RBP with TTR

Studies employing polarization of fluorescence (van Jaarsveld et al., 1973a) and equilibrium dialysis (Raz and Goodman, 1969) have shown that there is no 

Circular dichroic (CD) spectral studies have been conducted to examine the effects of the RBP-TTR interaction on the secondary structures of the two proteins. The CD spectra of mixtures of RBP and TTR in the 200- and 240-nm region were additive, suggesting that formation of the RBP-TTR complex results in very little if any alteration in the secondary structures of the two proteins (Rask et al, 1972 Gotto et al., 1972). In another study (Heller and Horwitz, 1973), it was reported that in the 240- to 300-nm region the CD spectra are not additive upon binding of holo-RBP to TTR, suggesting that some conformational changes in one or both proteins may occur on formation of the protein-protein complex. Further work is needed fully to resolve this question. 

The effects of a number of other perturbations on the interaction of RBP with TTR (and on the interactions of these proteins with their ligands retinol and thyroxine, respectively) have been explored. The other manipulations that have been examined include effects of changes in temperature and urea concentration and the effects of reduction and alkylation of disulfide bonds and of iodination (Raz et al., 1970) the effects of modifications of tyrosine, lysine, and tryptophan residues were also examined (Heller and Horwitz, 197S Horwitz and Heller, 1974b). Addition of 6 Af urea completely disrupted the RBP-TTR complex, markedly reduced the affinity of TTR for thyroxine, but did not interfere with the association of RBP with retinol. Iodination of isolated RBP decreased its affinity for TTR (Raz et al., 1970 Vahlquist, 1972 Vahlquist et al., 1973). However, it was found (Vahlquist, 1972 Vahlquist et al., 1973) that iodination of the RBP-TTR complex, followed by the dissociation of the complex and separate isolation of the two proteins, yielded iodinated RBP with full affinity for 

These observations suggest that one or more tyrosine residues of RBP may be present in the protein-protein binding domain. 

Estimates of the apparent association constant of human TTR for human holo-RBP have varied from 1.2 X 10 M (van Jaarsveld et al., 1973a) to approximately 8 X 10 M (Nilsson ef a/., 1975 Kopelman e/u/., 1976b Tragardh et al., 1980) to 1.3 X 10 (Fex et al., 1979). A number of studies using different methodologies have demonstrated that retinol-free apo-RBP also binds well to TTR (Raz et o/., 1970 Peterson and Rask, 1971 van Jaarsveld eta/., 1973a Fex et al., 1979), although one study (Heller and Horowitz, 1973) reported a lack of 

---

The assay described in this section involves incubation of I-RBP with TTR until equilibnum binding is achieved. The TTR-bound I-RBP is then separated from the unbound I-RBP by selective precipitation of the complex with 10% PEG 8000. At this PEG concentration, negligible amounts of free I-RBP are precipitated... 

In a placebo-controlled randomized supplementation trial (approved by the ethic commission of Ethiopia) in the rural area (AZOZO) district of Gondar Ethiopia from 220 households, 161 children (2-5 years of age) were selected at random for the study at a first visit to the local clinic, nutritional assessment, and stool examination (parasites or ova) were performed (Biesalski et ah, 1999). 141 children with parasites were treated with mebendazole. Heparin blood was obtained for assessment of vitamin A, RBP, and TTR (transthyretine) concentrations. 

Twenty-five children selected at random received aerosol treatment with RP 6000 vitamin A units per 2 weeks over 3 months being provided. Twenty-five further children served as controls receiving a placebo also aerosol delivered. The aerosol was administered through the mouth during breath inhalation with an adapter. No adverse effects or reactions were observed during inhalation and the children complied well with the treatment. Trained field workers performed the inhalation trials and blood sampling. In the study and control group. Heparin blood samples were collected before and at completion of the study for measurements of vitamin A, RBP, and TTR concentrations. 

Although our understanding of the retinoid system in fish is far from complete, it appears similar, but more complex, than that of the mammals. In fish, there are two forms of the retinoids, the standard retinoids as well as the didehydroretinoids. The fish have two additional receptors RXRS and RXRe. RA metabolites such as 4-OH RA bind to fish RARs with high affinity, while they do not in mammals. In fish, RBP and TTR circulate independent of one another. While we know that these differences exist, we have little idea of their implications on normal functions. Do the didehydroretinoids have the same functions as the standard retinoids What are the functions of RXRS and RXRs and what are their ligands Do RA metabolites play a role in gene expression or are they simply excreted These are just some of the questions that need to be addressed in future studies. 

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. 

Binding of RBP to TTR as well as to the receptor can be studied using RBP radiolabeled with Either the native or the recombinant form of RBP can be... 

