Biomedical subjects

J S Si

Publications and source records attributed to J S Si.

11 recordsLinked to original sources

PubMed1997

Structure-function relationships in interphotoreceptor retinoid-binding protein (IRBP).

PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) binds hydrophobic ligands in the retina. The polypeptide consists of 1230 amino acids in four 300 amino acid long repeats. We asked whether each of the four repeats can bind one retinoid or fatty acid analog. Our rationale was to make protein variants from the human cDNA bearing one or more of the repeats and examine binding capacities and dissociation constants. METHODS: Proteins were characterized by SDS-PAGE, western blotting, N-terminal sequencing, and CD spectroscopy. Binding properties with all-trans-retinol and 16-anthryloxy-palmitic acid (16-AP) were characterized by ligand fluorescence enhancement and curve fitting. RESULTS: Binding capacities varied according to the length of each protein. Each repeat possesses the capability of binding retinol and 16-AP. CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds a retinoid and fatty acid analog, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.

Animals↗

PubMed1997

A major cis activator of the IRBP gene contains CRX-binding and Ret-1/PCE-I elements.

PURPOSE: Interphotoreceptor retinoid binding protein (IRBP) is expressed exclusively and to high levels in photoreceptive cells. This study was an attempt to delineate the minimal regulated control region of the murine IRBP promoter involved in this expression pattern. METHODS: Fragments of the mouse IRBP 5' flanking region were tested for promoter activity in transient transfections of embryonic chick retina cells in primary culture. Electrophoretic mobility shift assays were used to identify specific cis-acting DNA elements within these fragments. RESULTS: Nested deletion analysis of a 1783 bp fragment of the murine IRBP 5' flanking region shows that high promoter activity is maintained with truncated fragments as short as 70 bp 5' to transcription start, but is lost with truncation to 45 bases. The 1783 bp promoter is active in cultures of retina cells but not brain cells or fibroblasts. The 70 bp fragment is active in retina and brain cells but not fibroblasts. Within retina cell cultures, the 1783 bp fragment is active in photoreceptor-like and amorphous or unidentifiable cells whereas the 70 bp is additionally active in multipolar neuron-like cells. The -70 to -45 interval contains Ret-1/PCE-I (AATTAG in the IRBP gene), a proposed retina-specific consensus sequence cis element, and a same-strand reversed copy of this sequence, GATTAA, the consensus binding element of the photoreceptor-specific trans-acting factor CRX. Mutation of either element suppresses promoter activity. Paralleling promoter tissue-specificity, the -70 to -45 fragment binds a sequence-specific protein complex found in retina and brain extracts but not fibroblasts. Mutation of both or either element inhibits this binding. CONCLUSIONS: These data suggest that a trans-acting complex binds a cis-element in the -70 to -45 sequence. This binding fully activates transcription but confers only partial tissue-specificity to IRBP gene expression.

Animals↗

PubMed1997

The 5' flanking regions of IRBP and arrestin have promoter activity in primary embryonic chicken retina cell cultures.

Primary cultures of embryonic chicken cells from various tissues were transiently transfected with plasmid vectors containing reporter genes linked to a 1.8 kb fragment of the mouse interphotoreceptor retinoid-binding protein (IRBP) 5' flanking region, a 1.5 kb fragment of the mouse arrestin 5' flanking region, or a 3.4 kb sequence of the bovine arrestin 5' flanking region. Promoter activity was evident in retina-derived cells, but not in fibroblasts or cells from whole brain. Transfection response also varied with transfection method, plasmid DNA concentration, post-transfection incubation time, and cell density. The data suggest that the primary embryonic chicken retinal cell culture system is a useful tool in studying photoreceptor-specific gene regulation.

Animals↗

PubMed1994

Light deprivation profoundly affects gene expression of interphotoreceptor retinoid-binding protein in the mouse eye.

