Immunohistochemical localization of bovine placental retinol-binding protein.

An immunogold staining method was used in combination with epipolarization microscopic detection to demonstrate the presence of bovine placental retinol-binding protein in bovine extraembryonic membranes. Amnion, chorion and allantois were fixed in Bouin fixation fluid and embedded in polyethylene glycol 1500. Sections (5 mm) were cut and transferred onto Digene silanated slides and immunostained using rabbit antiserum raised against bovine placental retinol-binding protein followed by goat anti-rabbit IgG labeled with 1 nm gold. Gold particles after silver enhancement were viewed and photographed under epipolarization microscopy. Epithelial cells of all three membranes (i.e. amniotic ectoderm, chorionic trophectoderm, and allantoic endoderm) were immunoreactive, while mesodermal cells, collagen, and blood cells were not. These data, together with our previous observation that these three placental membranes synthesize and secrete retinol-binding protein, indicate that epithelial cells lining the amnion, chorion and allantois are the major sources of this protein. The presence of retinol-binding protein in placental membranes and their fluids may be indicative of an important role for retinol in placental differentiation and development.     

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein

As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

The mechanism of uptake of retinol by plasma-membrane vesicles.

The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Retinol-binding protein is synthesized in the mammalian eye

As the chromophoric component of the visual pigment, retinol plays an essential role in vision. In the plasma, retinol is transported by retinol-binding protein (RBP) in complex with transthyretin (TTR, prealbumin). In previous work we demonstrated intraocular synthesis of TTR. To determine whether RBP is also synthesized in the eye, we performed Northern and Western blot analysis of rat eye, and detected both RBP mRNA and immunoreactive RBP. Regional Northern analysis of bovine eye localized RBP mRNA to ciliary body/iris and retina/RPE. Preliminary immunohistochemical studies revealed a widespread but heterogeneous distribution of RBP in rat eye. We postulate that ocular RBP and TTR are involved in the intraocular translocation of retinol.     

Differentiation-dependent expression of retinoid-binding proteins in BFC-1 beta adipocytes.

Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.     

Characterization and immunolocalization of bovine uterine retinol-binding protein.

Endometrial explants obtained from cows between Days 13 and 29 of pregnancy were cultured for 24 h in modified minimum essential medium in the presence of [35S]methionine or [3H]leucine. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Uterine luminal flushings were obtained from cyclic cows (Days 2-20 of estrous cycle) and early pregnant cows (Days 17-22). Endometrial tissues from cows on Days 17 and 29 of pregnancy were prepared for immunocytochemistry. A uterine secretory protein, which consisted of five isoelectric variants (pI 5.3-6.1) of identical molecular mass (23,000 Da), was shown to react immunologically with antiserum raised against bovine placental retinol-binding protein (bpRBP). Limited N-terminal sequence analysis of two major isoforms showed that the protein had nearly complete homology with bovine placental and plasma retinol-binding protein (RBP) over the first 25 amino acids. Through use of bpRBP antiserum, immunoreactive RBP was detected in uterine flushings collected from cows in the late luteal phase of the estrous cycle and early pregnancy by Western blotting, and in medium conditioned by uterine explants prepared at Days 13-29 of pregnancy by immunoprecipitation. Immunoreactive RBP was localized in endometrial surface and glandular epithelium on Days 17 and 29 of pregnancy by immunocytochemistry. These results demonstrate that RBP is a product of bovine uterine tissues. The uterine RBP may play an important role in vitamin A transport between maternal tissues and developing embryos.     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR—RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR—RBP concentrations determined by this method are highly correlated to holo-TTR—RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR—RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR—RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.     

Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein

RBP4 (plasma retinol-binding protein) is the 21?kDa transporter of all-trans retinol that circulates in plasma as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesized in the liver but is also produced by adipose tissue and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration.This paper reports the high resolution three-dimensional structures of human RBP4 naturally lacking bound retinol purified from plasma, urine and amniotic fluid. In all these crystals we found a fatty acid molecule bound in the hydrophobic ligand-binding site, a result confirmed by mass spectrometry measurements.In addition we also report the 1.5?Å resolution structures of human holo-RBP4 and of the protein saturated with palmitic and lauric acid and discuss the interaction of the fatty acids and retinol with the protein.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.     

