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.     

Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Separable binding proteins for retinoic acid and retinol in bovine retina.

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Cellular retinoic acid binding protein is associated with mitochondria

We report that immunohistochemical staining for cellular retinoic acid-binding protein (CRABP) was restricted to the cytoplasm of cortical cells in bovine adrenal. In contrast, staining for the similar protein, cellular retinol-binding protein (CRBP), was found throughout these cells. After transfections of CRABP and CRBP into cultured cells, immunofluorescence analyses again revealed cytoplasmic restriction only for CRABP, with a pronounced punctate appearance. Use of organelle-specific fluorochromes indicated that CRABP immunofluorescence overlaid exactly with the pattern of the mitochondrial-specific fluorochrome. Confirmation of this association came with subcellular fractionation of the adrenal cortex. CRABP, but not CRBP, co-sedimented with the mitochondria, a novel finding for a member of this superfamily of cellular lipid-binding proteins.     

Protein and ligand adaptation in a retinoic acid binding protein.

A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all-trans and 9-cis isomers, the structures of all-trans retinoic acid-ERABP and 9-cis retinoic acid ERABP were determined. Our results indicate that the all-trans isomer of retinoic acid adopts an 8-cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB-ERABP is also reported herein. To accommodate this all-trans analog, which cannot readily adopt a cis-like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo-proteins.     

Selective and specific ion binding on proteins at physiologically-relevant concentrations

Insoluble proteins dissolved in unsalted water appear to have no well-folded tertiary structures. This raises a fundamental question as to whether being unstructured is due to the absence of salt ions. To address this issue, we solubilized the insoluble ephrin-B2 cytoplasmic domain in unsalted water and first confirmed using NMR spectroscopy that it is only partially folded. Using NMR HSQC titrations with 14 different salts, we further demonstrate that the addition of salt triggers no significant folding of the protein within physiologically relevant ion concentrations. We reveal however that their 8 anions bind to the ephrin-B2 protein with high affinity and specificity at biologically-relevant concentrations. Interestingly, the binding is found to be both salt- and residue-specific.     

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.     

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.     

Fatty acid binding proteins from heart

Heart contains a fatty acid binding protein (FABP) concentration comparable to liver, when it is determined with a fatty acid-binding assay. The low concentration detected with anti-liver FABP antibodies is related to the different chemical forms and physiochemical properties of liver and heart FABP. The ratio of fatty acid bound per purified protein molecule is one or lower. Rat heart mitochondria oxidize FABP-bound fatty acids. The FABP content of rat heart is dependent on sex and diurnal cycle, but is not influenced by starvation or clofibrate feeding. It is also not different in the newborn rat. FABP was obtained from human heart in a yield of 11%. It shows similar binding characteristics to palmitic, oleic and arachidonic acid. The functional significance of the specific heart FABP is discussed in relation to myocardial fatty acid metabolism in normal and pathological conditions.     

Cellular retinoic acid binding protein I mediates rapid non-canonical activation of ERK1/2 by all-trans retinoic acid

All-trans retinoic acid (atRA), one of the active ingredients of vitamin A, exerts canonical activities to regulate gene expression mediated by nuclear RA receptors (RARs). AtRA could also elicit certain non-canonical activities including, mostly, rapid activation of extracellular signal regulated kinase 1/2 (ERK1/2); but the mechanism was unclear. In this study, we have found that cellular retinoic acid binding protein I (CRABPI) mediates the non-canonical, RAR- and membrane signal-independent activation of ERK1/2 by atRA in various cellular backgrounds. In the context of embryonic stem cells (ESCs), atRA/CRABPI-dependent ERK1/2 activation rapidly affects ESC cell cycle, specifically to expand the G1 phase. This is mediated by ERK stimulation resulting in dephosphorylation of nuclear p27, which elevates nuclear p27 protein levels to block G1 progression to S phase. This is the first study to identify CRABPI as the mediator for non-canonical activation of ERK1/2 by atRA, and demonstrate a new functional role for CRABPI in modulating ESC cell cycle progression.     

