Differential mechanisms of retinoid transfer from cellular retinol binding proteins types I and II to phospholipid membranes

Cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II) are known to differentially facilitate retinoid metabolism by several membrane-associated enzymes. The mechanism of ligand transfer to phospholipid small unilamellar vesicles was compared in order to determine whether differences in ligand trafficking properties could underlie these functional differences. Unidirectional transfer of retinol from the CRBPs to membranes was monitored by following the increase in intrinsic protein fluorescence that occurs upon ligand dissociation. The results showed that ligand transfer of retinol from CRBP-I was >5-fold faster than transfer from CRBP-II. For both proteins, transfer of the other naturally occurring retinoid, retinaldehyde, was 4-5-fold faster than transfer of retinol. Rates of ligand transfer from CRBP-I to small unilamellar vesicles increased with increasing concentration of acceptor membrane and with the incorporation of the anionic lipids cardiolipin or phosphatidylserine into membranes. In contrast, transfer from CRBP-II was unaffected by either membrane concentration or composition. Preincubation of anionic vesicles with CRBP-I was able to prevent cytochrome c, a peripheral membrane protein, from binding, whereas CRBP-II was ineffective. In addition, monolayer exclusion experiments demonstrated differences in the rate and magnitude of the CRBP interactions with phospholipid membranes. These results suggest that the mechanisms of ligand transfer from CRBP-I and CRBP-II to membranes are markedly different as follows: transfer from CRBP-I may involve and require effective collisional interactions with membranes, whereas a diffusional process primarily mediates transfer from CRBP-II. These differences may help account for their distinct functional roles in the modulation of intracellular retinoid metabolism.     

A rapid versatile microassay for cellular retinol-binding protein using Lipidex-1000 microcolumns

A new, rapid and versatile microassay for cellular retinol-binding protein has been developed based on separation of bound and free ligand by means of Lipidex-1000, a hydrophobic Sephadex derivative. This requires quantitative manipulation of retinol in aqueous solution. The tendency of retinol to adhere to glass and plastic surfaces was overcome by addition of the detergent Ammonyx LO, which yields a micellar dispersion. Detergent concentrations up to 10 mM did not interfere with binding of retinol to Lipidex-1000 or binding protein. The binding capacity of Lipidex-1000 was found to exceed 400 nmol of retinol per ml of gel. Retinal pigment epithelium (RPE) cells were used as a source for cRBP (cellular retinol-binding protein). The binding protein is saturated with ligand by incubation for 60 min at room temperature at concentrations of free retinol over 180 nM. Separation of protein-bound retinol from free retinol is achieved via Lipidex-1000: protein-bound (specific and nonspecific) retinol is not retained and is eluted by buffer with the protein fraction. Free retinol is retained by Lipidex and is subsequently recovered by elution with methanol. Total recovery of ligand approaches 100%. Analysis time is about 4 hr for a maximum of ca. 50 samples. Nonspecific protein binding can be determined equally effectively either by incubation with 3 mM PCMBS or by addition of a 100-fold molar excess of nonlabeled retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein,multiligand complexes by mass spectrometry

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.     

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.     

Cross talk between signaling and vitamin A transport by the retinol-binding protein receptor STRA6

The plasma membrane protein STRA6 transports vitamin A from its blood carrier retinol binding protein (RBP) into cells, and it also functions as a cytokine receptor which activates JAK/STAT signaling. We show here that, unlike other cytokine receptors, phosphorylation of STRA6 is not simply induced upon binding of its extracellular ligand. Instead, activation of the receptor is triggered by STRA6-mediated translocation of retinol from serum RBP to an intracellular acceptor, the retinol-binding protein CRBP-I. The observations also demonstrate that the movement of retinol from RBP to CRBP-I, and thus activation of STRA6, is critically linked to the intracellular metabolism of the vitamin. Furthermore, the data show that STRA6 phosphorylation is required for retinol uptake to proceed. Hence, the observations demonstrate that STRA6 orchestrates a multicomponent "machinery" that couples vitamin A homeostasis and metabolism to activation of a signaling cascade and that, in turn, STRA6 signaling regulates the cellular uptake of the vitamin. STRA6 appears to be a founding member of a new class of proteins that may be termed "cytokine signaling transporters."     

Molecular dynamics simulations of the whey protein beta-lactoglobulin.