Studies on the Cellular Uptake of Retinol Binding Protein and Retinol

The uptake and release of (125)I-RBP and of holoRBP labeled with [(3)H]retinol ((3)H-ROH) were studied in two cell lines which synthesize and secrete RBP, the HepG2 hepatocarcinoma cell line and the Caki-1 kidney adenocarcinoma cell line, and in HeLa cells that do not express the endogenous RBP gene. In all three cell lines a part of endocytosed (125)I-RBP is recycled to the extracellular medium and part is degraded. Nonspecific endocytosis of (125)I-RBP was estimated to be approximately 10% of total endocytosed (125)I-RBP. In HepG2 cells the (3)H-ROH from the [(3)H]retinol-RBP complex ((3)H-ROH-RBP) is recycled bound to RBP into serum-free chase medium. This (3)H-ROH recycling is blocked in HepG2 cells by cyclohexymide and by brefeldin A, an inhibitor of protein export from the main secretory route, and is absent in HeLa cells, which do not synthesize RBP. These data suggest that at least part of retinol taken up from exogenous holoRBP is delivered to newly synthesized RBP. (3)H-ROH recycled by HeLa cells is bound to serum albumin, as is a portion of that recycled by HepG2 cells. Transfer of (3)H-ROH from RBP to serum albumin does not occur in the absence of cells. We conclude that RBP is endocytosed through a specific pathway and that the RBP-associated retinol is transferred to newly synthesized RBP or to serum albumin.     

Isolation and characterization of a 94,000-dalton protein with thyrotropic activity from early bovine placenta

A thyrotropic protein was extracted and purified from the placenta of early bovine gestations. After protein extraction, the 45-60% ammonium sulfate precipitate of maternal and fetal bovine cotyledons was found to compete with thyroid stimulating hormone (TSH) for binding to thyroid cell membranes and to mediate TSH specific biological effects including the stimulation of cyclic AMP production, iodide uptake, and thyroxine secretion. The placental thyrotropin was further purified by gel and anion exchange chromatography, followed by binding to thyroid cell membranes and elution by mild acid treatment. 400 micrograms of isolated protein with 4.5 units of TSH-like binding activity/mg of protein was recovered from the placenta of a 90-day-old bovine gestation, representing 2 X 10(-4%) of its original wet weight. The placental thyrotropin appeared to be a 94,000-dalton protein with pI 6.0 and composed of two noncovalently associated chains of 50,000 and 44,000 daltons. The placental 94,000-dalton thyrotropin bound to TSH membrane receptors and induced specific TSH-mediated biological effects, but was structurally and immunologically distinct from TSH and hypophysical or placental gonadotropins.     

视黄醇结合蛋白的分离纯化

在变性条件下,应用Sephacryl-100凝胶过滤和Source-30Q阴离子交换两步分离,实现了分离纯化性质不稳定、易于降解的视黄醇结合蛋白(RBP)之目的。最后经过分步缓慢复性,获得具有生物活性的RBP,为其单克隆抗体制备及最终应用于临床营养评价和相关疾病的诊断创造了条件。     

Retinoids and retinoid-binding protein expression in rat adipocytes.

Adipose tissue has been reported to contain relatively high levels of the specific mRNA for retinol-binding protein (RBP) (Makover A., Soprano, D.R., Wyatt, M. L., and Goodman, D.S. (1989) J. Lipid Res. 30, 171-180). Studies were conducted to explore retinoid and retinoid-binding protein storage and metabolism in adipose tissue. In these studies, we measured RBP and cellular retinol-binding protein (CRBP) mRNA levels and retinoid levels in 6 adipose depots in male rats. Total RNA was isolated from inguinal, dorsal, mesenteric, epididymal, perinephric, and brown adipose tissue, and average RBP and CRBP mRNA levels were determined by Northern blot analysis. The relative levels of RBP mRNA in these 6 anatomically different adipose depots averaged, respectively, 6.3, 6.7, 16, 34, 37, and 21% of the level in a rat liver RNA standard. Retinoid levels in the 6 depots were similar and averaged approximately 6-7 micrograms of retinol eq/g of adipose tissue. Since adipose tissue contains several cell types, the cellular localizations of RBP and CRBP expression and retinoid storage were examined. RNA was prepared from isolated rat adipocytes and stromal-vascular cells. Cellular levels of the mRNAs for RBP, CRBP, apolipoprotein E (apoE), lipoprotein lipase, adipocyte P2, and adipsin were measured by Northern blot analysis. RBP was expressed almost exclusively in the adipocytes and only weakly in the stromal-vascular cells. Both CRBP and apoE mRNA levels were relatively high in the stromal-vascular cell preparations and only very low mRNA levels were found in the adipocytes. Lipoprotein lipase, adipsin, and adipocyte P2 mRNAs were found in substantial levels in both the adipocytes and stromal-vascular cells, but with higher levels present in the adipocytes. Cultured adipocytes synthesized RBP protein and secreted it into the medium. Only adipocytes (not stromal-vascular cells) contained retinol, at levels between 0.65-0.8 micrograms of retinol eq/10(6) cells. These studies demonstrate that adipocytes store retinoid and synthesize and secrete RBP, and suggest that rat adipocytes may be dynamically involved in retinoid storage and metabolism.     

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.