Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP-transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP-TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [3H]retinol-RBP or the [3H]retinol-RBP-TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [3H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP-TTR complex was nearly twofold greater than that from RBP alone. The uptake of [3H]retinol from protein-bound retinol was inhibited by an excess of either retinol-RBP or retinol-RBP-TTR complex. Moreover, retinol uptake through the RBP-TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP-TTR complex into both PC and NPC. The uptake of [3H]retinol (2 microM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

Retinol bound to cellular retinol-binding protein is a substrate for cytosolic retinoic acid synthesis

Retinol bound to cellular retinol-binding protein (CRBP) was found to be oxidized to retinoic acid by a soluble activity from calf liver. Cytosolic retinoic acid synthesis from retinol-CRBP was strictly dependent on the exogenous supply of either NAD or NADP. NAD-supported reactions carried out in the presence or in the absence of dimethyl sulfoxide yielded apparent Km and Vmax values for the retinol-CRBP complex of 3.5 +/- 0.6 microM, 611 +/- 49 pmol h-1 (mg of protein)-1, and 0.84 +/- 0.12 microM, 601 +/- 38 pmol h-1 (mg of protein)-1, respectively. The corresponding values for the oxidation of free retinol, dissolved in dimethyl sulfoxide, were 7.1 +/- 0.3 microM and 948 +/- 47 pmol h-1 (mg of protein)-1. Since the dissociation constant of the bovine retinol-CRBP complex is less than 10(-8) M, whereas the Km for retinol-CRBP is of the same order as the Km for free retinol, synthesis of retinoic acid from retinol-CRBP does not rely on prior dissociation of retinol. ApoCRBP proved to be a specific inhibitor of retinoic acid synthesis from CRBP-bound retinol. Its inhibitory effect was indistinguishable from the dilution of the radioactive retinol-CRBP substrate that was obtained by the addition of unlabeled holoCRBP. In contrast, the oxidation of CRBP-bound retinol was not inhibited by the addition of other retinoid binding proteins nor by the addition of either free retinol or retinol complexed with proteins distinct from CRBP. These results indicate that the protein moiety of holoCRBP is specifically recognized by the cytosolic enzyme system that catalyzes retinoic acid synthesis from CRBP-bound retinol.     

19F-NMR studies of retinol transfer between cellular retinol binding proteins and phospholipid vesicles

The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing. 19F-NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine-labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP. Differences in how readily protein-bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism.     

NMR studies of fluororetinol analogs complexed to two homologous rat cellular retinol-binding proteins

Comparative 19F-NMR studies of fluororetinol analogs with rat cellular retinol binding protein II (CRBP II) and rat cellular retinol-binding protein (CRBP) were performed to probe differences in the binding interactions of these two homologous proteins. Line shape analyses of 19F-NMR spectra of (E,E,Z,E)-6-fluoro-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetren-1-ol (ligand 1), (E,E,Z,E)-6-fluoro-9-(2,2' dimethyl-6-methylcyclohexenyl)-3,7- dimethyl-2,4,6,8-nonatetren-1-ol (ligand 2), (E,Z,E,E)-5-fluoro-9-(2,2'- dimethyl-6-methylcyclohexenyl)-3,7-dimethyl-2,4,6,8-nonatetren+ ++-1-ol (ligand 3), when complexed with CRBP II at temperatures ranging from 0-45 degrees C, revealed that the 19F resonances corresponding to the bound ligand were in slow chemical exchange between two resonance frequencies. This was further supported by a 2D-NOESY exchange experiment. The kex at 25 degrees C was estimated from spectral simulation and fitting analyses to be 887 s-1, 1010 s-1 and 771 s-1 for CRBP II complexed 1, 2, and 3, respectively. In contrast, only a single absorption was observed for bound ligands complexed with rat CRBP over this temperature range, suggesting that the conformational dynamics of retinol binding are different for these two closely homologous proteins.     

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor

BACKGROUND: The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. METHODS: We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. RESULTS: Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. CONCLUSIONS: All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.     

Streptozotocin-induced diabetes lowers retinol-binding protein and transthyretin concentrations in rats

Retinol-binding protein (RBP) and transthyretin (TTR) in the plasma, liver and kidney, retinol in plasma, and total vitamin A in the liver were measured in rats 6 weeks after diabetes mellitus had been induced by streptozotocin (STZ). The diabetic rats gained 83% less weight despite consuming 45% more feed than the non-diabetic controls. Plasma and kidney concentrations of RBP and TTR were significantly lower in diabetic than in the non-diabetic control rats. Unlike the retinol carrier proteins, plasma albumin concentrations remained unaffected. Plasma concentrations of retinol were decreased while its hepatic levels increased in the diabetic animals. The depressed circulatory levels of retinol may reflect an altered metabolism of its transport proteins.     

