Retinol-binding Protein Complex Research Articles

The Extracellular Protein, Transthyretin Is an Oxidative Stress Biomarker.

The extracellular protein, transthyretin is responsible for the transport of thyroxin and retinol binding protein complex to the various parts of the body. In addition to this transport function, transthyretin has also been involved in cardiovascular malfunctions, polyneuropathy, psychological disorders, obesity and diabetes, etc. Recent developments have evidenced that transthyretin has been associated with many other biological functions that are directly or indirectly associated with the oxidative stress, the common hallmark for many human diseases. In this review, we have attempted to address that transthyretin is associated with oxidative stress and could be an important biomarker. Potential future perspectives have also been discussed.

Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade

The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A-retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K(d)s ~2 nM and ~200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

Expression of the megalin C-terminal fragment by macrophages during liver fibrogenesis in mice

The low-density-lipoprotein receptor megalin (LRP2, gp330) is strongly expressed in the kidney, where it is responsible for the resorption of metabolites from primary urine. One of the main ligands is the complex of retinol and retinol binding protein. Megalin has been hypothesized to be part of the retinol storage system in liver. Considering the role of hepatic stellate cells in retinol storage and fibrogenesis we investigated mouse strains that developed different degrees of fibrosis after challenge with CCl 4. Immunoblotting revealed the invariable expression of the megalin C-terminal fragment independent of liver damage in all strains. However, only a specific cell population in centrilobular areas of fibrotic livers from DBA/2J mice, which were most susceptible for CCl 4-induced fibrogenesis in our study, was stained using megalin-specific antibodies. Double immunostaining indicated that a subset of hepatic macrophages might represent the megalin-expressing cells in fibrotic liver. Fluorescence activated cell sorting based isolation of hepatic macrophages and megalin specific expression analysis demonstrated the transcription of the whole megalin gene in liver macrophages. We argue that megalin might exhibit a proinflammatory effect by the uptake of retinoids in recruited monocytes, which thereby differentiate to liver macrophages and potentiate fibrogenesis by the release of proinflammatory mediators. Otherwise, megalin might be activated in macrophages during advanced fibrogenesis and act as a negative regulator of proinflammatory genes.

Free Energy Landscape of the Retinol/Serum Retinol Binding Protein Complex: A Biological Host−Guest System

Small molecule host-guest complexes have traditionally provided model systems for biological ligand recognition. Nonetheless, direct extrapolation of these results is precluded by the comparative simplicity of these supramolecular assemblies. If energetic behavior analogous to small molecule host-guest chemistry exists, it is unclear how this would manifest for protein-small molecule interactions. To answer this question, we employ the retinol/serum retinol binding protein (sRBP) system as an analogue of a classical host-guest complex. Using a combination of molecular dynamics simulations and free energy methods, we decompose the potential of mean force for retinol unbinding from the sRBP into constituent interactions. Our calculations reveal an unexpected mechanism of host-guest complexation. Desolvation is sufficient to drive formation of an intermediate binding state; however, a combination of electrostatic and van der Waals interactions pull the intermediate into a stable configuration. Association is accompanied by a change in the conformational flexibility of the portal domains of sRBP and subsequent "stiffening" of the holo sRBP, reflecting an "order-disorder" transition in the protein.

Cell-surface receptors for retinol-binding protein.

Target tissues for retinol have receptors to which retinol and retinol-binding protein complex (retinol-RBP) binds specifically. Following this binding, either the whole retinol-RBP is internalized into the cells of some tissues or only the retinol enters the cells in other tissues, with liberation of apo-RBP. However, uptake of free retinol is the preferred mode of cellular entry in keratinocytes.