Ambient light appears to play a role in regulating gene and protein expression of interphotoreceptor retinoid-binding protein (IRBP), a protein that facilitates the transport of retinoids between the neural retina and pigment epithelium in the visual cycle. Pregnant CD-1 mice were placed in the dark approximately 48 hr before parturition, and the pups were reared for 14 days under these conditions. Control animals were reared on a 12 hr light/12 hr dark cycle. Northern blotting of total RNA isolated from whole mouse eyes at post-natal days 7-14 (P7-P14) showed a marked reduction in IRBP message in the light-deprived animals to 10-20% of levels in control animals. Reprobing of the blots for opsin and S-antigen message showed a significant decrease of about 80-90% in opsin message at 5.1 kb but no change in S-antigen message in the eyes of light-deprived mice. Light microscopic examination of the light-deprived mouse retinas showed no apparent abnormalities in morphological development and immunocytochemistry demonstrated normal distribution and levels of IRBP protein. Immunochemical quantitation of IRBP protein confirmed that there was no reduction in light-deprived as compared to normal mouse eyes. Similarly, when adult mice were light-deprived for 14 days, a marked reduction in IRBP message was also observed with no decrease in the amount of IRBP protein. Thus, light deprivation causes a large decrease in IRBP message in the mouse eye, but IRBP protein is not decreased. The dramatic effect of light deprivation on IRBP mRNA and some opsin mRNAs, but not on S-antigen message and the fact that IRBP protein levels are relatively unaffected, suggest a complex pathway of light regulation of photoreceptor function previously not encountered. This may involve regulatory controls at levels including gene transcription, mRNA stability or protein degradation that may make use of a feedback control mechanism involving light- or dark-dependent signal transduction.

Animals↗

PubMed1994

An early decrease in interphotoreceptor retinoid-binding protein gene expression in Abyssinian cats homozygous for hereditary rod-cone degeneration.

Levels of interphotoreceptor retinoid-binding protein (IRBP) protein and message in retinas of Abyssinian cats homozygous for progressive rod-cone degeneration were determined at early ages, well before the onset of clinical retinal degeneration. IRBP gene expression was assessed by immunochemical quantitation of IRBP protein, and by Northern blotting and slot-blotting of total RNA using a human IRBP cDNA probe. Morphology was assessed by electron microscopy and immunocytochemistry. Levels of both IRBP protein and message in affected Abyssinian cat retinas were significantly reduced below normal as early as 4 weeks of age at the earliest stage of retinal disorientation. Opsin mRNA was more abundant in affected Abyssinian cat retinas than in control retinas. This was at least 1 year before the onset of clinical symptoms. The reduction in IRBP gene expression to levels significantly below normal well before the onset of retinal degeneration in affected Abyssinian cat retinas indicates that this represents a primary defect or at least an early problem that could itself cause adverse effects.

Animals↗

PubMed1994

Biochemical and biophysical properties of recombinant human interphotoreceptor retinoid binding protein.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds and transports retinoids and fatty acids in the interphotoreceptor space (IPS). To understand the relationship between the protein structure and its functions requires bulk quantities of human IRBP. The authors sought to produce recombinant human IRBP (rhIRBP), a perfect duplicate in amino acid sequence of the authentic human protein. This material could serve as a supply of the protein and later could be used to make mutants of the protein. The goals of the present study were to produce human IRBP in an expression system and to examine some of its biochemical properties. METHODS: A cDNA encoding human IRBP was cloned into the transplacement vector, pVL1392, and the plasmid was recombined with linearized baculovirus on cotransfection into Sf9 cells. Viruses containing the human IRBP cDNA were identified by polymerase chain reaction analysis. IRBP was secreted from virus-infected insect cells. rhIRBP was purified from cell medium and was examined by chromatography, N-terminal protein sequencing, immunologic techniques, and fluorometry. Eyecup and retina washes of human donor eyes provided a source of authentic human IRBP (IPS-IRBP). RESULTS: rhIRBP and IPS-IRBP exhibit similar elution profiles on concanavalin A, ion-exchange, and size exclusion chromatography. rhIRBP contains a five-amino-acid propeptide at the N-terminus as deduced from the cDNA sequence. Retinol binding of rhIRBP has been characterized by fluorometric titration. The dissociation constant is approximately 1.04 microM, close to that reported for bovine IRBP. By scanning fluorometry, the emission and excitation maxima are 479 nm and 339 nm, respectively. CONCLUSIONS: The baculovirus system provides an excellent method to produce and secrete human IRBP. The recombinant protein can be readily purified from cell culture medium. Its behavior in chromatography and in binding studies suggests that the recombinant protein is virtually identical to the authentic protein. This validates its use in place of IRBP from human donor eyes. Small, but significant, differences in biochemical properties in comparing human and bovine material highlight the significance of studying the human protein.

