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.     

Specificity of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin

We have reported previously that cellular retinol-binding protein (CRBP) is able to transfer retinol to specific binding sites in nuclei and chromatin. In this report, we have examined the specificity of the interaction of the protein moiety of retinol-CRBP (R-CRBP) with chromatin and nuclei in the transfer process. We first determined the ability of apo-CRBP, apo-serum retinol-binding protein (RBP), and apo beta-lactoglobulin (BLG), all capable of retinol binding, to compete with R-CRBP in the transfer of retinol to chromatin and nuclei. Apo-CRBP was an effective competitor but apo-RBP and apo-BLG showed no competitive ability. On the other hand, cellular retinol-binding protein type II (CRBP(II], whose amino acid sequence shows a considerable similarity to CRBP, did compete for the transfer of retinol from the R-CRBP complex, but less effectively than CRBP. These results demonstrate that the interaction of the protein moiety of the R-CRBP complex with nuclei and chromatin is quite specific.     

Identification and structural analysis of a zebrafish apo and holo cellular retinol-binding protein

Cellular retinol-binding proteins (CRBPs) are cytoplasmic retinol-specific binding proteins. Mammalian CRBPs have been thoroughly characterised previously. Here we report on the identification and X-ray structural analysis of the apo (1.7A resolution) and holo (1.4A resolution) forms of a zebrafish CRBP. According to amino acid sequence and structure analyses, the zebrafish CRBP that we have identified resembles closely mammalian CRBP II, suggesting that it is the zebrafish orthologue of this mammalian CRBP type. Zebrafish CRBP forms a tight complex with all-trans retinol, producing an absorption spectrum similar to those of mammalian holo-CRBPs, albeit slightly blue-shifted. The superposition of the alpha-carbon atoms of the liganded (complexed with retinol) and unliganded forms of zebrafish CRBP shows significant differences in correspondence of the betaC-betaD (residues 55-58) and betaE-betaF (residues 74-77) turns, providing evidence for the occurrence of conformational changes accompanying retinol binding/release. Remarkable and well-defined ligand-dependent conformational changes in the protein region comprising the two beta-turns affect both the main chain and the side-chains of several residues. The two beta-turns project towards the interior of the cavity devoid of ligand of the apoprotein. The side-chains of F57, Y60 and L77 change substantially their orientation and position in the apoprotein relative to the holoprotein. In the beta-barrel internal cavity of apo-CRBP they occupy some of the space that is otherwise occupied by bound retinol in holo-CRBP, and are displaced from these positions on ligand binding. These results indicate that a flexible area encompassing the betaC-betaD and betaE-betaF turns may serve as the ligand portal and that these turns undergo conformational changes associated with the not yet clarified mechanism of retinol binding and release in CRBPs.     

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group. Finally, neither all-trans-retinol nor retinoic acid bound to E. coli-derived rat intestinal fatty acid-binding protein, a homologous protein whose tertiary structure is known. Together, the data suggest that these three family members have acquired unique functional capabilities.     

Ligand binding and structural analysis of a human putative cellular retinol-binding protein

Three cellular retinol-binding protein (CRBP) types (CRBP I, II, and III) with distinct tissue distributions and retinoid binding properties have been structurally characterized thus far. A human binding protein, whose mRNA is expressed primarily in kidney, heart, and transverse colon, is shown here to be a CRBP family member (human CRBP IV), according to amino acid sequence, phylogenetic analysis, gene structure organization, and x-ray structural analysis. Retinol binding to CRBP IV leads to an absorption spectrum distinct from a typical holo-CRBP spectrum and is characterized by an affinity (K(d) = approximately 200 nm) lower than those for CRBP I, II, and III, as established in direct and competitive binding assays. As revealed by mutagenic analysis, the presence in CRBP IV of His(108) in place of Gln(108) is not responsible for the unusual holo-CRBP IV spectrum. The 2-A resolution crystal structure of human apo-CRBP IV is very similar to those of other structurally characterized CRBPs. The side chain of Tyr(60) is present within the binding cavity of the apoprotein and might affect the interaction with the retinol molecule. These results indicate that human CRBP IV belongs to a clearly distinct CRBP subfamily and suggest a relatively different mode of retinol binding for this binding protein.     

