Plasma Retinol Binding Protein RBP

Retinol is released from the Uver bound to an a-globulin, retinol binding protein (RBP) this serves to mainttun the vittunin in aqueous solution, protects it against oxidation, and also deUvers the vitamin to target tissues. RBP binds 1 mol of retinol per mol of protein. 

RBP forms a 1 1 complex with the tetreuneric thyroxine-binding prealbumin, transthyretin. This is importtuit to prevent urinary loss of retinol bound to the relatively small RBP (Mr 21,000), whichwouldbe filtered by the glomerulus trtuisthyretin has an Mr of 54,000 hence, the complex will not normally be filtered. However, moderate renal damage, or the increased permeability of the glomerulus in infection, may result in considerable loss of vitamin A bound to RBP-transthyretin. 

The treuisthyretin tetramer could theoretically bind 2 mol of holo-RBP, but does not, because holo-RBP is limiting. In vitamin A deficiency, the ratio of RBPitransthyretin falls, indicating that although binding to transthyretin is essential for secretion of holo-RBP from the liver, vitamin A is not essential for secretion of transthyretin. Other tissues secrete holo-RBP, but not trcmsthyretin it is assumed that this binds to transthyretin in the circulation. 

Metabolites of polychlorinated biphenyls bind to tbe thyroxine binding site of transthyretin and, in doing so, impair the binding of RBP. As a result of this, there is free RBP-bound retinol in plasma, which is filtered at the glomerulus and hence lost in the urine. This may account for the vitamin A depleting action of polychlorinated biphenyls (Brouwer and van den Berg, 1986). 

RBP is relatively rich in aromatic amino acids, which create a deep hydrophobic pocket that is specific for the / -ionone ring, polyene side chtdn, and poleir end group. In addition to all-trans-retinol, RBP binds retinrildehyde, retinoic acid, and 13-ds-retinol, butnot retinyl esters or / -carotene. RBP shows considerable steuctural homology with / -lactoglobulin from milk and other 

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Figure S.3 Schematic diagram of the structure of human plasma retinol-binding protein (RBP), which is an up-and-down P barrel. The eight antiparallel P strands twist and curl such that the structure can also be regarded as two p sheets (green and blue) packed against each other. Some of the twisted p strands (red) participate in both P sheets. A retinol molecule, vitamin A (yellow), is bound inside the barrel, between the two P sheets, such that its only hydrophilic part (an OH tail) is at the surface of the molecule. The topological diagram of this stmcture is the same as that in Figure 5.2. (Courtesy of Alwyn Jones, Uppsala, Sweden.)...

The first lipocalin whose 3-D structure was solved and refined at high resolution was the human plasma retinol-binding protein (RBP) [22, 23]. RBP acts as a natural transporter of vitamin A (retinol) in the blood of vertebrates. Upon complexation in a hydrophobic cavity with complementary shape, the poorly soluble terpenoid alcohol becomes packaged by the protein and protected from oxidation or double-bond isomerization. RBP is synthesized in the liver and directly loaded with fhe hgand in fhe hepatocyte, where retinol is stored. Furthermore, the holo-RBP forms a structurally defined ternary complex with transthyretin [24], also known as prealbumin. After delivery of the retinol ligand to a target tissue, fhe complex decomposes and fhe monomeric apo-RBP becomes filtered out by fhe kidney and degraded. 

The peculiar spectral properties of CRBP-bound retinol and RME are indicative of specific ligand-protein interactions (see Fig. 2) Instead, other holo-retinoid-binding proteins exhibit less characteristic spectra. For example the absorption spectrum of the complex of retinol with plasma retinol-binding protein (RBP) is characterized by a single, well-shaped peak centered at approx 328 nm On the other hand, the absorption spectra of some CRBP-bound retinoids, like CRBP-bound a l-trans retinal (12,13), do not resemble those of CRBP-bound retinol and RME. 

