Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components

Retinol (vitamin A alcohol) is involved in the proper differentiation of epithelia. The mechanism of this involvement is unknown. We have previously reported that purified cellular retinol-binding (CRBP) will mediate specific binding of retinol to nuclei isolated from rat liver. We now report that pure CRBP delivers retinol to the specific nuclear binding sites without itself remaining bound. Triton X-100-treated nuclei retain the majority of these binding sites. CRBP is also capable of delivering retinol specifically to isolated chromatin with no apparent loss of binding sites, as compared to whole nuclei. CRBP again does not remain bound after transferring retinol to the chromatin binding sites. When isolated nuclei are incubated with [3H]retinol- CRBP, sectioned, and autoradiographed, specifically bound retinol is found distributed throughout the nuclei. Thus, CRBP delivers retinol to the interior of the nucleus, to specific binding sites which are primarily, if not solely, on the chromatin. The binding of retinol to these sites may affect gene expression.     

Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II

Retinol bound to cellular retinol-binding protein (CRBP) was available for esterification by liver microsomes in the absence of exogenous acyl donors. Moreover, exogenous acyl-CoA gave little or no stimulation of ester production over what was observed with the endogenous acyl donor. In contrast, unbound retinol was esterified in an acyl-CoA-dependent reaction. The presence of two different enzyme activities, acyl-CoA-dependent and -independent, was demonstrated by differential sensitivities to several enzyme inhibitors. The enzyme reaction with retinol-CRBP and endogenous acyl donor produced retinyl esters normally found in vivo in liver. In addition, rates of esterification with this system were sufficient to maintain liver stores. Liver also contains cellular retinol-binding protein, type II (CRBP(II] during the perinatal period. Radioimmunoassay revealed highest levels of CRBP(II) in liver 3-4 days after birth. Examination of retinol esterification by microsomes from the liver of 3-day-old rats revealed a retinyl ester synthase activity with lower Km and higher Vmax than that found in the adult. The activity could use either retinol-CRBP or retinol-CRBP(II) and an endogenous acyl donor. The microsomes from 3-day-old liver had greater esterifying ability than microsomes from adult liver, perhaps due to the presence of two retinyl ester synthase enzymes.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

Studies of the spontaneous transfer of retinol from the retinol:retinol-binding protein complex to unilamellar liposomes

The transfer of retinol from its complex with the retinol-binding protein to cell surfaces was studied using unilamellar liposomes as a cell surface model. The transfer of retinol to liposomes at 37 degrees C was rapid and reached an apparent equilibrium within 60 min. The amount of retinol transferred to the liposomes at equilibrium was directly proportional to the starting concentration of retinol:retinol-binding protein over a wide range of retinol:retinol-binding protein concentrations and also directly proportional to the concentration of liposomal phospholipid in the system, when the concentration of retinol:retinol-binding protein was held constant. The transfer increased slightly with temperature. Transfer was increased by a factor of 1.8 at pH 4.5 compared to pH around 7. Prealbumin in amounts sufficient to complex all retinol:retinol-binding protein, decreased retinol transfer to liposomes indicating that prealbumin increases the affinity of retinol-binding protein for retinol. Addition of apo retinol-binding protein to the system decreased the transfer of retinol to liposomes considerably probably through competition with the liposomes for retinol. In similarly designed experiments delipidated bovine serum albumin competed much less with liposomes for retinol. The results show that spontaneous transfer of retinol from the retinol:retinol-binding protein complex to liposomal membranes occurs in vitro and suggests that a similar transfer may occur in vivo from retinol:retinol-binding protein to cell surface membranes.     

Studies of the spontaneous transfer of retinol from the retinol: retinol-binding protein complex to unilamellar liposomes

The transfer of retinol from its complex with the retinol-binding protein to cell surfaces was studied using unilamellar liposomes as a cell surface model. The transfer of retinol to liposomes at 37°C was rapid and reached an apparent equilibrium within 60 min. The amount of retinol transferred to the liposomes at equilibrium was directly proportional to the starting concentration of retinol: retinol-binding protein over a wide range of retinol:retinol-binding protein concentrations and also directly proportional to the concentration of liposomal phospholipid in the system, when the concentration of retinol:retinol-binding protein was held constant. The transfer increased slightly with temperature. Transfer was increased by a factor of 1.8 at pH 4.5 compared to pH around 7. Prealbumin in amounts sufficient to complex all retinol:retinol-binding protein, decreased retinol transfer to liposomes indicating that prealbumin increases the affinity of retinol-binding protein for retinol. Addition of apo retinol-binding protein to the system decreased the transfer of retinol to liposomes considerably probably through competition with the liposomes for retinol. In similarly designed experiments delipidated bovine serum albumin competed much less with liposomes for retinol. The results show that spontaneous transfer of retinol from the retinol:retinol-binding protein complex to liposomal membranes occurs in vitro and suggests that a similar transfer may occur in vivo from retinol:retinol-binding protein to cell surface membranes.     