RBP is synthesized and secreted by the liver and circulates in plasma mainly as a protein-protein complex with TTR. Normally, RBP is secreted almost entirely as the holoprotein, containing a molecule of bound retinol. TTR is also produced... 

Free uncomplexed RBP is small enough to be filtered by the renal glomeruli, whereas TTR and the RBP-TTR complex are not. Although very little RBP is normally present in the free state, the rates of glomerular filtration and renal metabolism of RBP are sufficiently large enough to constitute the major pathway of RBP catabolism. Patients with severe chronic renal disease show a reduced metabolic clearance rate and an elevated plasma concentration of RBP, with normal levels of TTR. 

An estimate can be made of the concentration of free uncomplexed RBP normally present in plasma in equilibrium with the circulating RBP-TTR complex. Using values for the total plasma concentration of RBP of 45 p.g/ml (2.14 pA/) and of TTR of 250 xg/ml (4.56 jiJl/) and an estimate of 10 for the association constant for the protein-protein complex, it can be calculated that the free RBP concentration is approximately 0.082 pAf or 3.8% of the total plasma RBP. If, as discussed above, the affinity of apo-RBP for TTR is less than that of holo-RBP, free RBP will comprise somewhat more than 3.8% of total plasma RBP and will be relatively enriched in apo-RBP as compared to the total RBP present in plasma. 

For cynomolgus monkeys (2.8-4.0 kg body weight), the biological half-times observed for RBP and TTR (using RBP with normal affinity for TTR) were 6.6 h and 22-23 h, respectively. Free RBP, without affinity for TTR, showed a biological half-time of only 1.9 h. 

These data indicate that the turnover of RBP in vivo is quite rapid, with a fairly high body synthetic (production) rate for a protein of such low plasma concentration. This rapid turnover rate underlies the potential usefulness of RBP measurements in helping to assess the functional state of the liver in patients with hepatic disease, or the nutritional status of patients with borderline or actual malnutrition (see discussion below under clinical studies). It is of interest to compare the turnover of RBP with that of other plasma proteins. It has been pointed out (F. R. Smith et al., 1975) that the respective half-lives (in days) and synthetic rates (in milligrams per kilogram per day) in normal adult human subjects are approximately 0.5 and 5 for RBP 2-3 and 8-9 for TTR (Vahlquist et al., 1973 Socolow et al., 1965) and 14 and 200 for albumin (Beeken et al., 1962). 

A number of studies have examined RBP levels (and usually TTR and vitamin A levels as well) in patients with various kinds of acute and chronic diseases of the liver (Smith and Goodman, 1971 Kindler, 1972 Prellwitz et al., 1974 Skredeeta/., 1975 Brissot er a/., 1978 Vahlquist a/., 1978a Russell et a/., 1978 McClain et al., 1979). In patients with clinically significant hepatic parenchymal disease, the plasma levels of vitamin A, RBP, and TTR have usually been found to be substantially depressed. The low levels of RBP and TTR presumably reflect a reduced rate of production of the proteins by the diseased liver. 

In 63 patients with liver disease, the levels of vitamin A, RBP, and TTR were all markedly decreased and were highly significantly correlated with each other over a wide range of concentrations (Smith and Goodman, 1971). Nineteen patients with acute hepatitis were studied with serial samples as the disease improved, the plasma levels of vitamin A, RBP, and TTR all increased. In these patients, the RBP concentrations correlated negatively with the values of standard tests of liver function (plasma bilirubin, glutamic-oxaloacetic transaminase, and alkaline phosphatase). It has been reported that the level of RBP is of value clinically in assessing the course of acute infectious hepatitis and, to a limited extent, in the differentiation of various forms of jaundice (Kindler, 1972). It seems clear that measurements of plasma RBP levels in patients with liver disease could, if available, be used as an index of hepatic parenchymal functional status and hence could serve as a useful clinical liver function test. 

Several studies have explored relationships between vitamin A transport and visual dark adaptation in patients with chronic liver disease. In one study (Vahl-quist et al., 1978a), patients with liver disease and low plasma RBP levels (below 20 xg/ml) were found to have impaired dark adaptation, suggesting that these patients had peripheral vitamin A deficiency symptoms secondary to their inability to mobilize vitamin A from the liver. In these patients, vitamin A therapy did not affect either the reduced dark adaptation ability or the low plasma RBP levels. In another study (McClain et al., 1979) many patients with alcoholic cirrhosis, with significantly depressed serum levels of zinc, vitamin A, RBP, and TTR, manifested impaired dark adaptation. Some of these patients did not correct their abnormal dark adaptation with vitamin A supplementation but did with zinc therapy. In a third study (Russell et al., 1978) vitamin A therapy of patients... 

