The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding.     

Ovomucoid and ovoinhibitor isolated from chicken egg white are immunologically cross-reactive

We report the first application of high pressure liquid chromatography (HPLC) in the rapid detection of cellular retinoic acid binding protein (CRABP) and cellular retinol binding protein (CRBP). Cytosols from cultured cells (3T6 and MCF-7) or from tumors (melanoma and ovarian) were labeled with [3H]retinoic acid (30 Ci/mmol) and [3H]retinol (43 Ci/mmol) and analyzed via HPLC employing a 60 cm TSK 3000 sw column. In each case CRABP and CRBP were readily detectable at an elution volume of 22.5 ml, consistent with their molecular weights of 14,600. Identity of the binding protein peaks was established by saturability, specificity, and selective inhibition of binding by an organomercurial. Thus, this method, which resolves CRABP and CRBP in crude mixtures from the majority of cytosolic proteins, should be a valuable tool in the evaluation of vitamin A-binding protein interactions and their biological significance.     

Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.     

Expression of the cellular retinoic acid binding proteins,type II and type I,in mature rat olfactory epithelium

Cellular retinoic acid binding proteins, types I and II (CRABP I and II), are cytosolic proteins that exhibit a binding preference for all-trans retinoic acid. As part of a larger study to determine whether retinoic acid plays a role in neurogenesis in vivo, we questioned whether CRABP II is present in rat postnatal olfactory epithelium (OE), a sensory tissue that continually replaces neurons throughout adult life. We have determined that both CRABP II and CRABP I proteins and the mRNAs that encode them are present in postnatal rat OE. Immunoreactivity with CRABP II and CRABP I antibodies was not observed in the nasal respiratory epithelium. Double immunolabeling experiments, conducted with antibodies showing specificity for each antigen, indicate that CRABP II and CRABP I are found in different cell types within the olfactory neuroepithelium. We also asked whether CRABP II is expressed in the postnatal rat retina, a neural tissue that is not known to show neuron replacement during adult life. CRABP type II immunoreactivity was not observed in the mature rat retina. The presence of CRABP II in postnatal OE and its absence from mature retina is consistent with previous reports indicating that the distribution of CRABP II in adult mammals is restricted to tissue systems that exhibit ongoing growth and differentiation throughout life.     

Expression of cellular retinoic acid binding protein (CRABP) in Escherichia coli. Characterization and evidence that holo-CRABP is a substrate in retinoic acid metabolism.

Cellular retinoic acid binding protein (CRABP) has been expressed efficiently in Escherichia coli from the cDNA of bovine adrenal CRABP and characterized, especially with respect to affinity for endogenous retinoids and a role for it in retinoic acid metabolism. The purified E. coli-expressed CRABP was similar to authentic mammalian CRABP in molecular weight (approximately 14,700), isoelectric point (4.76), absorbance maxima (apo-CRABP, 280 nm; holo-CRABP, 350 and 280 nm with the ratio A350/A280 = 1.8), and in fluorescence excitation (350 nm) and emission spectra (475 nm). The equilibrium dissociation constant, Kd, of E. coli-derived CRABP and all-trans-retinoic acid was 10 +/- 1 nM (mean +/- S.D., n = 4) by retinoid fluorescence and 7 +/- 1 nM (mean +/- S.D., n = 3) by quenching of protein fluorescence, but neither retinol nor retinal bound in concentrations as high as 7 microM. All-trans-cyclohexyl ring derivatives of retinoic acid (3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, 18-hydroxy-) had affinities similar to that of all-trans-retinoic acid, whereas 13-cis-retinoic acid and 4-oxo-13-cis-retinoic acid had approximately 25-fold lower affinity. Holo-CRABP was a substrate for retinoic acid catabolism in rat testes microsomes by three criteria: 1) the rate of retinoic acid metabolism with CRABP in excess of retinoic acid exceeded the rate supported by the free retinoic acid; 2) increasing the apo-CRABP did not decrease the rate as predicted if free retinoic acid were the only substrate; and 3) holo-CRABP had a lower Michaelis constant (1.8 nM) for retinoic acid elimination than did free retinoic acid (49 nM). These data indicate a direct role for CRABP in retinoic acid metabolism and suggest a mechanism for discriminating metabolically between all-trans- and 13-cis-retinoids.     