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type ii (CRBP(II] are cytoplasmic proteins that bind trans-retinol as an endogenous ligand. These proteins are structurally similar having greater than 50% sequence homology. Employing fluorescence, absorbance, and competition studies, the ability of pure preparations of CRBP(II) and CRBP to bind various members of the vitamin A family has been examined. In addition to trans-retinol, CRBP(II) was able to form high affinity complexes (K'd less than 5 X 10(-8) M) with 13-cis-retinol, 3-dehydroretinol, and all-trans-retinaldehyde. CRBP bound those retinol isomers with similar affinities, but did not bind trans-retinaldehyde. Neither protein bound retinoic acid nor 9-cis- and 11-cis-retinol. The spectra of 13-cis-retinol and 3-dehydroretinol, when bound, were shifted and displayed fine structure compared to their spectra in organic solution. However, the lambda max and fluorescent yield of a particular ligand were different when bound to CRBP(II) versus CRBP. It appears that CRBP(II) and CRBP bind trans-retinol, 13-cis-retinol, and 3-dehydroretinol in a planar configuration. However, the binding sites of CRBP(II) and CRBP are clearly distinct based on the observed spectral differences of the bound ligands and the observations that only CRBP(II) could bind trans-retinaldehyde. The ability of CRBP(II) to bind trans-retinaldehyde suggests a physiological role for the protein in accepting retinaldehyde generated from the cleavage of beta-carotene in the absorptive cell.     

Binding of retinol induces changes in rat cellular retinol-binding protein II conformation and backbone dynamics

The structure and backbone dynamics of rat holo cellular retinol-binding protein II (holo-CRBP II) in solution has been determined by multidimensional NMR. The final structure ensemble was based on 3980 distance and 30 dihedral angle restraints, and was calculated using metric matrix distance geometry with pairwise Gaussian metrization followed by simulated annealing. The average RMS deviation of the backbone atoms for the final 25 structures relative to their mean coordinates is 0.85(+/-0.09) A. Comparison of the solution structure of holo-CRBP II with apo-CRBP II indicates that the protein undergoes conformational changes not previously observed in crystalline CRBP II, affecting residues 28-35 of the helix-turn-helix, residues 37-38 of the subsequent linker, as well as the beta-hairpin C-D, E-F and G-H loops. The bound retinol is completely buried inside the binding cavity and oriented as in the crystal structure. The order parameters derived from the (15)N T(1), T(2) and steady-state NOE parameters show that the backbone dynamics of holo-CRBP II is restricted throughout the polypeptide. The T(2) derived apparent backbone exchange rate and amide (1)H exchange rate both indicate that the microsecond to second timescale conformational exchange occurring in the portal region of the apo form has been suppressed in the holo form.     

Retinoic acid rapidly induces lung cellular retinol-binding protein mRNA levels in retinol deficient rats

Retinol deficiency resulted in decreased mRNA levels for cellular retinol-binding protein (CRBP) in the lungs and the testes. The level of lung CRBP mRNA increased 2.3-fold one hour after oral administration of retinoic acid to retinol deficient rats. In contrast, testicular CRBP mRNA level was not influenced. Our data indicate that retinoic acid regulates CRBP mRNA level in the whole animal and this rapid effect suggests a role for CRBP in the mechanism of vitamin A action at genomic level.     

Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein

Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat(-/-) mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.     

Acyl-CoA-independent esterification of retinol bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine

Cellular retinol-binding protein (type II) (CRBP(II)), a newly described retinol-binding protein, is present in the small intestinal absorptive cell at high levels. Retinol (vitamin A alcohol) presented as a complex with CRBP(II) was found here to be esterified by microsomal preparations from rat small intestinal mucosa. The esterification observed utilized an endogenous acyl donor(s) and produced retinyl esters containing linoleate, oleate, palmitate, and stearate in a proportion quite similar to that previously reported for retinyl esters in lymph and isolated chylomicrons of rat. No dependence on endogenous or exogenous acyl-CoA could be demonstrated. The apparent Km for retinol-CRBP(II) in the reaction with endogenous acyl donor was 2.4 X 10(-7) M. Retinol presented as a complex with CRBP(II) was esterified more than retinol presented as a complex with cellular retinol-binding protein or retinol-binding protein, two other proteins known to bind retinol in vivo, but about the same as retinol presented bound to bovine serum albumin or beta-lactoglobulin. The ability of protein-bound retinol to be esterified was related to accessibility of the hydroxyl group, as judged by the ability of alcohol dehydrogenase to oxidize the bound retinol. However, whereas retinol bound to CRBP(II) was unavailable for esterification in any acyl-CoA-dependent reaction, retinol bound to bovine serum albumin was rapidly esterified in a reaction utilizing exogenous acyl-CoA. The results suggest that one of the functions of CRBP(II) is to accept retinol after it is absorbed or generated from carotenes in the small intestine and present it to the appropriate esterifying enzyme.     