Retinoic acid

What is retinoic acid? Retinoic acid is a lipophilic molecule of low molecular weight (300 Da). Retinoic acid is synthesized from vitamin A (retinol) and found in embryos and adult vertebrates. Because animals are unable to produce vitamin A, they obtain this vitamin in the form of carotenoids from plants or retinyl esters derived from animal products. How is retinoic acid produced? In vertebrates, vitamin A, in the form of retinyl esters, is stored mainly in the liver as well as in the lungs, kidneys and bone marrow. Retinol, bound to the retinol-binding protein (RBP), enters the circulatory system in order to reach cells that require retinoic acid. The retinol–RBP complex enters the cells through the RBP receptor (STRA6). Within the cells, retinol is metabolized in a reversible step to retinal by the retinol or alcohol dehydrogenases (RODHs or ADHs) and then in an irreversible manner to retinoic acid by the retinal dehydrogenases (RALDHs). In the cytoplasm, retinoic acid is associated with the cellular retinoic acid-binding protein (CRABP). And what does it do? Simply speaking, retinoic acid functions to regulate gene expression (Figure 1). In cells, retinoic acid enters the nucleus where it binds to heterodimers formed by two different classes of nuclear receptors: Retinoic acid receptors (RARs) and retinoid X receptor (RXRs). The receptor heterodimers are bound to a specific DNA sequence known as the retinoic acid-response element (RARE) and upon retinoic acid binding, recruits co-activators (including histone acetyltransferase (HAT)), leading to transcription activation of specific retinoic acid-regulated genes. How many RARs and RXRs are there? In most vertebrates, there are three RARs (α, β and γ) and three RXRs (α, β and γ), all of which can have multiple isoforms, leading to large numbers of possible receptor combinations. How is the retinoic acid ‘signal’ turned off? In the cytoplasm, retinoic acid can be catabolized by the cytochrome P450 enzyme (CYP26) into inactive compounds that are then excreted. In the absence of retinoic acid, the RAR/RXR heterodimers bound to their target sequences recruit the nuclear receptor co-repressor complex NCOR/Sin3A/HDAC (histone deacetylase), inhibiting transcription activation. What does retinoic acid do? In adults, vitamin A and its retinoid derivatives play significant roles in a number of processes, such as vision, learning and memory, immune function, reproduction as well as the maintenance of epithelial function and differentiation. In the hippocampus, retinoic acid plays important physiological roles in synaptic plasticity, learning and memory, and adult neurogenesis. In the retina, retinoic acid acts as a light-signaling neuromodulator and regulates gap junction-mediated coupling of retinal neurons. It has also been implicated in the control of sleep and the circadian clock. Why are developmental biologists so obsessed with retinoic acid? This simple molecule has been implicated in the control of many aspects of embryo development that include cranio-facial morphogenesis, neuronal differentiation, eye, limb, lung, kidney and heart development. The patterning of the antero-posterior embryo body axis is also directly regulated by retinoic acid, which controls the expression of several HOX genes through RAREs in their regulatory/enhancer regions. Are there any human diseases related to retinoic acid deficiency? Reduction of vitamin A levels in the diet can lead to a debilitating immune system, anemia or blindness. During pregnancy, insufficient levels of vitamin A lead to fetal vitamin A deficiency-induced syndrome. This results in developmental defects exhibiting many common features with those observed in the RAR/RXR mutations in mouse, including cranio-facial and eye abnormalities. Retinoic acid as an anticancer drug…? Acute promyelocytic leukaemia is caused by a chromosomal translocation that fuses the promyelocytic leukaemia gene (PML) on chromosome 15 with the RARα gene on chromosome 17. The PML-RARα fusion protein leads to a recruitment of co-repressor complexes that epigenetically silence gene expression. Differentiation therapy with retinoic acid is being used in combination with chemotherapy for the treatment of patients with acute promyelocytic leukaemia, resulting in a 70-80% cure rate. Retinoic acidVilhais-Neto et al.Current BiologyApril 08, 2008In Brief(Current Biology 18, R191–R192, March 11, 2008) Full-Text PDF Open Archive

A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin A

Vitamin A has diverse biological functions. It is transported in the blood as a complex with retinol binding protein (RBP), but the molecular mechanism by which vitamin A is absorbed by cells from the vitamin A-RBP complex is not clearly understood. We identified in bovine retinal pigment epithelium cells STRA6, a multitransmembrane domain protein, as a specific membrane receptor for RBP. STRA6 binds to RBP with high affinity and has robust vitamin A uptake activity from the vitamin A-RBP complex. It is widely expressed in embryonic development and in adult organ systems. The RBP receptor represents a major physiological mediator of cellular vitamin A uptake.

