Determination of binding affinities of retinoids to retinoic acid-binding protein and serum albumin

Binding affinities of retinoic acid and its synthetic analogues to intracellular retinoic acid-binding protein, which is a possible candidate for mediating their biological function, and to serum albumin, the plasma transport protein, were evaluated. A quantitative method involving elimination of interfering serum albumin by immunoprecipitation was developed to measure the binding efficiency of these retinoids, some of which are active in modifying epithelial differentiation and preventing tumorigenesis. Two cyclopentenyl analogues of retinoic acid and 13-cis-retinoic acid showed, like retinoic acid, a binding efficiency of 100% for the cellular binding protein. With the phenyl, dichlorophenyl and trimethylmethoxyphenyl analogues of retinoic acid, the binding efficiency increased as the substituents on the aromatic ring increased; thus the trimethylmethoxyphenyl analogue binds almost as efficiently as retinoic acid itself. However, the trimethylmethoxyphenyl analogue with a sulphur atom on the side chain has a much decreased binding affinity. The correlation noticed between the binding efficiency of these retinoids and their biological activity in differentiation and/or in the control of tumorigenesis particularly enhances the confidence in the present method of determining the relative binding efficiencies. None of the vitamins, hormones and cofactors tested, showed appreciable affinity for the retinoic acid-binding site. Studies on binding of retinoic acid and its analogues to serum albumin indicate that no correlation exists between binding affinity for albumin and their biological potency.     

Novel retinoid-binding proteins from filarial parasites.

The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid-binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding-protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions.     

Purification and crystallization of a retinoic acid-binding protein from rat epididymis. Identity with the major androgen-dependent epididymal proteins

The retinoic acid binding activity in the lumen of the rat epididymis (Ong, D., and Chytil, F. (1988) Arch. Biochem. Biophys. 267, 474-478) has been purified to homogeneity. The protein exists in two forms, one form having an additional three amino acids at the amino terminus. The amino acid sequence of the protein was determined to 20 amino acids and proved to be identical to that of the major androgen-dependent proteins from rat epididymis as deduced from the cDNA sequence. These proteins are thought to play a role in sperm maturation, perhaps, it can be suggested now, by delivering retinoic acid to the sperm. The retinoic acid-binding protein has sequence homology to the serum retinol-binding protein and is predicted to have the same overall fold of the polypeptide chain. The epididymal retinoic acid-binding protein has been crystallized from 39 to 43% saturated ammonium sulfate, 10 mm Tris, pH 8.0. The crystals are space group P2(1), with a = 39.4, b = 58.9, c = 65.4 a, beta = 105 degrees 16 min.     

Effects of dietary retinoic acid on cellular retinol- and retinoic acid-binding protein levels in various rat tissues

A study was conducted to explore the effects of retinoic acid, fed to retinol-deficient rats, on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). Sensitive and specific radioimmunoassays were employed to measure the levels of both CRBP and CRABP. Two groups of six male rats each were fed a purified retinoid-deficient diet supplemented with either: i) retinyl acetate (control group); or ii) retinoic acid (30 mg/kg diet) (retinol deficient-retinoic acid group). The retinoic acid supplementation was begun after 38 days on the retinoid-deficient diet alone, and was continued for 52-54 days. Analysis of the data indicated that only the CRBP level of the proximal epididymis in the retinol-deficient/retinoic acid group differed significantly from (was lower than) the corresponding control level, at the 1% confidence level. CRABP tissue levels did not differ significantly between the two groups. Thus, a moderately large intake of retinoic acid, as the only source of retinoids, had very little effect on the tissue distribution or levels of either its own cellular binding protein (CRABP) or of CRBP. This study provides further information showing that the tissue levels of the cellular retinoid-binding proteins are highly regulated and maintained in rats, even in the presence of marked changes in retinoid nutritional status.     

Purification and properties of cellular retinoic acid-binding protein from neonatal rat skin

Cellular retinoic acid-binding protein (CRABP) was detected in cytosolic extracts of dermis and epidermis of neonatal rat skin using high-performance size-exclusion liquid chromatography and was more abundant in dermal tissue. CRABP was purified 1000-fold from an acid-precipitated, 50,000 x g supernatant of neonatal rat skin by ion-exchange chromatography on DEAE-Sephacel, followed by chromatofocussing and hydrophobic-interaction chromatography. The protein had an apparent Mr of 14,800. In chromatofocussing experiments the apoprotein and holoprotein gave different elution profiles, indicating a charge difference between the two forms. The ability of various retinoids to compete with all-trans-retinoic acid for binding to CRABP was assayed: 4-oxoretinoic acid and two synthetic retinoids were effective competitors, but 13-cis-retinoic acid, 3,4-didehydroretinoic acid and the acid derivative of etretinate competed poorly. The binding protein had a Kd for all-trans-retinoic acid of 8 nM using a dextran-charcoal assay, but a higher value was obtained using high-performance size-exclusion liquid chromatography. The holoprotein dissociated rapidly at room temperature and had a half-life of 4.7 min. At 0 degrees C, the holoprotein had a half-life of 200 min.     

Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography

A method to measure the endogenous levels of apo and holo cellular retinoic acid-binding proteins was developed using calf testis cytosol as the source of retinoic acid-binding protein. [3H]Retinoic acid-retinoic acid-binding protein complexes were assayed by high-performance size-exclusion chromatography. Preincubation of cytosol with 10 mM p-hydroxymercuribenzoate at 4 degrees C resulted in complete inhibition of retinoic acid binding to apo retinoic acid-binding protein. In addition, total dissociation of preformed holo retinoic acid-binding protein complexes was noted within 20 min after mercurial addition. Thus, p-hydroxymercuribenzoate converted the total pool of cellular retinoic acid-binding protein (apo plus holo) to mercurial-protein complexes unable to bind retinoic acid in vitro. Mercurial inhibition of retinoic acid-retinoic acid-binding protein complex formation was totally reversed upon the addition of 50 mM dithiothreitol. Total cytosolic retinoic acid-binding protein was determined from specific retinoic acid binding after treatment with p-hydroxymercuribenzoate and dithiothreitol. Apo cellular retinoic acid-binding protein concentration was measured by determining specific radioligand binding prior to p-hydroxymercuribenzoate treatment, and correcting for exchange of endogenously bound retinoid with exogenous tritiated retinoic acid. Holo cellular retinoic acid-binding protein concentration was derived from the difference between total and apo retinoic acid-binding protein concentrations. Using this method, we have demonstrated that retinoid-responsive EJ and T24 human bladder carcinoma cell lines and AT3A and AT3B rat pancreatic acinar carcinoma cell lines lack detectable levels of either apo or holo cellular retinoic acid-binding protein. These results established that retinoid inhibition of transformed bladder and acinar cell proliferation in culture was mediated by a cellular retinoic acid-binding protein-independent mechanism.     

An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required. The presence of the retinoid binding proteins CRBP and CRABP could not be detected in MA-10 Leydig cell cytosol indicating that the stimulatory action of retinoids on progesterone production is not mediated through these cellular binding proteins. Both previous and present findings suggest that retinoids play an important role in the regulation of Leydig cell steroidogenesis and that MA-10 Leydig tumor cells may represent an ideal in vitro cell system to study the mechanism of action of retinoids in Leydig cell steroidogenesis.     

Functional studies of newly synthesized benzoic acid derivatives: identification of highly potent retinoid-like activity

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid-like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid-like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)-transformed 3T3 cells in serum-free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth-stimulating activity of these compounds on MSV-transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.     

Cellular retinoid-binding proteins in reginerating rat liver: demonstration of a novel cellular retinoid-binding protein

Changes in the levels of liver cellular aetinol- and retinoic acid-binding proteins were studied after partial (about 70%) hepatectomy for 14 days in the rat. It was found that a novel binding protein designated F-type appears transiently in liver cytosol 3 days after the operation. The appearance of this protein coincides with the peak level of the alpha 1-fetoproteain. In contrast, cellular retinoic acid-binding protein was detected only the first day after hepatectomy, whereas no significant change was observed in the level of the cellular retinol-binding protein during the entire observation period. [3H]Retinol or [3H]retinoic acid complexed with serum retinol-binding protein injected intravenously into vitamin A-deficient rats 1 day after the hepatectomy was recovered 5 min or 20 min later bound specifically to cellular retinol- or retinoic acid-binding protein, respectively. The results presented here strongly suggest that each of the three cellular retinoid-binding proteins plays a distinct role in cell proliferation and differentiation.     