Pig epidermal growth factor precursor contains segments that are highly conserved among species

The aim of the present work was to study the binding of [125I]-BLGA (beta-lactoglobulin variant A) to the plasma membrane fraction of hybrid cells. This binding increased as a function of time with on-rate and off-rate constant at 4.47 +/- 0.18 x 10(6) M-1 min-1 and 0.17 +/- 0.07 min-1, respectively (n = 3). The saturation study showed a single binding site type corresponding to a Kd at 8.26 +/- 2.98 nM and 14.02 +/- 2.61 x 10(12) sites per mg of the plasma membrane protein (n = 3). Competitive of binding BLGA was observed with BLGA, complexed with retinol and also with RBP (retinol-binding protein). Gel filtration of [125I]-BLGA incubated with Triton X-100 solubilized membrane showed the formation of a ligand-receptor complex. Cross-linking of the tracer to plasma membrane showed a complex with a M(r) at 69 kDa, suggesting a receptor M(r) of 51 kDa, as seen by autoradiography of SDS-PAGE.     

Comparative studies of human and chicken retinol-binding proteins and prealbumins

Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.     

Retinol transport into the cell nucleus in vitro

The retinol interaction with intact cellular nuclei, nuclear envelope and chromatin was investigated. We have shown that the Cellular Retinol-Binding Protein (CRBP) plays a very important role in such interaction. Retinol can specifically interacts with nuclei, nuclear envelope and chromatin only when it presents as a complex with CRBP. The obtained data allowed us to suggest that the process of retinol delivery to hypothetical nuclear receptors include of at least the following stages: 1) specific binding with nuclear envelope; 2) penetration into nuclei; 3) specific binding with chromatin receptors. CRBP is a necessary component at all this stages. Also we show that CRBP has not a species or tissue specificity.     

beta-carotene 15,15'-dioxygenase activity and cellular retinol-binding protein type II level are enhanced by dietary unsaturated triacylglycerols in rat intestines

The purpose of this study was to examine effects of dietary triacylglycerols on beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) activity and cellular retinol-binding protein [CRBP (II)] in rats. Six groups of eight rats (7-wk old) were fed one of the following diets: standard (STD; 2.5% soybean oil), saturated (SFA; 15% hydrogenated soybean oil), monounsaturated (MUFA; 15% olive oil), polyunsaturated (PUFA; 15% soybean oil) or clofibrate (CLF; 2.5% soybean oil + 0.2% clofibrate) for 3 wk. The dioxygenase specific activities of the intestinal homogenates in the MUFA and PUFA groups fed the high fat diets were 2.4 times that of the STD group fed a low fat diet (P < 0.01), whereas the activities of the SFA and CLF groups were not significantly different from that of the STD group. The level of CRBP (II) in the intestine of the PUFA group was 1. 3-fold that of the STD group (P < 0.05), whereas there were no significant differences among the other groups. In a second experiment, the dioxygenase activity of rat intestine was followed over 3 wk of feeding the STD and PUFA diets. After the PUFA diet was consumed for 1 d, the activity was enhanced to 2.7 times the baseline level and remained thereafter at that high level, whereas the activity of the STD group remained at the low baseline level. Thus, dietary polyunsaturated triacylglycerols enhanced both beta-carotene 15,15'-dioxygenase activity and CRBP (II) level in rat intestine. These results suggest that the dioxygenase and CRBP (II) are regulated by the same mechanism involving long-chain fatty acids and their metabolites.     

The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization

The interaction between retinol-binding proteins and prealbumins of human and chicken was studied by fluorescence polarization techniques. The binding affinity between chicken plasma retinol-binding protein and chicken prealbumin was essentially the same as between the respective human proteins. Human urine retinol-binding protein displayed a similar affinity, though possibly slightly smaller than that of the human plasma protein, toward human prealbumin. Retinol-binding proteins and prealbumins of human and chicken have been found to cross-interact displaying an affinity similar to that displayed by the proteins of the same species. Solution of a binding equation which assumes identical, independent sites, indicated that the number of binding sites on prealbumin for retinol-binding protein is somewhat less than 2 with the human system, and in the neighborhood of 4 with the chicken system. A possible interpretation suggests that prealbumin possesses four identical binding sites for retinol-binding protein, one for each subunit, but that the binding is of a negative cooperative nature. A major share of the negative cooperativity is likely to result from steric hindrance induced by already bound retinol-binding protein molecules, which have a sizable volume compared to the volume of the prealbumin molecule. The cooperativity is likely to be more pronounced with the human system. Rotational relaxation times derived from Perrin plots suggest that 1:1 molecular complexes of retinol-binding proteins with prealbumins have a compact structure.     

Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.     

Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation

The expression of cellular retinoic-acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP), as well as their relationship to retinoic acid (RA) synthesis and degradation were examined in the developing mouse cerebellum and choroid plexus of the fourth ventricle. The choroid plexus, which expresses the RA-synthesizing retinaldehyde dehydrogenase RALDH-2, is likely to represent a diffusion source of RA for the closely apposed cerebellum, regulating its development. We found CRBP to be expressed in the choroid plexus and, in an in-vitro assay, addition of recombinant CRBP to RALDH-2 increased RA synthesis from retinaldehyde, with the amount of increase depending on the CRBP/retinaldehyde ratio. A technique that characterizes RA-binding proteins according to their isoelectric point showed both CRABP I and CRABP II to be present in the cerebellum and P19 cells, and only CRABP II to be present in the choroid plexus. With this technique, CRABP I could also be detected in the HL60 cell line. In addition to the two known acidic RA-binding proteins CRABP I and II, the cerebellum expressed a third RA-binding protein distinguishable by its neutral isoelectric point; the same binding protein was also detected in the olfactory bulb, kidney and testes. We used the RA-binding technique to follow the rate of elimination of bound RA from the cerebellum. A systemic injection of 0.3 micromols RA into postnatal day-1 mice was almost completely removed after 8 hours. These results suggest mechanisms by which the retinoid-binding protein may regulate the equilibrium of RA synthesis and catabolism in the cerebellum and choroid plexus.     

Genetically probing the regions of ribose-binding protein involved in permease interaction

The ribose-binding protein (RBP) of Escherichia coli, located in the periplasm, binds to ribose and mediates transport and chemotaxis. The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz. Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene. The expression of ompC can be increased by an interaction of ribose-bound RBP with Trz. A transport defect, either in the binding protein or in the membrane permease, causes a signalling-constitutive Lac+ phenotype of Trz even in the absence of ribose. This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high-affinity transport system. By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction. Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar-binding properties were not substantially affected. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor.     

Physical determinants of intermembrane protein transfer

Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.     

Dilated fallopian tubes: MR imaging characteristics

A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.     

The use of benevolent power in nursing care

In response to estrogen the rat cervical epithelium undergoes squamous metaplastic changes, progressing from a resting state through a proliferating, secretory stage and finally to a cornified stage before sloughing or being reabsorbed. The transition from a secretory to a cornified epithelium is preceded by a dramatic reduction in the expression of the cellular retinol binding protein (CRBP). The associations among retinoids (retinol and retinoic acid), CRBP expression, and estrogen-induced keratinocyte differentiation were explored in cultured cervical epithelial cells. Retinoids supported proliferation of cervical epithelial cells expressing basal keratins. Alone, estrogen had no effect on proliferation and enhanced expression of keratins characteristic of stratified cervical epithelial cells. When added together, estrogen prevented retinoid effects on proliferation, whereas retinoids prevented the estrogen-enhanced expression of differentiation-associated cytokeratins. When CRBP expression was repressed by elevating intracellular cyclic AMP levels, the ability of retinol, but not retinoic acid, to block estrogen-induced changes in keratin expression was severely compromised. These results support a critical role for CRBP in cervical cell responsiveness to circulating retinoids (primarily retinol). We hypothesize that retinol inhibits estrogen-induced keratinization of the cervical epithelium, and the drop in CRBP level results in transient vitamin A deficiency within cervical epithelial cells, permitting the orderly transition from the secretory to the cornified stage.     

Monitoring adenoviral p53 transduction efficiency by yeast functional assay

The pathogenesis of ossification of the posterior longitudinal ligament (OPLL) remains to be elucidated, though etiologic factors for OPLL have been identified. High levels of serum retinol and retinol binding protein (RBP) have been observed in patients with diffuse idiopathic skeletal hyperostosis (DISH). OPLL is often associated with DISH. In this study, the levels of serum retinol and RBP were determined in 70 patients with OPLL in the cervical spine, and compared with those in normal subjects. Bone metabolic markers of serum intact osteocalcin, urinary pyridinoline and deoxypyridinoline were examined as well. Among female patients, level of serum RBP was significantly higher in those in their 60's, and those with mixed type OPLL. Level of serum RBP was significantly higher in both sexes, and retinol was exhibited higher in female patients, if they were associated with DISH. Patients with OPLL exhibited no abnormal bone metabolic marker levels. These findings suggest that vitamin A may play a role in the development of OPLL.