Amino Acid Sequence↗

PubMed1992

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas.

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) was quantitatively assessed in retinas of normal and rd mutant mice using short-term organ culture with [35S]methionine. Retinas were studied at ages P9-P12, time points prior to and immediately after the onset of the degeneration of the rd retina. Soluble proteins of the retinal pellet and the incubation medium were subjected to SDS-polyacrylamide gel electrophoresis. Analysis of labeled protein bands utilized a radioactivity scanning system to quantify [35S]methionine incorporation into newly synthesized IRBP. The synthesis and secretion into the incubation medium of IRBP by rd mouse retinas was comparable to normal retinas at P9-P10 but decreased by more than 50% by P12. IRBP mRNA levels were evaluated in retinas of normal and rd mice ages P7-P14. Although IRBP mRNA expression increased in the rd mouse through P10, it decreased markedly thereafter. Previously reported immunocytochemical studies suggested that IRBP was not secreted in the rd mouse retina. The results of this study indicate, however, that rd mouse retinas, when removed from the eye, have the capacity to synthesize and secrete IRBP.

Animals↗

PubMed1992

A molecular perspective on mammalian evolution from the gene encoding interphotoreceptor retinoid binding protein, with convincing evidence for bat monophyly.

The evolutionary relationships of the various orders of placental mammals remain an issue of uncertainty and controversy. Molecular studies of mammalian phylogeny at the DNA level that include more than just a few orders are still relatively meager. Here we report results on mammalian phylogeny deduced from the coding sequence of the single-copy nuclear gene for the interphotoreceptor retinoid binding protein (IRBP). Analysis of 13 species representing eight eutherian orders and one marsupial yielded results that falsify the hypothesis that megachiropteran bats are "flying primates," only convergently resembling microchiropteran bats. Instead, in agreement with more traditional views, as well as those from other recent molecular studies, the results strongly support a monophyletic Chiroptera (micro- and megabats grouped together). The IRBP results also offer some rare molecular support for the Glires concept, in which rodents and lagomorphs form a superordinal grouping. Also in congruence with other recent molecular evidence, IRBP sequences do not support the view of a superorder Archonta that includes Chiroptera along with Dermoptera (flying lemur), Scandentia (tree shrew), and Primates. IRBP was not however, without its shortcomings as a molecular phylogenetic system: high levels of homoplasy, evident in the marsupial outgroup, did not allow us to properly root the tree, and several of the higher level eutherian clades were only weakly supported (e.g., a Carnivora/Chiroptera clade and an Artiodactyla/Carnivora/Chiroptera clade). We suggest that these shortcomings may be diminished as the phylogenetic density of the data set is increased.

Amino Acid Sequence↗

PubMed1991

The molecular biology of IRBP: application to problems of uveitis, protein chemistry, and evolution.

In closing, molecular biology has helped us in the study of uveitis, in defining the parts of the IRBP molecule that cause EAU/EAP. Simple northern and Southern blotting experiments can answer significant questions in the study of gene structure and expression and in relating these to genetic eye diseases. Molecular biology has provided answers to several long-standing cell biology question such as: where is IRBP synthesized, where is the gene expressed, and how is the IRBP polypeptide processed? In considering how mother nature invented the IRBP gene, the gene structure suggests some interesting alternative models, and causes us to speculate about how large proteins may have evolved. Finally, the determination of the protein sequence helps to put us in the position to ask and answer questions about the function of IRBP. It also allows us to begin to determine the consequences of a nonfunctional IRBP. We don't know the answers to all these questions yet, but the structural analyses and the isolation of these genes and cDNAs, presented here, united with other powerful biological techniques should provide the answers.

Amino Acid Sequence↗

PubMed1989

Cloning of cDNAs encoding human interphotoreceptor retinoid-binding protein (IRBP) and comparison with bovine IRBP sequences.

We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.

Amino Acid Sequence↗

PubMed1989

Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone.

We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.

Amino Acid Sequence↗