULTS: IRBP mRNA was first observed in the pineal at 24 hours post-fertilization (hpf) and in the ventral retina at 50 hpf. Immunoreactive IRBP was first observed at 72 hpf. Remarkably, IRBP was expressed not only by photoreceptors but also by the adult and embryonic RPE. In embryos, expression in both retina and RPE began in a ventronasal patch and spread to involve the entire eye. In general, early IRBP expression was dominated by photoreceptors, but then RPE expression spread beyond the limit of photoreceptor expression. Double in situ hybridizations suggests that cones express IRBP mRNA before they express a specific opsin, while rods may express rod opsin prior to IRBP. CONCLUSIONS: The temporal and spatial patterns of IRBP expression by the RPE and retina are consistent with a role in retinal development and suggest coordination of RPE and photoreceptor differentiation.

Animals↗

PubMed1998

Soluble expression in E. coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin: correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases.

The exchange of all-trans retinol and 11-cis retinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed of regions coded for by each of IRBP's four exons. Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins. The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography. Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct. All-trans retinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bound to X4IRBP at 2.20+/-0.29 sites with a KD=1.25+/-0.39. One of the two sites was localized to Exons(2+3) and had a KD=0.26+/-0.13 micron. This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a KD=1.94+/-0.20 micron. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini.

Amino Acid Sequence↗

PubMed1998

Sebaceous carcinoma. Tumor progression through mutational inactivation of p53.

BACKGROUND: Sebaceous carcinoma may masquerade for years as an inflammatory condition. In many cases, this may be because of the presence of longstanding intraepithelial disease (e.g., dysplasia or carcinoma in situ), which eventually progresses to invasive carcinoma recognized through tumefaction and a worsening clinical presentation. The mechanism for this tumor progression is unknown. In the Far East, human papilloma virus (HPV) has been suggested to play a role in the development of sebaceous carcinoma by inactivating tumor suppressor gene p53. Here, the authors explore the molecular basis of the progression of ocular sebaceous carcinoma. METHODS: Cases of sebaceous carcinoma seen at the University of Virginia, Department of Ophthalmology, during the period from 1989 to 1996 were analyzed for HPV infection by in situ hybridization and polymerase chain reaction. The expression of p53, p21WAF-1, Bcl-2, and epithelial membrane antigen was examined by immunohistochemistry. In one of the cases, frozen tumor was available, allowing exons 5 through 9 of the p53 gene to be sequenced. RESULTS: Seven cases were identified, all of which were from women. All were negative for HPV. In cases in which disease was restricted to dysplasia (carcinoma in situ), p53 but not p21WAF-1 was negative. In contrast, cases that contained a component of invasive or metastatic carcinoma showed striking hyperexpression of nuclear p53 in all of the malignant cells. In one of these cases, a G:C-->T:A transversion was found in the p53 gene. This mutation, characteristic of bulky carcinogens, substituted phenylalanine for cysteine 277, a residue that participates in hydrogen bonding to the p53 DNA binding consensus sequence. CONCLUSIONS: Mutational inactivation of p53 may be involved in the progression of sebaceous carcinoma.

Adult↗

PubMed1998

Fourth module of Xenopus interphotoreceptor retinoid-binding protein: activity in retinoid transfer between the retinal pigment epithelium and rod photoreceptors.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.

Animals↗

PubMed1998

Ultrastructural changes in rabbit ciliary body after extraocular mitomycin C.