Invertebrate intracellular fatty acid binding proteins

Fatty acid binding proteins are multigenic cytosolic proteins largely distributed along the zoological scale. Their overall identity at primary and tertiary structure is conserved. They are involved in the uptake and transport of hydrophobic ligands to different cellular fates. The precise functions of each FABP type remain imperfectly understood, since sub-specialization of functions is suggested. Evolutionary studies have distinguished major subfamilies that could have been derived from a common ancestor close to vertebrate/invertebrate split. Since the isolation of the first invertebrate FABP from Schistocerca gregaria in 1990, the number of FABPs isolated from invertebrates has been increasing. Differences at the sequence level are appreciable and relationships with vertebrate FABPs are not clear, and lesser among invertebrate proteins, introducing some uncertainty to infer functional relatedness and phylogenetic relationships. The objective of this review is to summarize the information available on invertebrate FABPs to elucidate their mutual relationships, the relationship with their vertebrate counterparts and putative functions. Structure, gene structure, putative functions, expression studies and phylogenetic relationships with vertebrate counterparts are analyzed. Previous suggestions of the ancestral position concerning the heart-type of FABPs are reinforced by evidence from invertebrate models.     

Interference of retinoic acid binding to its binding protein by omega-6 fatty acids

Cellular retinoic acid-binding protein (CRABP) is the putative mediator of the biological effects of retinoic acid in the control of epithelial differentiation and tumorigenesis. Omega-6 fatty acids such as linoleic acid and arachidonic acid, precursors of prostaglandin synthesis, caused inhibition of retinoic acid binding to CRABP. These fatty acids, however, possessed lower affinity than retinoic acid for the binding protein. Omega-3 fatty acids, such as eicosapentaenoic acid and docosohexaenoic acid, did not cause such inhibition in the binding of retinoic acid. Whereas retinoic acid was a potent modulator of differentiation of F9 embryonal carcinoma cells, neither omega-3 nor omega-6 fatty acids showed any significant differentiation potential. Competition by omega-6 fatty acids with retinoic acid for CRABP may neutralize the binding protein-mediated biological functions of retinoic acid, and could thereby enhance tumor production.     

Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-hydroxyethoxy)retinoic acid

An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.     

Pharmacokinetic assessment of teratologically effective concentrations of an endogenous retinoic acid metabolite

Retinoic acid is a natural vitamin A derivative that undergoes oxidative metabolism in the body to yield several metabolites, which apparently represent the products of a detoxification pathway. To assess if such metabolic conversions diminished teratogenic potency, one of the major metabolites (4-oxo-all-trans-retinoic acid) was tested for its teratogenic activity in pregnant ICR mice and further investigated for its pharmacokinetic features to determine if it accumulated in the embryo in concentrations sufficient to elicit a teratogenic response. Administration of single oral doses (10, 25, 50, or 100 mg/kg) of the compound to ICR mice on day 11 of gestation (plug day = day 0) produced dose-dependent frequencies of serious fetal anomalies of the type usually associated with the use of retinoic acid and other retinoids. The metabolite was equivalent in teratogenic potency to retinoic acid, and, in the instance of cleft palate frequency, it was even more active. Concentrations of 4-oxo-all-trans-retinoic acid and its 13-cis isomer were measured in the maternal plasma and whole embryos at 30 min to 10 hr after administration of the lowest (10 mg/kg) and the highest (100 mg/kg) teratogenic dose of 4-oxo-all-trans-retinoic acid by means of high-performance liquid chromatography methodology. Distribution of the compound in the maternal system and transfer to the embryo occurred rapidly with either dose. Peak concentration in the maternal plasma and the embryo persisted for 3-4 hr after the higher dose but not with the lower dose; however, elimination kinetics for the two dose levels were similar.(ABSTRACT TRUNCATED AT 250 WORDS)