Rapid characterization of cellular retinoid-binding proteins by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed for the analysis of cellular retinol and retinoic acid-binding proteins. The chromatographic analysis of the retinol-retinol-binding protein complex or the retinoic acid-retinoic acid-binding protein complex requires 15 min. The use of high-specific-radioactivity retinoid ligand (30-40 Ci/mmol) allows routine detection of 25 fmol of retinoid-binding protein/mg of cytosolic protein. Thus, this method provides a rapid alternative to sucrose density gradient sedimentation analysis of the cellular retinoid-binding proteins. High-performance size-exclusion chromatography is well suited to screening novel tissues, tumors, and cell lines for the presence of retinoid-binding proteins and to the further characterization of these cellular proteins. This method was applied to the characterization of cellular retinol- and retinoic acid-binding proteins in fetal rabbit tissues. Both retinol- and retinoic acid-binding proteins were detected in fetal rabbit brain, intestine, kidney, and lung at a gestational age of 28 days. Neither retinoid-binding protein was detected in 28-day-old fetal rabbit placenta. Specific retinoic acid binding in fetal intestinal cytosol decreased as a function of increasing gestational age.     

The relationship among retinoid structure, affinity for retinoic acid-binding protein, and ability to respecify pattern in the regenerating axolotl limb

To further our understanding of the action of retinoids on the respecification of pattern in the regenerating axolotl limb we have studied the relative potencies of a range of synthetic and natural retinoids administered locally to the blastema. Alterations in the polar end group of the retinoic acid (RA) molecule to produce esters, the alcohol, or the aldehyde abolish the ability of the molecule to respecify pattern. On the other hand, alterations of the ring or side chain to produce the synthetic retinoids arotinoid and TTNPB considerably increases the potency of the molecule to respecify pattern--TTNPB is at least 100X more potent than retinoic acid. To examine the role of cellular retinoic acid-binding protein (CRABP) in the respecification process we determined the relative binding affinities of these retinoids for CRABP. These data correlated well with the respecification series: retinoids which showed no affinity for CRABP did not respecify pattern and those which did show affinity for CRABP did respecify pattern. Furthermore the most potent retinoid, TTNPB, has a higher affinity for CRABP than RA itself. This suggests that CRABP may be playing an important role in the action of RA on pattern formation in the regenerating limb.     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.     

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.     

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.     

Structure and function of cytoplasmic retinoid binding proteins

We examined the ligand protein interactions of two highly homologous cellular retinol binding proteins, CRBP and CRBP-II, and two highly homologous cellular retinoic acid binding proteins, CRABP-I and CRABP-II. While the crystal structures of all four have been determined, nuclear magnetic resonance studies provide a means for observing dynamic aspects of ligand protein interactions of these proteins in solution. The cellular functions of these proteins are less well understood. We have modeled retinoid flux between cytoplasmic retinoid proteins and model membranes and with nuclear receptors. Based on our in vitro studies, we propose that certain retinoids may indirectly influence retinoid signaling by displacing endogenous retinoids from the cytoplasmic proteins to the nuclear receptors.     

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.     

Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.     

Retinoic acid-binding protein: a plasma membrane component

Soluble protein extracts from lyophilized plasma membranes prepared from chick embryo skin and transplantable murine carcinomas were found to contain a specific retinoic acid-binding component. This binding component showed high affinity for biologically active analogs of retinoic acid. The plasma membrane component exhibited similar physicochemical properties to those of the retinoic acid-binding protein described earlier in the cytosol. The protein exhibited mercurial-sensitive thiol functions in ligand binding; the mercurial-inhibition was reversed on treatment with thiol compounds. The plasma membrane binding component may be involved in the cellular uptake of retinoic acid.     

Purification and properties of retinol- and retinoic acid-binding proteins from a transplantable mouse colon tumor

Cellular retinol-binding protein and retinoic acid-binding protein, the possible mediators of the action of retinoids in epithelial differentiation and control of tumorigenesis, have been reproducibly purified from mouse colon tumor 26, and some of their properties were studied. The main steps of purification involved acid-precipitation, DEAE-Sephadex, CM-cellulose and Sephadex G-100 chromatography. About 2 mg of the binding proteins were isolated from 60 g tumor. The purified preparations showed only two protein bands on polyacrylamide gel electrophoresis. The two binding proteins were partially resolved by sedimentation equilibrium technique; but was completely separable by preparative electrophoresis in the presence of sodium dodecyl sulfate. The retinol- and retinoic acid-binding proteins are presumably monomers with molecular weights of 15,500 and 14,600, respectively, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. On gel filtration however, both the binding proteins retarded to the same molecular size of 17,800. On preparative columns, both the proteins expressed the same isoelectric pH, 4.5. Both proteins of the tumor possessed functional thiol groups. The mercurial inhibition of the binding capacity of the proteins for their ligands was reversible upon treatment with thiol compounds.     

Metabolism of retinoids by embryonal carcinoma cells

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.