Domains of cellular retinoic acid-binding protein I (CRABP I) expression in the hindbrain and neural crest of the mouse embryo.

We describe here the distribution of cellular retinoic acid-binding protein I (CRABP I) in the head of the early mouse embryo from day 8 to day 13 of gestation, using both in situ hybridisation to localise mRNA and immunocytochemistry to localise protein. The distribution of mRNA and protein was found to be identical. CRABP I first appeared in part of the presumptive hindbrain of the presomite embryo and then became localised to rhombomeres 2, 4, 5 and 6. The only other area of expression in the cephalic neuroepithelium was in a part of the midbrain roof. The neural crest and its mesenchymal derivatives, the branchial arches, expressed CRABP I and crest could be seen streaming from the neuroepithelium of individual rhombomeres into particular branchial arches. This suggested a fate map could be constructed describing the rhombomeric origin of branchial arch mesenchyme. Later in development, axons throughout the hindbrain expressed CRABP I. The results are considered in terms of the role of retinoic acid in the specification of neuronal phenotype in the hindbrain and in axon outgrowth.     

Identification of proteins differentially expressed during chondrogenesis of mesenchymal cells

We performed comparative proteome analysis of mesenchymal cells and chondrocytes to identify proteins differentially expressed during chondrogenesis. Nine such proteins were identified. Type II collagen, matrilin-1, carbonic anhydrase-II (CA-II), 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2, and aldo-keto reductase were increased during chondrogenesis, whereas cellular retinoic acid binding protein-I (CRABP-I), CRABP-II, cytoplasmic type 5 actin, and fatty acid binding protein were decreased or almost disappeared. Expression of type II collagen, matrilin-1, PAPS synthetase-2, and CA-II was regulated by extracellular signal-regulated protein kinase, protein kinase C, and p38 kinase, signaling molecules known to regulate chondrogenesis.     

Proteomic identification of differentially expressed genes in neural stem cells and neurons differentiated from embryonic stem cells of cynomolgus monkey (Macaca fascicularis) in vitro

Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from primate ES cells to neural cells. We therefore sought to identify the proteins involved in neurogenesis, from Macaca fascicularis ES cells (CMK6 cell line) to neural stem (NS) cells to neurons using two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), and liquid chromatography-tandem mass spectrometry (LC-MS-MS). During the differentiation of highly homogeneous ES cells to NS cells, we identified 17 proteins with increased expression, including fatty acid binding protein 7 (FABP7), collapsin response mediator protein 2 (CRMP2), and cellular retinoic acid binding protein 1 (CRABP1), and seven proteins with decreased expression. In the differentiation of NS cells to neurons, we identified three proteins with increased expression, including CRMP2, and 10 proteins with decreased expression. Of these proteins, FABP7 is a marker of NS cells, CRMP2 is involved in axon guidance, and CRABP1 is thought to regulate retinoic acid access to its nuclear receptors. Western blot analysis confirmed the upregulation of FABP7 and CRABP1 in NS cells, and the upregulation of CRMP2 in NS cells and neurons. RT-PCR results showed that CRMP2 and FABP7 mRNAs were also upregulated in NS cells, while CRABP1 mRNA was unchanged. These results provide insight into the molecular basis of monkey neural differentiation.     

Lipid binding activities of the P2 protein in peripheral nerve myelin

Lipid binding activities of the P2 protein in peripheral nerve myelin were examined using retinoic acid, retinol and oleic acid as ligands. The P2 protein showed the specific binding affinity to both of retinoic acid and retinol. The binding site of these ligands was suggested to be similar. In addition, the high binding activity of the P2 protein with oleic acid was also observed. The ligands specificities of the P2 protein are clearly different from those of cellular retinoic acid binding protein (CRABP), cellular retinol binding protein (CRBP), and Z protein. In amino terminal sequence, however, the P2 protein contained considerable homologous structure to these lipid binding proteins. Therefore, the P2 protein and these lipid binding proteins may belong to a family of structurally related proteins evolved from a common ancestral gene.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus

Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously.     