The Modified-Relative-Dose-Response Values in Serum and Milk Are Positively Correlated over Time in Lactating Sows with Adequate Vitamin A Status

The modified-relative-dose-response (MRDR) test, which requires a blood sample after dosing with 3,4-didehydroretinyl acetate (DRA), has been used to determine vitamin A (VA) status of individuals and groups worldwide. Less invasive methods using milk are in development in a swine model. Swine are a good choice for studying VA metabolism because their gastrointestinal anatomy, morphology, physiology, and VA requirements are similar to those of humans. In this study, DRA was used as a VA tracer in lactating sows to follow the metabolism of newly ingested VA. Lactating sows (n = 6) were administered 35 micromol DRA after overnight food deprivation. Blood and milk were collected at 0, 1.5, 3, 5, 7, 9, 24, and 48 h; livers were obtained at the time of killing. Samples were analyzed for didehydroretinol (DR), retinol (R), and didehydroretinyl esters (DRE). Serum DR:R was compared with that in milk and other VA indicators. DRE rapidly increased in serum, corresponding to chylomicra, whereas DR increased at a slower rate corresponding to the holo-DR:retinol-binding protein complex released from the liver. An estimated 10-20% of the dose was irreversibly lost in milk over 48 h. The mean MRDR value was 0.018 +/- 0.013 at 5 h and the mean liver VA was 0.73 +/- 0.21 micromol/g, both signifying sufficient stores. Milk and serum DR:R values were directly correlated (r = 0.64, P < 0.0001). Thus, DR:R values in milk may be a potential alternative to serum in determining VA status in lactating women. Future work is required in VA-deficient sows and women of varying VA status to determine DR trafficking and to compare DR:R values in milk with those in serum.

Biochemical basis for retinol deficiency induced by the I41N and G75D mutations in human plasma retinol-binding protein

Retinol-binding protein (RBP) is the retinol-specific carrier protein present in plasma, where it circulates almost entirely bound to thyroxine-binding transthyretin (TTR). Recently, depressed plasma retinol and RBP levels in carriers of the I41N and G75D RBP point mutations have been reported. We show here that although recombinant human N41 and D75 RBPs can form complexes with retinol and TTR in vitro, the retinol-mutated RBP complexes are significantly less stable than human normal holo-RBP, as revealed by the markedly facilitated retinol release by mutated holo-RBPs to phospholipid membranes, in accordance with the location of mutated residues inside the RBP retinol-binding cavity. Taken together, the data are consistent with the I41N and G75D point mutations being the cause of an altered interaction of retinol with RBP, resulting in a remarkably reduced stability of the retinol–RBP complex, which in turn can lead to the lowering of plasma retinol and RBP levels.

Characterization of the molten globule state of retinol‐binding protein using a molecular dynamics simulation approach

Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic conditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation conformers, with a radius of gyration at least 1.1 A greater than that of the native state, contain on average 37%beta-sheet secondary structure. In these conformers the central regions of the two orthogonal beta-sheets that make up the beta-barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the beta-sheets, and large variations in side chain packing even in the protein core. Significant conformational changes are seen in the simulation conformers for residues 85-104 (beta-strands E and F and the E-F loop). These changes give an opening of the retinol-binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface.

Structure and function of hepatic stellate cells.

Hepatic stellate cells (vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, Ito cells) exist in the space between parenchymal cells and sinusoidal endothelial cells of the hepatic lobule and store 80% of retinoids in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of retinoid homeostasis; they express specific receptors for retinol-binding protein (RBP), a binding protein specific for retinol, on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis. Hepatic stellate cells in arctic animals such as polar bears and arctic foxes store 20-100 times the levels of retinoids found in humans or rats. In pathological conditions such as liver fibrosis, hepatic stellate cells lose retinoids, and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped stellate cells to that of fibroblasts or myofibroblasts. The three-dimensional structure of ECM components was found to regulate reversibly the morphology, proliferation, and functions of the hepatic stellate cells. Molecular mechanisms in the reversible regulation of the stellate cells by ECM imply cell-surface integrin binding to ECM components, followed by signal transduction processes and then cytoskeleton assembly. Stellate cells also exist in extrahepatic organs such as pancreas, lung, kidney, and intestine. Hepatic and extrahepatic stellate cells form the stellate cell system.

