Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.

Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.

The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. Due to the tight binding of retinol within a substantially apolar binding site, an intense fluorescence emission characterizes the RBP-bound vitamin. The addition of TTR to the retinol-RBP complex (holoRBP) causes a marked increase in the fluorescence anisotropy of the RBP-bound retinol within the system, due to the formation of the holoRBP-TTR complex, which allows the interaction between the two proteins to be monitored. The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

BACKGROUND: Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling. RESULTS: The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each approximately 300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 microM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites. However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules. CONCLUSION: Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle.

Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate.

Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell-mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)-specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor-kappaB (NF-kappaB), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor alpha and interleukin-1beta were suppressed in eyes of PDTC-treated EAU mice. However, when T cells from PDTC-treated EAU mice, Ag-presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell-proliferative response and cytokine production. Pretreatment of Ag-primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector-phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF-kappaB pathway in the lesion could be a novel target for the successful control of uveoretinitis.

Integrating protein structures and precomputed genealogies in the Magnum database: examples with cellular retinoid binding proteins.

BACKGROUND: When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. RESULTS: The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1) multiple sequence alignments, 2) mapping of alignment sites to crystal structure sites, 3) phylogenetic trees, 4) inferred ancestral sequences at internal tree nodes, and 5) amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. CONCLUSION: We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural bioinformatics resources that are useful for identifying experimentally testable hypotheses about the molecular basis of protein behaviors and functions, as illustrated with the examples from the cellular retinoid binding proteins.

Complexes between recombinant intracellular carriers of vitamin A and their specific ligands investigated by electrospray-mass spectrometry.

The intracellular carriers of vitamin A, cellular retinol-binding protein type I, cellular retinol-binding protein type II and cellular retinoic acid-binding protein type I are members of the intracellular lipid-binding proteins family, in which the ligand-binding cavity is located in the interior of a barrel-like structure. The dissociation constants of the specific complexes in water solutions around neutrality are very low (in the 0.1 to 10 nM range). Because of their high stability, they represent ideal systems to verify the adequacy of electrospray ionization-mass spectrometry in the analysis of non-covalent protein-ligand complexes. The electrospray interface parameters were varied to detect the presence of species not present in solution but generated as artefacts during transfer of complexes from the condensed state to the gas-phase. The results clearly indicate that mass-spectrometry data reflect the situation present in solution only if the electrospray conditions are carefully selected. In particular, the values of cone voltage and temperature compatible with persistence of the complexes in the gas phase were determined for each vitamin A carrier. Lack of correlation between complex stability in solution and in the gas phase is attributable to the specific and differential effects of the two environments on protein conformation and ligand-protein interactions.

A novel retinol-binding protein in the retina of the swallowtail butterfly, Papilio xuthus.

Retinoid-binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol-binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol-binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand-binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplasmic matrix. All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes. Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.

Structure and backbone dynamics of Apo- and holo-cellular retinol-binding protein in solution.

Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo- and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo- and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligand-binding site. These comprise alpha-helix II, the subsequent linker to beta-strand B, the hairpin turn between beta-strands C and D, and the betaE-betaF turn. The internal backbone dynamics, obtained from 15N relaxation data (T1, T2, and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the betaC-betaD and betaE-betaF turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment.

Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues.

Retinoids are important regulatory signaling molecules during embryonic development. The molecular properties of rainbow trout (Oncorhynchus mykiss) retinol-binding protein (rtRBP), the specific retinol carrier in vertebrate plasma, were studied to elucidate its role in transporting retinols to developing fish oocytes. A 954-nucleotide rtRBP cDNA was cloned from the liver coding for a 176-amino-acid (aa) mature protein, with an estimated molecular mass of 20,267 Da. The nucleotide sequence suggests a putative 16-aa signal peptide and shows all the aa residues that were previously identified as critical for the retinol binding pocket. Five of the eight amino acid residues that are associated with the interaction of RBP and transthyretin in mammalian and non-mammalian species are conserved. The deduced aa sequence of rtRBP shows 60-66% identity with zebrafish, chicken, mouse, rat, horse, bovine, and human RBPs and 56% identity with Xenopus RBP. Northern blot analysis revealed a approximately 1.1-kb hepatic mRNA transcript. RBP is highly expressed in the liver, but low levels were also detected in the spleen, kidney, ovary, and brain. In the rainbow trout, 17beta-estradiol treatment led to a decrease in the RBP mRNA signal relative to that of the controls. The efficacy of the 17beta-estradiol treatment was verified by an induction of vitellogenin (VTG) mRNA expression in the liver and occurrence of VTG in the plasma.

Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide.

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.

Gecko iota-crystallin: how cellular retinol-binding protein became an eye lens ultraviolet filter.

Eye lenses of various diurnal geckos contain up to 12% iota-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. iota-Crystallin could be either the gecko's housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with iota-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the iota-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant iota-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (approximately 1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. iota-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

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.

The atom assignment problem in automated de novo drug design. 4. Tests for site-directed fragment placement based on molecular complementarity.

Three previous papers in this series have outlined an optimization method for atom assignment in drug design using fragment placement. In this paper the procedure is rigorously tested on a selection of five ligand-protein co-crystals. The algorithm is presented with the molecular graph of the ligand, and the electrostatic/hydrophobic potential of the site, with the aim of creating a placement on the molecular graph which is as electrostatically complementary or hydrophobically similar to the site as possible. Various designer options were tested, including, where appropriate, hydrogen bonding and a restricted number of halogens. In most cases, the placement obtained was at least as good as the native ligand, if not significantly better.