In a study of 26 patients with chronic renal disease of varying etiologies (Smith and Goodman, 1971), plasma RBP and vitamin A levels were found to be markedly elevated [116 9 (SEM) p.g/ml and 98 9 pg/dl, respectively], while TTR levels remained normal. Both the molar ratio of RBPzTTR (1.06 0.10), and that of RBP vitamin A (1.92 0.22) were markedly elevated as compared to normals (or to patients with liver disease). In many patients, RBP was present in molar excess as compared with TTR. The presence of a relatively large proportion of free RBP, not complexed to TTR, in some patients was confirmed by gel filtration. On average, circulating RBP was less saturated with retinol than normal. 

The effects of protein-energy malnutrition (PEM), and its treatment, on the plasma retinol transport system have been investigated in a large number of studies during the past decade. Patients with PEM have decreased plasma concentrations of RBP, TTR, and vitamin A. Two major factors can contribute to these low plasma concentrations. First, patients with PEM manifest a defective hepatic production of RBP because of a lack of substrate (calories, amino acids from dietary protein) needed for RBP synthesis. Thus, PEM per se is associated with impaired production of RBP and TTR and defective vitamin A mobilization from the liver. Second, however, PEM is often accompanied by inadequate... 

Levels of RBP, TTR, and vitamin A were measured in 14 patients with hyperthyroidism and in 7 with hypothyroidism (Smith and Goodman, 1971). Both RBP and TTR levels were significantly lower than normal in hyperthyroid patients. In hypothyroid patients, plasma vitamin A was increased significantly RBP levels were somewhat higher than normal, but the difference was not statistically significant. In both hyper- and hypothyroidism, approximately normal molar ratios of RBP TTR and of RBP vitamin A were observed. The significance of these observations in thyroid disorders is not clear. 

CRBP is distinct from RBP, both immunologically (Ross and Goodman, 1979 Bashor and Chytil, 1975) and by a number of other criteria. It has a lower molecular weight and fails to complex with TTR (Ross and Goodman, 1979 Ong and Chytil, 1978a). CRBP will not bind retinaldehyde, retinoic acid, and retinyl acetate, although these ligands will bind to RBP (Horwitz and Heller, 1974). CRBP has spectroscopic properties that differ from those of RBP. Like free retinol, holo-RBP has a smooth absorption band centered at 330 nm. In CRBP, however, the retinol absorption band is shifted bathochromically to 350... 

RBP and retinol exist in the circulation bound as a ternary complex to TTR. TTR consists of a tetramer made up of four identical subunits, with a total molecular weight of 55 kDa. Binding of RBP-ROH to TTR prevents renal filtration of the smaller RBP (MW 21 kDa) and retinol [61]. It was originally proposed, based on the observation that plasma RBP levels drop in vitamin A-deficiency whereas those of TTR do not, that RBP must complex with TTR in the circulation after both proteins are independently secreted from the hepatocyte [63]. However, recent studies by several independent research groups have suggested that the RBP-TTR complex is formed within the ER of hepatocytes prior to secretion [55, 56, 64-68]. 

Both RBP and TTR have a relatively short half-life ( 0.5 and 2-3 days, respectively) and, therefore, they must be synthesized continuously to maintain normal plasma levels. Plasma retinol, RBP, and TTR are reduced in states of impaired protein synthesis, which may be due to an inadequate intake of protein or energy or to impairments in metabolism. Plasma RBP and TTR are sometimes used as clinical indicators of visceral protein synthesis. During infection and/or inflammation, plasma retinol, RBP, and TTR fall transiently, even though liver vitamin A reserves are adequate, due to altered protein synthesis during the acute-phase response. Because multiple nutritional and metabolic disturbances can lead to a similar decrease in plasma retinol, RBP, and TTR, laboratory values must be interpreted with caution. 

When vitamin A stores are adequate, the liver secretes retinol bound to retinol-binding protein (RBP) into the circulation to provide tissues with a constant supply of vitamin A. In the circulation, the retinol-RBP complex is found bound to another circulating protein of hepatic origin, transthyretin (TTR). TTR also binds thyroid hormone and consequently plays a role in the transport of both vitamin A and thyroid hormone. The molecular size of the retinol-RBP complex is quite small, and the formation of the... 

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]. 

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). 

RBP can be expressed as a fusion protein with a variety of tags added on to Its termini. We have expressed RBP as a streptag-fusion protein (5). This has two advantages. First, commercially available streptavidin-affinity resins can be used to purify the protein (instead of TTR-resm). Second, the apo-or inactive... 

---