Retinol Analysis in Dried Blood Spots by HPLC

There are many advantages to measuring vitamin A in dried blood spots (DBS) from a finger prick as compared to plasma collected by venipuncture. The advantages include easier collection, transport and storage; accessibility to younger and more remote populations; and decreased risk of disease transmission. We describe a method for the extraction of retinol from DBS for analysis by HPLC and initial comparison to plasma retinol. The effects of various buffers, detergents, antioxidants and chelators were evaluated to establish the most effective approach to elute the retinol: retinol binding protein (holo-RBP) complex from the blood collection cards. The process involves ultrasonic agitation to elute holo-RBP into a phosphate buffer containing an antioxidant and metal chelator. The holo-RBP complex was denatured by the addition of ethanol containing additional antioxidants permitting the extraction of free retinol into hexane. Following solvent evaporation, the extract was dissolved in methanol for HPLC analysis. The initial measured retinol levels in freshly collected DBS declined for 6-10 d whether stored at 25, 4 or -20 degrees C, but remained consistent thereafter (homeostatic). By incorporating a "recovery/volume adjustment" factor, measured retinol values in homeostatic DBS were adjusted to the equivalent of plasma retinol. For 17 normal adults, the correlation coefficient was 0.90 between plasma retinol and adjusted DBS retinol in samples that had been stored at -70 degrees C for < 9 mo. The use of this new sample matrix for vitamin A assessment will allow access to previously unavailable populations.

The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP.

Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. However, the concentration of RBP in the plasma is limiting, and the complex isolated from serum is composed of TTR and RBP in a 1 to 1 stoichiometry. We report here the crystallographic structure at 3.2 A of the protein-protein complex of human RBP and TTR. RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry: Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.

Crystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein (RBP) complex bound to an anti-RBP Fab.

A macromolecular complex of human transthyretin, human retinol-binding protein and an anti-retinol-binding-protein Fab was crystallized by vapour diffusion in sitting drops. Diffraction from these crystals at cryogenic temperatures was consistent with the space group C222, with cell parameters a = 159.34, b = 222.40 and c = 121.27 A. Crystals diffracted to a resolution limit of 3.36 A using synchrotron radiation. Based on a 2:2:1 stoichiometry for the Fab-retinol-binding-protein-transthyretin complex and the presence of one such complex per asymmetric unit, a reasonable Vm coefficient of 2.74 A3 Da-1 could be estimated.

Molecular mechanisms in the reversible regulation of morphology, proliferation and collagen metabolism in hepatic stellate cells by the three‐dimensional structure of the extracellular matrix

Hepatic stellate cells (vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, Ito cells) exist in the perisinusoidal space of the hepatic lobule and store 80% of the body's retinoids as retinyl palmitate in lipid droplets in the cytoplasm. Under physiological conditions, these cells play pivotal roles in the regulation of retinoid homeostasis; they express specific receptors for retinol-binding protein (RBP), a binding protein specific for retinol, on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis. However, in pathological conditions such as liver fibrosis, these cells lose retinoids and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan and adhesive glycoproteins. The morphology of these cells also changes from star-shaped stellate cells to that of fibroblasts or myofibroblasts. The three-dimensional structure of ECM components was found to regulate reversibly the morphology, proliferation and functions of hepatic stellate cells. Molecular mechanisms in the reversible regulation of stellate cells by ECM imply cell surface integrin binding to ECM components followed by signal transduction processes and then cytoskeleton assembly.

Dissection of multi-protein complexes using mass spectrometry: Subunit interactions in transthyretin and retinol-binding protein complexes

Complexes formed between transthyretin and retinol-binding protein prevent loss of retinol from the body through glomerular filtration. The interactions between these proteins have been examined by electrospray ionization combined with time-of-flight mass analysis. Conditions were found whereby complexes of these proteins, containing from four to six protein molecules with up to two ligands, are preserved in the gas phase. Analysis of the mass spectra of these multimeric species gives the overall stoichiometry of the protein subunits and provides estimates for solution dissociation constants of 1.9 +/- 1.0 x 10(-7) M for the first and 3.5 +/- 1.0 x 10(-5) M for the second retinol-binding protein molecule bound to a transthyretin tetramer. Dissociation of these protein assemblies within the gas phase of the mass spectrometer shows that each retinol-binding protein molecule interacts with three transthyretin molecules. Mass spectral analysis illustrates not only a correlation with solution behavior and crystallographic data of a closely related protein complex but also exemplifies a general method for analysis of multi-protein assemblies.

