Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. Acetylation with N-acetylimidazole and alkaline titration.

The behavior of holo-retinol-binding protein (RBP) from human plasma at alkaline pH was examined by absorption and circular dichroism measurements. Between pH 7.5 and 11.7 the ionization of the phenolic hydroxyl groups is reversible. However, there is a gradual irreversible loss of retinol as the pH is raised. After 4 hours at pH 11.7, 13 percent of retinol is lost from retinol-RBP. Alkaline titration of apo-RBP was time-independent and reversible between pH 7.5 and 11.7. The titration data of the phenolic hydroxyl groups in apo-RBP could be fitted with a single theoretical ionization curve of 8.6 phenolic groups having an apparent pK of 11. Acetylation of retinol-RBP with 10-fold molar excess of N-acetylimidazole over tyrosine resulted in the acetylation of all lysine residues and in the acetylation of 0.9 to 1.3 tyrosyl residues per molecule (out of eight). Acetylation of retinol-RBP, APO-RBP, and retinol-RBP-prealbumin complex with 50-fold molar excess of N-acetylimidazole resulted, again, with all of the lysine residues being acetylated and between 1.8 and 2.8 tyrosyl residues per molecule being acetylated. The acetylation did not affect the interaction between retinol and RBP. However, acetylation disrupted the normal binding between retinol-RBP and prealbumin. Deacetylation of tyrosyl residues with hydroxylamine failed to restore the normal binding of retinol-RBP to prealbumin. This excludes the acetylated tyrosyl-residues from being involved in the binding between the two proteins.     

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.     

Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type ii (CRBP(II] are cytoplasmic proteins that bind trans-retinol as an endogenous ligand. These proteins are structurally similar having greater than 50% sequence homology. Employing fluorescence, absorbance, and competition studies, the ability of pure preparations of CRBP(II) and CRBP to bind various members of the vitamin A family has been examined. In addition to trans-retinol, CRBP(II) was able to form high affinity complexes (K'd less than 5 X 10(-8) M) with 13-cis-retinol, 3-dehydroretinol, and all-trans-retinaldehyde. CRBP bound those retinol isomers with similar affinities, but did not bind trans-retinaldehyde. Neither protein bound retinoic acid nor 9-cis- and 11-cis-retinol. The spectra of 13-cis-retinol and 3-dehydroretinol, when bound, were shifted and displayed fine structure compared to their spectra in organic solution. However, the lambda max and fluorescent yield of a particular ligand were different when bound to CRBP(II) versus CRBP. It appears that CRBP(II) and CRBP bind trans-retinol, 13-cis-retinol, and 3-dehydroretinol in a planar configuration. However, the binding sites of CRBP(II) and CRBP are clearly distinct based on the observed spectral differences of the bound ligands and the observations that only CRBP(II) could bind trans-retinaldehyde. The ability of CRBP(II) to bind trans-retinaldehyde suggests a physiological role for the protein in accepting retinaldehyde generated from the cleavage of beta-carotene in the absorptive cell.     

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。     

Study on the correlation between urinary retinol-binding protein and nonalcoholic fatty liver disease

BackgroundNonalcoholic fatty liver disease (NAFLD) affects human health worldwide. Our objective was to explore the correlation between urinary retinol-binding protein (URBP) and NAFLD.MethodsThis cross-sectional study included 445 NAFLD patients and 911 healthy controls. The URBP level and other parameters were measured.ResultsThe URBP level (expressed by the RBP/creatinine ratio) was higher in the NAFLD patients compared with the non-NAFLD patients. The urinary RBP/creatinine ratio was an independent risk factor for NAFLD after univariate and multivariate regression analysis, with the or values of 2.271 (1.795-2.872, P < 0.001) and 2.338 (1.775-3.080, P < 0.001), respectively. The prevalence of the urinary RBP/creatinine ratio (groups 1, 2, 3, 4) was 20.0%, 17.3%, 27.3%, and 35.4%, respectively (P < 0.001), and the prevalence of NAFLD in the high urinary RBP/creatinine ratio group was significantly higher than that in the low urinary RBP/creatinine ratio group.ConclusionsOur results revealed that the urinary RBP/creatinine ratio was an independent risk factor for NAFLD.     

Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II

Retinol bound to cellular retinol-binding protein (CRBP) was available for esterification by liver microsomes in the absence of exogenous acyl donors. Moreover, exogenous acyl-CoA gave little or no stimulation of ester production over what was observed with the endogenous acyl donor. In contrast, unbound retinol was esterified in an acyl-CoA-dependent reaction. The presence of two different enzyme activities, acyl-CoA-dependent and -independent, was demonstrated by differential sensitivities to several enzyme inhibitors. The enzyme reaction with retinol-CRBP and endogenous acyl donor produced retinyl esters normally found in vivo in liver. In addition, rates of esterification with this system were sufficient to maintain liver stores. Liver also contains cellular retinol-binding protein, type II (CRBP(II] during the perinatal period. Radioimmunoassay revealed highest levels of CRBP(II) in liver 3-4 days after birth. Examination of retinol esterification by microsomes from the liver of 3-day-old rats revealed a retinyl ester synthase activity with lower Km and higher Vmax than that found in the adult. The activity could use either retinol-CRBP or retinol-CRBP(II) and an endogenous acyl donor. The microsomes from 3-day-old liver had greater esterifying ability than microsomes from adult liver, perhaps due to the presence of two retinyl ester synthase enzymes.     

Photo-affinity label for cellular retinol-binding protein

A number of retinoid derivatives have been synthesized for use as labels for cellular retinol-binding protein. Introduction of substituents abolished the binding of the derivatives to the protein, except in the case of the photo-reactive derivative, 4-azidoretinol. This compound was found to compete successfully with all-trans-retinol for binding to cellular retinol-binding protein, with a high relative binding affinity. Irradiation of a complex of 4-azidoretinol and a semi-purified preparation of cellular retinol-binding protein from liver resulted in a firm attachment stable to SDS-gel electrophoresis. It is therefore suggested that the irradiated product is held together covalently. A method for the synthesis of 4-azidoretinol is described.     

The three-dimensional structure of retinol-binding protein

The complex of retinol with its carrier protein, retinol-binding protein (RBP) has been crystallized and its three-dimensional structure determined using X-ray crystallography. Its most striking feature is an eight-stranded up-and-down beta barrel core that completely encapsulates the retinol molecule. The retinol molecule lies along the axis of the barrel with the beta-ionone ring innermost and the tip of the isoprene tail close to the surface.     

视黄醇结合蛋白的分子生物学

视黄醇结合蛋白 (RBP)是维生素A(VitA)的运载蛋白 ,结构上为疏水小分子结合蛋白家族的成员。视黄醇结合蛋白在肝脏中合成并释放入血液后 ,通过与视黄醇 (ROH)、甲状腺素运载蛋白 (TTR)及细胞表面受体相互作用 ,在VitA的储存、代谢、转运到周围靶器官中发挥重要功能。本文从基因结构与表达、构效关系及临床意义等方面介绍了视黄醇结合蛋白的分子生物学。     

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.     

Homology and structure-function correlations between alpha 1-acid glycoprotein and serum retinol-binding protein and its relatives

Although the serum protein alpha 1-acid glycoprotein (AGP) or orosomucoid has been extensively studied, its relationships with other proteins have been controversial and its precise physiological function has remained unclear. It is shown here that AGP is significantly similar in amino acid sequence and in the locations of introns in its structural gene to members of a protein superfamily that includes serum retinol-binding protein (RBP), beta-lactoglobulin (LG), alpha 2u-globulin, and protein HC (alpha 1-microglobulin). The view that the three-dimensional structure of AGP is closely similar to the published structures of RBP and LG is supported by its homology with these proteins, similarities in disulfide bond arrangements, and its secondary structure profile, predicted from the amino acid sequence. The relationship of AGP with this particular protein family indicates that its well-characterized ability to bind lipophilic drugs and certain steroids is a reflection of its true biological role. It is proposed that AGP and the other members of this extensive group of proteins should be designated lipocalins to reflect a common ability to bind lipophiles by enclosure within their structures in a manner that minimizes solvent contact.     