PURPOSE: To study the ultrastructural changes in ciliary body epithelium of the rabbit eye after subconjunctival injections of mitomycin C. METHODS: One eye of six New Zealand white rabbits was given a subconjunctival injection at the 12-o'clock position with 0.005, 0.02, 0.08, 0.1, 0.12, or 0.16 mg mitomycin C. The fellow eye was given a subconjunctival injection of balanced salt solution. Two weeks after treatment, the eyes were enucleated, and the ciliary body was exposed and submerged in fresh 4% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 degrees C. Electron microscopy of the ciliary body was performed at two sites: the injection site (12-o'clock position) and 180 degrees away (6-o'clock position). RESULTS: At dosages of 0.1 mg and higher, ciliary body epithelial cells beneath the injection site were thinned. There were vacuoles and expansion of intracellular and intercellular spaces. Plasma membrane infoldings were disrupted, and the apical membrane was thinned. Mitochondria and nuclei were normal. Ciliary body epithelium at 6-o'clock position showed only mild architectural distortion of the plasma membrane infoldings. Eyes that received lower doses of mitomycin C (0.005 mg, 0.02 mg, and 0.08 mg) and balanced salt solution showed normal ciliary body epithelium at the injection site and 180 degrees away. CONCLUSIONS: Subconjunctival injection of mitomycin C in the rabbit produces dose-dependent localized ultrastructural changes of the ciliary body epithelium.

Animals↗

PubMed1996

Interphotoreceptor retinoid-binding protein (IRBP): expression in the adult and developing Xenopus retina.

Apposition of the neural retina and pigment epithelium is critical to photoreceptor development and function. Interphotoreceptor retinoid-binding protein (IRBP) is a major component of the extracellular matrix separating these epithelia in the African clawed frog Xenopus laevis (Gonzalez-Fernandez et al., [1993], J. Cell Sci. 105:7-21). In the adult retina, IRBP appears to mediate the transport of hydrophobic molecules, particularly retinoids and fatty acids, within the hydrophilic extracellular domain. In this paper, we compare the distribution of IRBP and its mRNA in adult and embryonic Xenopus retina. Xenopus IRBP antisense RNA, labeled with tritium or digoxigenin, was used for in situ hybridizaton studies. For immunohistochemistry, we used an antiserum against Xenopus IRBP expressed in Escherichia coli. In the adult, we found that IRBP is synthesized at similar levels by both rods and cones. The protein is restricted to the interphotoreceptor matrix, with lesser amounts in the pigment epithelial cytoplasm. In the embryo, expression of the mRNA for IRBP is restricted to the central retina, where photoreceptor differentiation has taken place. By contrast, the protein is distributed throughout the embryonic subretinal space. Therefore, the presence of IRBP precedes photoreceptor differentiation. In summary, IRBP is synthesized by both rods and cones and may be internalized by the pigment epithelium. In the embryo, IRBP is synthesized by the central retina and diffuses through the matrix, reaching the undifferentiated peripheral retina. In view of its ligand-binding properties, diffusion of IRBP may provide the peripheral neural retina with a vehicle to transport retinoids and docosahexaenoic acid (molecules critical to normal retinal development) from the pigment epithelium.

Animals↗

PubMed1996

Zebrafish interphotoreceptor retinoid-binding protein: differential circadian expression among cone subtypes.

Retinoid trafficking between the photoreceptors and pigmented epithelium is probably mediated by interphotoreceptor retinoid-binding protein (IRBP), a 124-145 kDa glycolipoprotein in mammals and amphibians. In these animals, IRBP is composed of four homologous regions (modules) 300 amino acids in length. We have determined the primary structure of zebrafish IRBP and its expression pattern by northern analysis, reverse transcriptase-polymerase chain reaction and in situ hybridization under a variety of lighting conditions. Zebrafish IRBP is half the size (66.3 kDa) of mammalian IRBP because it is composed of only two modules, similar to goldfish IRBP. The first half of the zebrafish protein is most similar to the first module of mammalian IRBP and the second half to the fourth module of mammalian IRBP. This suggests that during the evolution of the ray-finned fish (Actinopterygii), the middle two modules were lost. Each of the modules contains conserved hydrophobic domains which may form the ligand-binding pocket. The expression of zebrafish IRBP mRNA is sevenfold higher in the middle of the light period (at mid-light) than in the middle of the dark period (at mid-dark). This rhythm persists for 2 days under conditions of constant light or constant darkness, then dampens to an intermediate level by 8 days of constant conditions. At mid-light, IRBP mRNA is expressed by all cone types and to a lesser extent by the rods. At mid-dark, the mRNA is restricted to the ultraviolet-sensitive short single cones. These data suggest that IRBP expression is regulated by circadian and light-driven mechanisms that act differentially on the various photoreceptor subtypes in the zebrafish retina.