The effect of dibutyryl cyclic AMP and butyrate on F9 teratocarcinoma cellular retinoic acid-binding protein activity

F9 teratocarcinoma cells contain a cellular retinoic acid-binding protein (CRABP) that may mediate the retinoic acid-induced differentiation of this cell line. Specific [3H]retinoic acid binding to CRABP in F9 stem cell cytosol is protein-dependent, reaches equilibrium within 4 h at 4 degrees C, and yields 643 +/- 105 fmol of [3H]retinoic acid per mg of protein with an apparent dissociation constant of 9.2 +/- 1.1 nM. When F9 stem cells are grown in the presence of either dibutyryl cyclic AMP or sodium butyrate, CRABP activity is stimulated 2-4-fold. The effect of these drugs on CRABP activity is both time and concentration-dependent, resulting in an increase in the number of binding sites for [3H]retinoic acid with no change in their affinity. The new [3H]retinoic acid-binding sites have a sedimentation coefficient of 2 S and are not displaced by excess retinol. When F9 stem cells are grown in the presence of cyclic 8-bromo-AMP or cholera toxin, no increase in CRABP activity is observed. We conclude that the stimulation of CRABP activity by dibutyryl cyclic AMP may result from the action of butyrate. In addition, the stimulation of retinoic acid-induced F9 cell differentiation by cyclic AMP analogs (Strickland, S., Smith, K.K., and Marotti, K.R. (1980) Cell 21, 347-355) and the inhibition of this differentiation by butyrate (Levine R. A., Campisi, J., Wang, S.-Y., and Gudas, L. J. (1984) Dev. Biol. 105, 443-450) are not correlated with increases or decreases, respectively, in the level of CRABP activity.     

Saturation analysis of cellular retinoid binding proteins: application to retinoic acid resistant human neuroblastoma cells and to human tumors

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28-43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10(-6) M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.     

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori)

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition. Conversely, the cells were sensitive to atRA treatment upon knocking down the BmCRABP expression. Subcellular localization revealed that BmCRABP is a cytoplasm protein, even when treated with atRA, the CRABP still remained in the cytoplasm. These data demonstrated that the function of BmCRABP have an effect on the physiological function of atRA.     

Transporter-to-trap conversion: a disulfide bond formation in cellular retinoic acid binding protein I mutant triggered by retinoic acid binding irreversibly locks the ligand inside the protein

Transport proteins must bind their ligands reversibly to enable release at the point of delivery, while irreversible binding is usually associated with the extreme cases of ligand sequestration. Protein conformational dynamics is an important modulator of binding kinetics, as increased flexibility in the regions adjacent to the binding site may facilitate both association and dissociation processes. Ligand entry to, and exit from, the internal binding site of the cellular retinoic acid binding protein I (CRABP I) occurs via a flexible portal region, which functions as a dynamic aperture. We designed and expressed a CRABP I mutant (A35C/T57C), in which a small-scale conformational switch caused by the ligand binding event triggers formation of a disulfide bond in the portal region, thereby arresting structural fluctuations and effectively locking the ligand inside the binding cavity. At the same time, no formation of the disulfide bond is observed in the apo form of the mutant, and most characteristics of the mutant, including protein stability, are very similar to those of the wild-type protein in the absence of retinoic acid. The mutation does not alter the kinetics of retinoic acid binding to the protein, although the disulfide formation makes the binding effectively irreversible, as suggested by the absence of retinoic acid transfer from the holo form of the mutant to lipid vesicles in the absence of a reducing agent. Taken together, these data suggest that the disulfide bond formation in the portal region arrests large-scale structural fluctuations, which are required for retinoic acid release from the protein. The unique properties of the CRABP I mutant described in this work can be used to inspire and guide a design of nanodevices for multiple tasks ranging from sequestering small-molecule toxins in both tissue and circulation to nutrient deprivation of pathogens.