Molecular dynamics simulations have been used to model the motions and conformational behavior of the whey protein bovine beta-lactoglobulin. Simulations were performed for the protein by itself and complexed to a single retinol ligand located in a putative interior binding pocket. In the absence of the retinol ligand, the backbone loops around the opening of this interior pocket shifted inward to partially close off this cavity, similar to the shifts observed in previously reported molecular dynamics simulations of the uncomplexed form of the homologous retinol binding protein. The protein complexed with retinol does not exhibit the same conformational shifts. Conformational changes of this type could serve as a recognition signal allowing in vivo discrimination between the free and retinol complexed forms of the beta-lactoglobulin molecule. The unusual bending of the single alpha-helix observed in the simulations of retinol binding protein were not observed in the present calculations.     

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.     

Solvent-induced ligand dissociation and conformational states of Cellular Retinol-Binding Protein type I

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation. Acidic aqueous solutions and water-alcohol mixtures were selected, among the best described denaturing solvents, to investigate their effects on the stability of the carrier-ligand complex and the conformational state of the protein upon ligand release. Circular dichroism (CD) and fluorescence spectroscopy were used to probe protein secondary and tertiary structure, compactness and retinol dissociation. While in purely aqueous media retinol dissociation parallels the acid-induced denaturation of the carrier, in water-alcohol mixtures it occurs in a range of co-solvent content lower than that required for protein denaturation. In light of these results, it is suggested that local solvent properties in vivo might modulate protein conformation and flexibility and thus play a fundamental role in the control of retinol exchange between carrier and membrane-bound donors and acceptors.     

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.     

Immunohistochemical localization of retinoid binding proteins at the materno-fetal interface of the porcine epitheliochorial placenta

Retinol and retinoic acid that are potent modulators of gene expression are vital for development and growth of the conceptus. Apart from being transported across the placenta, retinol and retinoic acid may also be active in the placenta per se. Three proteins involved in 1) serum transport of retinol (retinol binding protein [RBP]), 2) cellular transport and metabolism of retinol (cellular RBP [CRBP] I), and 3) retinoic acid (cellular retinoic acid binding protein [CRABP] I), respectively, have been located by immunohistochemistry during gestation in the porcine placenta. This is a diffuse epitheliochorial placenta composed of areolar-gland subunits, where transport of larger molecules takes place, and interareolar regions, where gas-exchange and trophoblast absorption of hemotroph occur. Immunoreactive-RBP (ir-RBP) as well as CRBP I (ir-CRBP) was detected in uterine glands and in areolar trophoblasts, suggesting that RBP-retinol is secreted by the glands and absorbed by the trophoblasts. Both proteins were present also at the interareolar regions, with ir-CRBP in both the uterine epithelium and the apposing trophoblasts, but ir-RBP only in the former. The localization of ir-CRABP was, in contrast, strictly limited to interareolar trophoblasts. Together these findings suggest that 1) the areolar gland subunits are important for transport of retinol and retinol-RBP, and 2) retinoid binding proteins are involved in the development and growth of the porcine placenta.     

Studies on phospholipase activities in Neurospora crassa conidia

Cytosol retinyl ester lipoprotein complex from rat liver was capable of transferring its unesterified retinol component to serum aporetinol-binding protein. In the presence of serum albumin and aporetinol-binding protein, 68% of retinyl ester was hydrolyzed and up to 30% of unesterified retinol was transferred from cytosol retinyl ester lipoprotein complex to serum aporetinol-binding protein in 24 h at 30 °C. The reconstituted retinol-retinol-binding protein complex showed biochemical and biophysical properties similar to native retinol-retinol-binding protein. Both native and reconstituted retinol-retinol-binding proteins had identical uv, CD, and fluorescence spectra as well as binding affinity to prealbumin. Treatment of cytosol retinyl ester lipoprotein with sulfhydryl reagent, with 1 n NaCl, or with diisopropyl fluorophosphate (0.14 mm) abolished the hydrolysis of retinyl ester; however, the activity of retinol transfer from cytosol retinyl ester lipoprotein complex to serum retinol-binding protein was still unaffected. The activity of retinol transfer was proportional to the amount of retinol content in the complex and the amount of aporetinol-binding protein. These experiments suggest that the cytosol retinyl ester lipoprotein complex serves three major functions: (i) as a storage form of retinyl ester and retinol; (ii) as an enzyme for hydrolyzing its own retinyl ester ligand; and (iii) as a medium for transfer of unesterified retinol to serum retinol-binding protein.     