Vitamin A uptake from retinol-binding protein in a cell-free system from pigment epithelial cells of bovine retina. Retinol transfer from plasma retinol-binding protein to cytoplasmic retinol-binding protein with retinyl-ester formation as the intermediate step

We have investigated the steps by which retinol, released from plasma retinol-binding protein (RBP), enters the cells and is accumulated for the most part as a retinyl-ester, only a small fraction of it being present as a complex with cytoplasmic retinol-binding protein (CRBP). For this purpose, we have developed a cell-free system composed of plasma membrane-enriched fractions from bovine retinal pigment epithelium which selectively incorporates exogenous vitamin A when presented as a retinol-RBP complex. Upon incubation in the presence of [3H]retinol-RBP, isolated plasma membrane fractions take up and esterify retinol. A 4-fold reduction of total vitamin A incorporation is observed in conditions which specifically inhibit retinyl-ester formation, thus indicating that the two processes of retinol uptake and esterification are functionally coupled. Evidence is presented that retinol bound to a plasma membrane receptor sharing functional and structural similarities with CRBP is the actual substrate for esterification. Vitamin A accumulation seems to require retinol esterification to allow the recycling of a limited number of free, plasma membrane-associated, retinol receptors. Mobilization of retinol stored as a membrane-bound retinyl-ester is mediated by a membrane-associated hydrolase activity selectively controlled by the level of apo-CRBP which acts as a carrier for the released retinol. Up to 90% of membrane-bound vitamin A is released upon incubation in the presence of apo-CRBP (11 microM) with concomitant formation of retinol-CRBP. The overall process, in which retinol never needs to leave its binding proteins, allows the accumulation of vitamin A in the form of a membrane-bound retinyl-ester and its regulated mobilization as a retinol-CRBP complex.     

Cellular retinol-binding protein (type two) is abundant in human small intestine

Human small intestine was found to contain a retinol-binding protein similar to the gut-specific cellular retinol-binding protein, type two [CRBP (II)], described in the rat. This newly detected human protein was immunochemically distinct from human cellular retinol binding protein previously described but immunochemically similar to rat CRBP (II). The partially purified protein bound retinol and exhibited fluorescence excitation and emission spectra distinct from those spectra for retinol bound to pure human CRBP but similar to the spectra for retinol bound to rat CRBP (II). Human CRBP (II) could be localized to the villus-associated enterocytes by immunohistochemistry, using antiserum against rat CRBP (II). The protein was abundant representing 0.4% of the total soluble protein in a jejunum mucosal extract. This protein may play an important role in the absorption and necessary intestinal metabolism of vitamin A.     

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.     

The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13C, 15N-apo-CRBP II and 15N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A. Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the betaC-betaD turn and the betaE-betaF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the betaC-betaD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the betaE-betaF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1H exchange rates and 15N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the microseconds to ms time-scale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the microseconds to ms time-scale which result in increased access to the binding cavity.     

Crystallization of rat cellular retinol binding protein II. Preliminary X-ray data obtained from the apoprotein expressed in Escherichia coli

Rat cellular retinol-binding protein II (CRBP II) is a member of a family of cytoplasmic proteins which bind hydrophobic ligands. CRBP II is thought to participate in the intestinal absorption and intracellular metabolism of retinoids. We have previously described the crystallization of a homologous rat intestinal fatty acid-binding protein (I-FABP) isolated from Escherichia coli containing a suitably constructed prokaryotic expression vector (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J., J. Biol. Chem. 262, 5428-5430). We have now efficiently expressed rat CRBP II in E. coli. The E. coli-derived protein, which does not contain any bound retinoid, has been purified and crystals grown from solutions of polyethylene glycol 4000. Crystals of apo-CRBP II are triclinic, space group P1, a = 36.8 A, b = 64.0 A, c = 30.4 A; alpha = 92.8 degrees, beta = 113.5 degrees, gamma = 90.1 degrees. Each unit cell contains two molecules of the 134-residue apoprotein. X-ray diffraction data suggest that the unit cell parameters of crystalline apo-CRBP II resemble those of I-FABP. Comparison of the tertiary structures of E. coli-derived rat I-FABP and CRBP II should provide insights about how these proteins evolved to bind different hydrophobic ligands.     

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

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

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.     