Absorption of Retinol from the Retinol:Retinol-Binding Protein Complex by Small Intestinal Gut Sheets from the Rat

Absorption of retinol by the absorptive cells of the small intestine is a necessary first step in the metabolism of vitamin A. Previous studies had suggested that absorption of retinol from the retinol:retinol-binding protein complex (retinol:RBP) might be receptor mediated. We investigated the specificity of this process by determining the absorption of retinol from retinol:RBP by small intestinal sheets obtained from the suckling rat. We found that the absorption of retinol from retinol:RBP was a saturable process. Unlike the absorption of free retinol, absorption of retinol from retinol:RBP was not inhibited byN-ethylmaleimide. The absorption was specifically competable by unlabeled retinol:RBP and by both apo- and holocellular retinol-binding protein, but not by β-lactoglobulin. This suggests that a specific mechanism is present for the absorption of retinol bound to RBP.

2909 Abnormal cellular acylcarnitine metabolism in chronic fatigue syndrome studied by PET

In this paper, we present a fast minimicroassay of serum vitamin A by capillary zone electrophoresis with laser-excited fluorescence detection. A 60 cm × 50 μm I.D. fused-silica capillary was used for the separation, and the polymer coating was burned off 20 cm from the cathodic end to form a detection window. The buffer system consisted of 50 mM sodium phosphate plus 10 mM sodium chloride at pH 7.8. A helium—cadmium laser set at 325 nm was used for excitation, and the fluorescence of the vitamin A—retinol-binding protein complex was monitored at 465 nm using a photodiode. The stray and scattered radiation were removed by two special filters. Using this system, about 8 nl of serum sample were injected for direct analysis without any sample preparation. The analysis time for each sample was less than 6 min, and subfemtomole levels of vitamin A in human or animal blood could be easily detected. Therefore, the method is potentially useful for finger-prick vitamin A analysis, especially for babies and young children.

Development of an artificial retina for light sense restration

In this paper, we present a fast minimicroassay of serum vitamin A by capillary zone electrophoresis with laser-excited fluorescence detection. A 60 cm × 50 μm I.D. fused-silica capillary was used for the separation, and the polymer coating was burned off 20 cm from the cathodic end to form a detection window. The buffer system consisted of 50 mM sodium phosphate plus 10 mM sodium chloride at pH 7.8. A helium—cadmium laser set at 325 nm was used for excitation, and the fluorescence of the vitamin A—retinol-binding protein complex was monitored at 465 nm using a photodiode. The stray and scattered radiation were removed by two special filters. Using this system, about 8 nl of serum sample were injected for direct analysis without any sample preparation. The analysis time for each sample was less than 6 min, and subfemtomole levels of vitamin A in human or animal blood could be easily detected. Therefore, the method is potentially useful for finger-prick vitamin A analysis, especially for babies and young children.

Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin

The retinol carrier retinol-binding protein (RBP) forms a complex with the thyroid hormone binding protein transthyretin in the plasma of a number of vertebrate species. The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with transthyretin have been investigated by means of fluorescence anisotropy studies. The presence of two independent and equivalent RBP binding sites per transthyretin molecule has been established for proteins purified from species distant in evolution. Although the natural ligand retinol participates in the interaction between retinol-RBP and transthyretin, its binding to RBP is not a prerequisite for protein—protein interaction. The dissociation constants of human transthyretin binding liganded and unliganded forms of human RBP were determined to be: all- trans retinol-RBP, K d ∼ 0.2 μM; apoRBP, K d − 1.2 μM; all- trans retinoic acid-RBP, K d − 0.8 μM; all- trans retinyl methyl ether-RBP, K d ∼ 6 μM. The complex of RBP with the synthetic retinoid fenretinide, which bears the bulky hydroxyphenyl end group, exhibits negligible affinity for transthyretin. The replacement of RBP-bound retinol with synthetic retinoids affects RBP-transthyretin recognition to an extent that appears to be well correlated with the nature and steric hindrance of the groups substituting the retinol hydroxyl group, consistent with their location at the interface between the contact areas of RBP and transthyretin.