The retinol-retinoic acid metabolic pathway is impaired in the lumbar spine of a rat model of congenital kyphoscoliosis

Although congenital scoliosis is defined as a genetic disease characterized by a congenital and abnormal curvature of the spinal vertebrae, our knowledge of the genetic underpinnings of the disease is insufficient. We herein show that the downregulation of the retinol-retinoic acid metabolism pathway is involved in the pathogenesis of congenital scoliosis. By analyzing DNA microarray data, we found that the expression levels of genes associated with the retinol metabolism pathway were decreased in the lumbar spine of Ishibashi rats (IS), a rat model of congenital kyphoscoliosis. The expression of Adh1 and Aldh1a2 (alcohol dehydrogenase), two enzymes that convert retinol to retinoic acid in this pathway, were decreased at both the gene and protein levels. Rarα, a receptor of retinoic acid and bone morphogenetic protein 2, which play a central role in bone formation and are located downstream of this pathway, were also downregulated. Interestingly, the serum retinol levels of IS rats were higher than those of wild-type control rats. These results indicate that the adequate conversion from retinol to retinoic acid is extremely important in the regulation of normal bone formation and it may also be a key factor for understanding the pathogenesis of congenital scoliosis.     

Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.

Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and transactivation activity of 13,14-dihydroretinoic acid

The metabolism of vitamin A is a highly regulated process that generates essential mediators involved in the development, cellular differentiation, immunity, and vision of vertebrates. Retinol saturase converts all-trans-retinol to all-trans-13,14-dihydroretinol (Moise, A. R., Kuksa, V., Imanishi, Y., and Palczewski, K. (2004) J. Biol. Chem. 279, 50230-50242). Here we demonstrate that the enzymes involved in oxidation of retinol to retinoic acid and then to oxidized retinoic acid metabolites are also involved in the synthesis and oxidation of all-trans-13,14-dihydroretinoic acid. All-trans-13,14-dihydroretinoic acid can activate retinoic acid receptor/retinoid X receptor heterodimers but not retinoid X receptor homodimers in reporter cell assays. All-trans-13,14-dihydroretinoic acid was detected in vivo in Lrat-/- mice supplemented with retinyl palmitate. Thus, all-trans-13,14-dihydroretinoic acid is a naturally occurring retinoid and a potential ligand for nuclear receptors. This new metabolite can also be an intermediate in a retinol degradation pathway or it can serve as a precursor for the synthesis of bioactive 13,14-dihydroretinoid metabolites.     

Identification of retinol as one of the protein HC chromophores

Protein HC (alias alpha 1-microglobulin) contains so far unidentified yellow-brown fluorescent chromophores. Several preparations of human protein HC were extracted with hexane. Most of the extracts contained a substance which, upon reversed-phase HPLC, co-eluted with all-trans- retinol and had an absorption spectrum identical to that of retinol. The substance was also, like retinol, destroyed by exposure to ultraviolet light and acid pH. These observations strongly support the proposal that protein HC is a member of the newly defined lipocalin protein superfamily. The highest retinol-protein HC molar ratio of the investigated protein HC preparations was 1.6 x 10(-3) indicating that retinol is not the only ligand bound to protein HC. This was confirmed by comparing the absorption spectrum of protein HC before and after hexane extraction.     

Vitamin A uptake from foods

PURPOSE OF REVIEW: To review our current understanding of vitamin A uptake from foods. RECENT FINDINGS: There are advancements in understanding the molecular processes involved in vitamin A uptake and the regulation of these processes. A number of genes involved in vitamin A transport and metabolism have been recently identified. The identification of mutations in human genes and targeted disruption of mouse genes have provided further insight as to how these genes contribute to meeting nutritional needs. SUMMARY: The rate limiting steps in the lymphatic absorption of vitamin A involve intracellular processing of vitamin A within the enterocyte. The key steps appear to be related to chylomicron formation and secretion and are closely coupled with fat absorption. The genes encoding serum retinol binding protein, cellular retinol binding protein I and cellular retinol binding protein II have been disrupted by homologous recombination in mice. Studies of these knockout mice indicate that extrahepatic uptake of postprandial vitamin A may play a particularly important role in the maternal-offspring transfer of vitamin A. Further studies of the transfer of maternal dietary vitamin A have important implications for assessing the upper limits of maternal vitamin A supplementation.