Comparison of the tissue-specific expression and developmental regulation of two closely linked rodent genes encoding cytosolic retinol-binding proteins

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are two highly homologous cytoplasmic proteins that bind all-trans-retinol. We have recently demonstrated that the mouse genes encoding CRBP and CRBP II are closely linked on chromosome 9 and that both human genes are located on chromosome 3 (Demmer, L.A., Birkenmeier, E.H., Sweetser, D.A., Levin, M.S., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J., and Gordon, J.I. (1987) J. Biol. Chem. 262, 2458-2467). We have now used RNA blot hybridization analysis to assess the degree to which these genes are coordinately expressed in fetal, suckling, weaning, and adult rat tissues. Both genes exhibit different developmental patterns of expression in liver, intestine, lung, kidney, testes, and placenta. In the intestine, CRBP mRNA was detected during the 16th day of gestation--prior to the development of a well-differentiated absorptive epithelium--and remained essentially unchanged throughout the peri- and postpartum periods. By contrast, the pattern of intestinal CRBP II mRNA accumulation closely parallels the times of first appearance, and subsequent proliferation, of the intestinal absorptive columnar epithelium, supporting the hypothesis that CRBP II is involved in the intestinal uptake or intracellular trafficking of this hydrophobic vitamin. In the fetal liver, both genes were expressed by gestational day 16. Whereas the concentration of hepatic CRBP mRNA increased markedly during the suckling and early weaning periods, CRBP II mRNA levels fell abruptly immediately after birth. These peripartum changes were not paralleled by remarkable alterations in the steady state levels of hepatic retinol. Marked changes in the expression of CRBP in the liver and of CRBP II in the intestine were also documented in pregnant and lactating female rats. These differences in CRBP/CRBP II gene expression strongly suggest that their proteins serve different physiological functions. The peripartum liver may provide a useful model for dissecting the relative roles played by these homologous proteins in retinoid metabolism as well as the factors which modulate activation and repression their genes.     

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Holocellular retinol binding protein as a substrate for microsomal retinal synthesis

Holocellular retinol binding protein (holo-CRBP) was substrate for retinal synthesis at physiological pH with microsomes prepared from rat liver, kidney, lung, and testes. Four observations indicated that retinal synthesis was supported by holo-CRBP directly, rather than by the unbound retinol in equilibrium with CRBP. First, the rate of retinal synthesis with holo-CRBP exceeded the rate that was observed from the concentration of unbound retinol in equilibrium with CRBP. Second, NADP was the preferred cofactor only with holo-CRBP, supporting a rate about 3-fold greater than that of NAD. In contrast, with unbound retinol as substrate, similar rates of retinal formation were supported by either NAD or NADP. Third, the rate of retinal synthesis was not related to the decrease in the concentration of unbound retinol in equilibrium with holo-CRBP caused by increasing the concentration of apo-CRBP. Fourth, the rate of retinal synthesis increased with increases in the concentration of holo-CRBP as a fixed concentration of unbound retinol was maintained. This was achieved by increasing both apo-CRBP and holo-CRBP, but keeping constant the ratio apo-CRBP/holo-CRBP. Retinal formation from holo-CRBP displayed typical Michaelis-Menten kinetics with a Km about 1.6 microM, less than the physiological retinal concentration of 4-10 microM in the livers of rats fed diets with recommended vitamin A levels. The Vmax for retinal formation from holo-CRBP was 14-17 pmol min-1 (mg of protein)-1, a rate sufficiently high to generate adequate retinal to contribute significantly to retinoic acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli

Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol. In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol. Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation. To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli. The purified E. coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine. Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence. They revealed that E. coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1. Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent. The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment. These studies suggest that E. coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions.     

Increased neonatal mortality in mice lacking cellular retinol-binding protein II

Cellular retinol-binding protein II (CRBP II) is a member of the cellular retinol-binding protein family, which is expressed primarily in the small intestine. To investigate the physiological role of CRBP II, the gene encoding CRBP II was inactivated. The saturable component of intestinal retinol uptake is impaired in CRBP II(-/-) mice. The knockout mice, while maintained on a vitamin A-enriched diet, have reduced (40%) hepatic vitamin A stores but grow and reproduce normally. However, reducing maternal dietary vitamin A to marginal levels during the latter half of gestation results in 100% mortality/litter within 24 h after birth in the CRBP II(-/-) line but no mortality in the wild type line. The neonatal mortality in heterozygote offspring of CRBP II(-/-) dams (79 +/- 21% deaths/litter) was increased as compared with the neonatal mortality in heterozygote offspring of wild type dams (29 +/- 25% deaths per litter, p < 0.05). Maternal CRBP II was localized by immunostaining in the placenta at 18 days postcoitum as well as in the small intestine. These studies suggest that both fetal as well as maternal CRBP II are required to ensure adequate delivery of vitamin A to the developing fetus when dietary vitamin A is limiting.