Molecular properties of bovine interphotoreceptor retinol-binding protein

Interphotoreceptor retinol-binding protein (IRBP) is a large retinol-carrying glycoprotein, located only in the interphotoreceptor (or subretinal) space of vertebrate eyes. It has recently been purified to apparent homogeneity. The present report presents its sedimentation, spectroscopic, and binding properties. The molecular weight of bovine IRBP, determined by sedimentation equilibrium, is 133,000. The sedimentation coefficient is 5.8S. The Stokes radius, 56 A, obtained from gel-filtration chromatography, is much larger than that expected for a globular protein of the same molecular weight. These results indicate that IRBP is asymmetric (it can modeled as a prolate ellipsoid of revolution with axial ratio of about 8:1) and explain the overestimates of molecular weight obtained in previous studies based on size-exclusion methods. The molar absorption coefficients for IRBP (at 280 nm) and for bound retinol are both unaffected by ligand dissociation. Fluorescence of the holoprotein displays neither fine structure nor energy transfer from tryptophan to bound retinol. Circular dichroism suggests a secondary structure containing approximately 15% alpha-helix and approximately 20% beta-structure, unchanged by the presence of ligand. The binding of retinol creates a positive, extrinsic Cotton effect at 330 nm, proportional to the amount of retinol bound. The apparent dissociation constant for all-trans-retinol is 1.3 X 10(-6) M. This relatively loose binding implies that, if required during the visual cycle, IRBP should be able to transfer its ligand to other binding proteins in the neural retina and retinal pigment epithelium.     

Cross talk between signaling and vitamin A transport by the retinol-binding protein receptor STRA6

The plasma membrane protein STRA6 transports vitamin A from its blood carrier retinol binding protein (RBP) into cells, and it also functions as a cytokine receptor which activates JAK/STAT signaling. We show here that, unlike other cytokine receptors, phosphorylation of STRA6 is not simply induced upon binding of its extracellular ligand. Instead, activation of the receptor is triggered by STRA6-mediated translocation of retinol from serum RBP to an intracellular acceptor, the retinol-binding protein CRBP-I. The observations also demonstrate that the movement of retinol from RBP to CRBP-I, and thus activation of STRA6, is critically linked to the intracellular metabolism of the vitamin. Furthermore, the data show that STRA6 phosphorylation is required for retinol uptake to proceed. Hence, the observations demonstrate that STRA6 orchestrates a multicomponent "machinery" that couples vitamin A homeostasis and metabolism to activation of a signaling cascade and that, in turn, STRA6 signaling regulates the cellular uptake of the vitamin. STRA6 appears to be a founding member of a new class of proteins that may be termed "cytokine signaling transporters."     

Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent M_r is 250 000 by gel-filtration chromatography, and 225 000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent M_r is 140 000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10⁻⁶ M, as measured by fluorimetric titration.     

Liver takes up retinol-binding protein from plasma

Retinol is transported in plasma bound to a specific transport protein, retinol-binding protein. We prepared 125I-tyramine cellobiose-labeled rat retinol-binding protein and studied its tissue uptake 1, 5, and 24 h after intravenous injection into rats. The liver was the organ containing most radioactivity at all time points studied. After 5 and 24 h, 30 and 22% of the injected dose were recovered in liver, respectively. After separating the liver into parenchymal and nonparenchymal cells in the 5-h group, we found that both cell fractions contained approximately the same amount of radioactivity (per gram of liver). Most of the retinol-binding protein radioactivity in the nonparenchymal cell fraction was in the stellate cells. The implication of these results for a possible transfer mechanism for retinol between parenchymal and stellate cells is discussed.     

Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent Mr is 250000 by gel-filtration chromatography, and 225000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent Mr is 140000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10(-6) M, as measured by fluorimetric titration.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.