The first example is the plasma-borne retinol-binding protein, RBP, which is a single polypeptide chain of 182 amino acid residues. This protein is responsible for transporting the lipid alcohol vitamin A (retinol) from its storage site in the liver to the various vitamin-A-dependent tissues. It is a disposable package in the sense that each RBP molecule transports only a single retinol molecule and is then degraded. 

The other major class of extracellular LBPs of mammals is the lipocalins (Flower, 1996). These are approximately 20 kDa, P-sheet-rich proteins, performing functions such as the transport of retinol in plasma or milk, the capture of odorants in olfaction, invertebrate coloration, dispersal of pheromones, and solubilizing the lipids in tears (Flower, 1996). The retinol-binding protein (RBP) of human plasma is found in association with a larger protein, transthyretin, the complex being larger than the kidney threshold and thus not excreted, although the RBP itself may dissociate from the complex to interact with cell surface receptors in the delivery of retinol (Papiz et al., 1986 Sundaram et al., 1998). 

Release from the liver When needed, retinol is released from fie liver and transported to extrahepatic tissues by the plasma retax -binding protein (RBP). The retinol-RBP complex attaches to spe cific receptors on the surface of the cells of peripheral tissues, permitting retinol to enter. Many tissues contain a cellular letaiol-binding protein that carries retinol to sites in the nucleus where the vitamin acts in a manner analogous to steroid hormones. 

In the intestinal mucosal cells, /3-carotene is cleaved via an oxygenase (an enzyme that introduces molecular 02 into organic compounds) to frans-retinal (aldehyde form of trans-retinol, as shown in Table 6.2), which in turn is reduced to frans-retinol, vitamin Av Retinol is then esterified with a fatty acid, becomes incorporated into chylomicrons, and eventually enters the liver, where it is stored in the ester form until it is required elsewhere in the organism. The ester is then hydrolyzed, and vitamin Ax is transported to its target tissue bound to retinol-binding protein (RBP). Since RBP has a molecular weight of only 20,000 and would be easily cleared by the kidneys, it is associated in the bloodstream with another plasma protein, prealbumin. 

Plasma Retinol Binding Protein Measurement of plasma concentrations of RBP may give some additional information. Indeed, it has been suggested that because retinol is susceptible to oxidation on storage of blood samples, measurement of RBP may be a better indication of the state of vitamin A status. In adequately nourished subjects, about 13% of immunologi-caUy reactive RBP in plasma is present as the apo-protein, whereas in vitamin A-deficient children, the proportion of apo-protein may rise to 50% to 90% of... 

The thyroidogenic effects and corresponding biochemical mechanisms of PCBs and other OHS were recently reviewed by Brouwer et al. [44]. The selective retention of certain OH-PCB congeners in blood (Sect. 5.2.2 and 5.3.2) is concomitant with effects observed on the plasma levels of thyroid hormones. Thyroxine is transported in plasma by a protein complex consisting of TTR and retinol binding protein (RBP). Rats administered CB-77 were shown to have reduced plasma levels of both thyroxine and retinol [196]. A major metabolite of CB-77, 4-OH-3,3, 4, 5-tetrachlorobiphenyl, was identified as the active compound [40]. The same hydroxy-PCB metabolite was found to be retained in mouse fetal soft tissue [191,197]. 

Retinol-binding protein (RBP) Liver M.W. 21,000 10-12 hours N 3.5-9.0 mg/dl Circulates in plasma in 1 1 complex with transthyretin, transports retinol and thyroxine, plasma levels influenced by glomerular filtration rate, retinol and zinc status, considered to be too sensitive and therefore has limited value... 

During absorption some /3-carotene is also converted to retinoid (Dimitrov et al, 1988 Olson, 1989 van Vliet et al, 1992 Scita et al, 1993) and transferred via a plasma chylomicron (renmant) retinyl ester compartment to a liver retinyl ester compartment. From here it is released in a plasma retinol-binding protein-retinol (RBP-ROH) compartment for transfer to target tissues. Eventually it is lost irreversibly from the RBP-ROH compart-... 