Changes in equine endometrial retinol-binding protein RNA during the estrous cycle and early pregnancy and with exogenous steroids.

A cDNA library was constructed from poly(A) RNA obtained from Day 14 nonbred equine endometrium. A cDNA probe for porcine retinol-binding protein (RBP) was used to screen the library, and a complete cDNA sequence (1133 bp, excluding the poly(A) tail) was obtained. Endometrial biopsies were obtained from cycling, nonbred mares at Days 0, 1, 4, 8, 10, 11, 13, and 15 and from pregnant mares at Days 11, 13, 15, and 17 after ovulation (n = 2 mares each day). Endometrial biopsies were also taken from 18 noncycling anestrous mares after the following treatments: C (vehicle control for 1 day, n = 3), E (estradiol-17 valerate, 5 mg/day for 6 days, n = 3), P (progesterone, 250 mg/day for 6 days, n = 4), ESP (E for 6 days followed by, P for 6 days, n = 4), and ELP (E for 6 days followed by P for 12 days, n = 4). Northern blot analyses were performed on total RNA (30 micrograms) using cDNA probes to equine (e) RBP and human glyceraldehyde-3 phosphate dehydrogenase (G3PDH). The RBP RNA levels (normalized to G3PDH) from nonbred mares were low during early diestrus and increased after Day 10, and RBP RNA levels from pregnant mares were similar to those of nonbred mares for corresponding days. E tended to decrease endometrial RBP RNA; and P, ESP, and ELP increased it compared to C. There were no significant differences among P, ESP, and ELP RBP RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine VEG proteins and tear prealbumins.

Small soluble proteins, belonging to the lipocalin family are secreted in large amounts by tongue von Ebner's glands and lachrymal glands. In humans, the lingual protein, called VEG, and the lachrymal protein, called tear prealbumin, have shown identical cDNA sequences. In the pig, we have purified homodimeric proteins with subunits of 17 kDa, both from von Ebner's glands and from lachrymal glands. In both cases, the proteins can be resolved into two isoforms on a chromatofocusing column. Partial aminoacid sequences and full cDNA sequences have been obtained for the more abundant forms purified from both tissues. The two proteins appear to be identical, as in humans. The reason why the same protein is expressed in different tissues, as well as its physiological function, still remain to be clarified.

Induction of unresponsiveness in adult rats by immunodominant and nondominant peptides.

Intravenous (i.v.) administration of antigens in aqueous solution is an efficient procedure for inducing unresponsiveness in adult animals. Using this procedure, we have compared the unresponsiveness-inducing capacity of an immunodominant and a nondominant peptide derived from the sequence of a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). The immunodominant peptide, R16, is highly immunogenic in the Lewis rat, while the nondominant peptide, R4, is much less immunogenic and requires higher doses to induce immune response. Pretreatment of Lewis rats with peptides R4 or R16 effectively induced unresponsiveness, shown by the reduced lymphocyte responses following the challenge with the same peptide in adjuvant emulsion. A correlation was found between the doses of each peptide needed to induce immunity or unresponsiveness: both of these doses had to be higher for peptide R4 than for R16. Remarkably, the minimal unresponsiveness-inducing doses of both peptides were lower than those used for the challenge. Pretreatment with the immunodominant peptide, R16, readily inhibited the response to this peptide by lymphocytes from rats challenged with whole IRBP and excessive doses of R16 even affected the response of these rats against whole IRBP. In contrast, R4, which is not recognized by cells of rats challenged with whole IRBP, had no effect on the responses of these rats. The data thus underscore the relationship between immunodominance and unresponsiveness-inducing capacity of peptides and support the notion that the processes of unresponsiveness induction are related to those that produce immunity.

Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli.

Comparative 19F NMR studies were performed on rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBPII) to better understand their role in intracellular retinol metabolism within the polarized absorptive epithelial cells (enterocytes) of the intestine. Efficient incorporation of 6-fluorotryptophan (6-FTrp) into these homologous proteins was achieved by growing a tryptophan auxotroph of Escherichia coli, harboring prokaryotic expression vectors with either a full-length rat CRBPII or CRBP cDNA on defined medium supplemented with the analog. It is possible to easily distinguish resonances corresponding to 6-FTrp-apoCRBP, 6-FTrp-CRBP-retinol (or retinal), 6-FTrp-apoCRBPII, and 6-FTrp-CRBPII-retinol (or retinal). We were thus able to use 19F NMR spectroscopy to monitor transfer of all-trans-retinol and all-trans-retinal between CRBPII and CRBP in vitro. Retinol complexed to CRBPII is readily transferred to CRBP, whereas retinol complexed to CRBP is not readily transferred to CRBPII. We estimated that the Kd for CRBP-retinol is approximately 100-fold less than the Kd for CRBPII-retinol. Transfer of all-trans-retinal occurs readily from CRBPII to CRBP and from CRBP to CRBPII. Results from competitive binding studies with retinol and retinal indicated that there is a much larger difference between the affinities of CRBP for retinol and retinal than between the affinities of CRBPII for these two ligands. However, the differences in binding specificities reflect differences in how the two proteins interact with retinol, rather than with retinal. 19F NMR analysis of recombinant isotopically labeled proteins represents a sensitive new and useful method for monitoring retinoid flux between the CRBPs in vitro.