Mapping of human cellular retinol-binding protein to chromosome 3

The gene for human cellular retinol-binding protein (CRBP) has been assigned to chromosome 3, based on an analysis of genomic DNA from human-hamster somatic cell hybrids using a cDNA probe for CRBP.     

Photo-affinity label for cellular retinol-binding protein

A number of retinoid derivatives have been synthesized for use as labels for cellular retinol-binding protein. Introduction of substituents abolished the binding of the derivatives to the protein, except in the case of the photo-reactive derivative, 4-azidoretinol. This compound was found to compete successfully with all-trans-retinol for binding to cellular retinol-binding protein, with a high relative binding affinity. Irradiation of a complex of 4-azidoretinol and a semi-purified preparation of cellular retinol-binding protein from liver resulted in a firm attachment stable to SDS-gel electrophoresis. It is therefore suggested that the irradiated product is held together covalently. A method for the synthesis of 4-azidoretinol is described.     

Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. Acetylation with N-acetylimidazole and alkaline titration.

The behavior of holo-retinol-binding protein (RBP) from human plasma at alkaline pH was examined by absorption and circular dichroism measurements. Between pH 7.5 and 11.7 the ionization of the phenolic hydroxyl groups is reversible. However, there is a gradual irreversible loss of retinol as the pH is raised. After 4 hours at pH 11.7, 13 percent of retinol is lost from retinol-RBP. Alkaline titration of apo-RBP was time-independent and reversible between pH 7.5 and 11.7. The titration data of the phenolic hydroxyl groups in apo-RBP could be fitted with a single theoretical ionization curve of 8.6 phenolic groups having an apparent pK of 11. Acetylation of retinol-RBP with 10-fold molar excess of N-acetylimidazole over tyrosine resulted in the acetylation of all lysine residues and in the acetylation of 0.9 to 1.3 tyrosyl residues per molecule (out of eight). Acetylation of retinol-RBP, APO-RBP, and retinol-RBP-prealbumin complex with 50-fold molar excess of N-acetylimidazole resulted, again, with all of the lysine residues being acetylated and between 1.8 and 2.8 tyrosyl residues per molecule being acetylated. The acetylation did not affect the interaction between retinol and RBP. However, acetylation disrupted the normal binding between retinol-RBP and prealbumin. Deacetylation of tyrosyl residues with hydroxylamine failed to restore the normal binding of retinol-RBP to prealbumin. This excludes the acetylated tyrosyl-residues from being involved in the binding between the two proteins.     

Extraction and recombination studies of the interaction of retinol with human plasma retinol-binding protein

Methods have been developed for the removal of retinol from human plasma retinol-binding protein (RBP), so as to form the retinol-free apoprotein, and for the recombination of apo-RBP with retinol to again form the holoprotein. Retinol is removed from RBP by gently shaking a solution of RBP with heptane under controlled conditions. During the shaking, retinol is gradually extracted from the RBP and into the heptane phase. The reassociation of apo-RBP with retinol is achieved by exposing a solution of apo-RBP to Celite coated with a thin film of retinol, followed by isolation of the RBP by gel filtration on Sephadex G-100. This procedure results in the recombination of apo-RBP with an amount of retinol almost identical with that previously removed by extraction. The two-phase extraction procedure was used to explore some of the factors which affect the interaction of retinol with RBP. The retinol-RBP complex was most stable in the lower portion of the pH range 5.6 to 10. The rate of removal of retinol from the RBP-prealbumin complex (the form in which RBP normally circulates in plasma) was markedly less than the rate of its removal from RBP alone. The interaction of retinol with RBP appears to be stabilized by the formation of the RBP-prealbumin complex. The recombination procedure was employed to examine the specificity of the binding of retinol to RBP, by determining whether compounds other than all-trans-retinol would effectively bind to apo-RBP. Apo-RBP did not bind cholesterol, but displayed a slight affinity for phytol. The affinity of RBP for beta-carotene was minimal, whereas both retinyl acetate and retinal were bound about one-third as effectively as all-trans-retinol. In contrast, retinoic acid bound to apo-RBP almost as effectively as did retinol. Each of two isomers of retinol, 13-cis and 11,13-di-cis-retinol, bound to apo-RBP to some extent. The 13-cis isomer appeared to bind somewhat less effectively than did the 11,13-di-cis isomer. The binding of retinol to RBP is highly but not absolutely specific.