Amino Acid Sequence↗

PubMed1995

Goldfish cones secrete a two-repeat interphotoreceptor retinoid-binding protein.

Vitamin A and fatty acids are critical to photoreceptor structure, function, and development. The transport of these nutrients between the pigment epithelium and neural retina is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP, a 133-kDa (human) glycolipoprotein, is the major protein component of the extracellular matrix separating these two cell layers. In amphibians and mammals, IRBP consists of four homologous repeats of about 300 amino acids which form two retinol and four fatty acid-binding sites. Here we show that IRBP in teleosts is a simpler protein composed of only two repeats. Western blot analysis shows that goldfish IRBP is half the size (70 kDa) of IRBP in higher vertebrates. Metabolic labeling studies employing Brefeldin A taken together with in situ hybridization studies and the presence of a signal peptide show that goldfish IRBP is secreted by the cone photoreceptors. The translated amino acid sequence has a calculated molecular weight of 66.7 kDa. The primary structure consists of only two homologous repeats with a similarity score of 52.5%. The last repeats of human and goldfish IRBPs are 69.1% similar with hydrophobic regions being the most similar. These data suggest that two repeats were lost during the evolution of the ray-finned fish (Actinopterygii), or that the IRBP gene duplicated between the emergence of bony fish (Osteichthyes) and amphibians. Acquisition of a multirepeat structure may reflect evolutionary pressure to efficiently transport higher levels of hydrophobic molecules within a finite space. Quadruplication of an ancestral IRBP gene may have been an important event in the evolution of photo-receptors in higher vertebrates.

Amino Acid Sequence↗

PubMed1994

Expression and characterization of the fourth repeat of Xenopus interphotoreceptor retinoid-binding protein in E. coli.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 microM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 microM. The expression of recombinant IRBP fragments as fusion proteins in prokaryotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

Animals↗

PubMed1993

Differential expression of interphotoreceptor retinoid-binding protein, opsin, cellular retinaldehyde-binding protein, and basic fibroblastic growth factor.

The mRNA for rat interphotoreceptor retinoid-binding protein (IRBP) consists of 5.2 kb and 6.4 kb transcripts which are expressed early during retinal development. Here, we characterized rat IRBP cDNA and genomic clones, determined the mechanism generating the two transcripts and compared their expression to mRNAs for opsin, cellular retinaldehyde-binding protein (CRAlBP) and basic fibroblast growth factor (bFGF). We found that human and rat IRBPs have a high degree of sequence homology (86% amino acid identity over the fourth repeat). RNA-PCR studies and Northern blot analysis, showed that the 6.4 kb mRNA has a longer 3'-untranslated region (UTR) than the 5.2 kb message. Both IRBP transcripts, but not the mRNA for opsin are present in the neonatal and adult pineal. During development, the mRNAs for IRBP and opsin reach one-half of their maximal levels by P5 and P11, respectively. The ratio of the two IRBP mRNAs remains constant throughout life. In contrast, the ratio of the longest to the shortest opsin mRNA decreases between E19 and 1 year of age. Immunohistochemistry demonstrated a marked increase in the amount of IRBP in the interphotoreceptor matrix between P1 and P9 corresponding to the enhanced expression of this mRNA. Up-regulation of opsin mRNA expression during the second postnatal week corresponds to the emergence of the outer segments. The temporal expression of CRAlBP is similar to IRBP while bFGF is not expressed until after photoreceptor differentiation is complete. In summary, two 3'-transcription termination sites explain the difference in IRBP mRNA sizes. The different temporal and tissue specific patterns of IRBP and opsin expression suggest that separate mechanisms control the expression of these two genes. The expression patterns of IRBP and CRAlBP are consistent with the role of vitamin A as a morphogen and bFGF in neuronal maintenance.

Age Factors↗