Rat cellular retinol binding Protein c-RBP Bovine plasma retinol-binding protein (bRBP)... 

Vitamin A is found in the blood in the form of retinol bound to a specific protein called the retinol binding protein (RBP). RBP has been purified from human plasma it has a molecular weight of 21,000 and binds one molecule of retinol per molecule of protein and moves as an a 1 protein on electrophoresis. 

Vitamin A is transported m the plasma as retinol bound to a carrier protein, called retinol-binding protein (RBP), which itself forms a complex with the thyroxine-binding protein, known as transthyretin (TTR). This complex exists in equilibrium with free holo-RBP, which can then interact with a specific cell-surface receptor, thereby inducing the release of its retinol to the target cell. Thus, RBP possesses at least three molecular-recognition properties it binds retinol and it interacts with both TTR and the cell-surface receptor (for reviews, see refs. I and 2). 

To study the interaction of retinol-binding protein (RBP) with its plasma carrier, transthyretin (TTR), spectrofluorimetry, and circular dichroism have previously been used. Both these techniques require milligram quantities of the proteins and this sets limitations on the use of these techniques for the study of RBP-TTR interactions using recombinant proteins. The Escherichia coll expression system described in Chapter 11 does not readily produce milligram quantities of RBP for routine use. For this reason, we have developed a highly sensitive method which employs radioiodinated I-RBP (unpublished). The method requires only microgram quantities of protein. This chapter describes a method to radioiodinate RBP without loss of activity and protocols for its use in the study of its interaction with TTR. 

The predominant retinoid in the fasting circulation is retinol, all of which is bound to its specific plasma transport protein, retinol-binding protein (RBP) [1,2]. Although retinol accounts for approximately 95 to 99% of all retinoid in the circulation, other retinoids also are present. Fasting human and rodent blood contains very low levels of both sAhtrans- and 13-cw-retinoic acid (approximately 0.2 to 0.7% of those of retinol) [3], as well as low levels of retinyl esters in lipoprotein fractions, particularly very low-density lipoproteins (VLDL) and low density lipoproteins (LDL) [4]. Soluble glucuronides of both retinol and retinoic acid are also detectable in the circulation of humans and rodents [5], as are provitamin A carotenoids like P-carotene... 

Retinol binding protein (RBP) in plasma, serum, and urine can be analyzed using different immunoassays, e.g., latex immunoassay [63], ELISA [59], monoclonal antibody-based fluorescence immunoassay [68] or immunonephelometry [69,70]. For principles of latex immunoassay and ELISA, see Section 3.2. 

Recently, a new mouse model was developed that allows us to examine photoresponses in an entirely opsin-free mouse model. Opsins require their chromophore, 11-cis-retinal, for function, and therefore, one obvious approach to create an opsin-free model would be to deprive mice of dietary vitamin A, rendering aU opsins nonfunctional, including rod and cone opsins as well as known and yet to be discovered novel opsins. This approach is not feasible in normal mice, because vitamin A is required during development, and adult mice have stored sufficient vitamin A in their Hver to last for the lifetime of the animals, even if they were put on a vitamin-A-free diet. The creation of the plasma retinol-binding protein mutant rbp) enables us to deplete ocular retinal in mice. The Rbp protein transports retinal from storage in the Hver to peripheral tissues. With the Rbp mutation, peripheral tissues such as the eye can be vitamin A depleted in 6 to 8 months. Therefore, we used the vitamin-A-depleted rhp mouse as an opsin-free model for circadian photoreception. 

Plasma retinol is transported by a specific 21-kDa transport protein, retinal binding protein (RBP). Most RBP is produced in the liver, but some extra-hepatic organs also synthesize it. Each molecule of RBP binds one molecule of all-tra s-retinol nonco-valently. In plasma, the retinol-RBP complex (holo-RBP) forms a larger complex with a cotransport... 

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