Cellular Retinol-binding Protein Type I Research Articles

Genetic dissection of retinoid esterification and accumulation in the liver and adipose tissue

Approximately 80-90% of all retinoids in the body are stored as retinyl esters (REs) in the liver. Adipose tissue also contributes significantly to RE storage. The present studies, employing genetic and nutritional interventions, explored factors that are responsible for regulating RE accumulation in the liver and adipose tissue and how these influence levels of retinoic acid (RA) and RA-responsive gene expression. Our data establish that acyl-CoA:retinol acyltransferase (ARAT) activity is not involved in RE synthesis in the liver, even when mice are nutritionally stressed by feeding a 25-fold excess retinol diet or upon ablation of cellular retinol-binding protein type I (CRBPI), which is proposed to limit retinol availability to ARATs. Unlike the liver, where lecithin:retinol acyltransferase (LRAT) is responsible for all RE synthesis, this is not true for adipose tissue where Lrat-deficient mice display significantly elevated RE concentrations. However, when CrbpI is also absent, RE levels resemble wild-type levels, suggesting a role for CrbpI in RE accumulation in adipose tissue. Although expression of several RA-responsive genes is elevated in Lrat-deficient liver, employing a sensitive liquid chromatography tandem mass spectrometry protocol and contrary to what has been assumed for many years, we did not detect elevated concentrations of all-trans-RA. The elevated RA-responsive gene expression was associated with elevated hepatic triglyceride levels and decreased expression of Pparδ and its downstream Pdk4 target, suggesting a role for RA in these processes in vivo.

Cellular Retinol-Binding Protein Type I (CRBP-I) Regulates Adipogenesis

Adipogenesis is governed by a well-documented cascade of transcription factors. However, less is known about non-transcription factors that govern early stages of adipogenesis. Here we show that cellular retinol-binding protein type I (CRBP-I), a small cytosolic binding protein for retinol and retinaldehyde, is specifically restricted to preadipocytes in white adipose tissue. The absence of CRBP-I in mice (CRBP-I-KO mice) leads to increased adiposity. Despite increased adiposity, CRBP-I-KO mice remain more glucose tolerant and insulin sensitive during high-fat-diet feeding. 3T3-L1 cells deficient in CRBP-I or mouse embryonic fibroblasts derived from CRBP-I-KO mice had increased adipocyte differentiation and triglyceride (TG) accumulation. This was due to increased expression and activity of PPAR gamma, while other transcription factor pathways in early and late differentiation remained unchanged. Conversely, the overexpression of CRBP-I in 3T3-L1 cells results in decreased TG accumulation. In conclusion, CRBP-I is a cytosolic protein specifically expressed in preadipocytes that regulates adipocyte differentiation in part by affecting PPAR gamma activity.

Linkages between hepatic vitamin A storage and metabolism and hepatic steatosi

Mice lacking lecithin:retinol acyltransferase (LRAT) and cellular retinol‐binding protein, type I (CRBPI) (Lrat−/−/CrbpI−/− mice) fed a chow diet or the AIN‐93M diet accumulate no hepatic retinyl ester stores and have low hepatic retinol levels compared to matched wild type, CrbpI−/− and Lrat−/− mice. Further study showed that CrbpI−/− and Lrat−/−/CrbpI−/− mice spontaneously accumulate significantly more (> 2‐fold) hepatic fat than wild type mice. This suggested a link between low hepatic vitamin A levels and development of fatty liver. Consequently, we assessed gene expression levels by qRT‐PCR for a number of vitamin A responsive genes and a number of genes involved in mediating hepatic fat metabolism. Cyp26A1 and RARβ, which are responsive to retinoic acid, were upregulated in livers of CrbpI−/− and Lrat−/−/CrbpI−/− mice. Genes involved in hepatic fat metabolism, including PPARγ and PPARβ/δ, were also altered. We suggest that the impaired ability of CrbpI−/− and Lrat−/−/CrbpI−/− mice to store vitamin A results in increased retinoic acid synthesis, which elevates retinoic acid responsive gene expression and dysregulates hepatic fat metabolism and accumulation. (Supported by R01 DK079221)

Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase

Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-trans-retinoic acid, however, initial assays suggested that RDH10 prefers NADP(+) as a cofactor, undermining its role as an oxidative enzyme. Here, we present evidence that RDH10 is, in fact, a strictly NAD(+)-dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates. RDH10 has a relatively high apparent K(m) value for NAD(+) (~100 microm) but the lowest apparent K(m) value for all-trans-retinol (~0.035 microm) among all NAD(+)-dependent retinoid oxidoreductases. Due to its high affinity for all-trans-retinol, RDH10 exhibits a greater rate of retinol oxidation in the presence of cellular retinol-binding protein type I (CRBPI) than human microsomal RoDH4, but like RoDH4, RDH10 does not recognize retinol bound to CRBPI as a substrate. Consistent with its preference for NAD(+), RDH10 functions exclusively in the oxidative direction in the cells, increasing the levels of retinaldehyde and retinoic acid. Targeted small interfering RNA-mediated silencing of endogenous RDH10 or RoDH4 expression in human cells results in a significant decrease in retinoic acid production from retinol, identifying both human enzymes as physiologically relevant retinol dehydrogenases. The dual cis/trans substrate specificity suggests a dual physiological role for RDH10: in the biosynthesis of 11-cis-retinaldehyde for vision as well as the biosynthesis of all-trans-retinoic acid for differentiation and development.

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.

Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber's congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits approximately 2000-fold lower K(m) values for NADP(+) and NADPH than for NAD(+) and NADH and recognizes both retinoids and lipid peroxidation products (C(9) aldehydes) as substrates. The k(cat) values of RDH12 for retinaldehydes and C(9) aldehydes are similar, but the K(m) values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (k(cat)/K(m) approximately 900 min(-)(1) microM(-)(1)), followed by 11-cis-retinal (450 min(-)(1) mM(-)(1)) and 9-cis-retinal (100 min(-)(1) mM(-)(1)). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products.

Spatio-temporal distribution of cellular retinol-binding protein gene transcripts (CRBPI and CRBPII) in the developing and adult zebrafish (Danio rerio).

We have cloned and determined the nucleotide sequence of the cDNA coding for a cellular retinol-binding protein type I (CRBPI) from zebrafish. The deduced amino acid sequence of the zebrafish CRBPI showed highest sequence identity ( approximately 59%) to the mammalian CRBPIs of the intracellular lipid-binding protein (iLBP) multigene family. Phylogenetic analysis clustered the zebrafish CRBPI to the CRBPI clade. The zebrafish CRBPI gene (rbp1) and CRBPII gene (rbp2) both consist of four exons separated by three introns, identical to all other iLBP genes in vertebrates. Two transcription start sites were identified in the rbp1 promoter and a single transcription start site was identified for rbp2. Radiation hybrid mapping assigned the zebrafish rbp1 gene to linkage group 16 and conserved syntenic genes were found by comparative analysis of mammalian orthologous rbp1 genes. RT-PCR detected mRNA transcripts in the adult intestine, liver, brain, ovary and testis for rbp1 gene and in the intestine and liver for rbp2 gene. Whole mount in situ hybridization of zebrafish embryos revealed rbp1 mRNA expression in the developing zebrafish central nervous system at specific sites that are known to have abundant retinoic acid distribution and significant retinoic acid action. Whole mount in situ hybridization also showed that the zebrafish rbp2 mRNA was localized specifically in the embryonic intestinal bulb and the developing intestine during the larval stage, implying a novel function for the rbp2 gene product during organogenesis and development of the zebrafish intestine.

Uptake and esterification of vitamin A by RCS rat retinal pigment epithelial cells in primary culture

We investigated the capacity of Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells to take up all- trans-retinol (ROL) (vitamin A) and to metabolize it into retinyl esters (RE). Cultures of RPE cells were established from RCS and control newborn rats. All- trans-ROL was delivered to the apical surface of the RPE monolayer. Retinoids were analyzed by high-performance liquid chromatography. The cellular retinol-binding protein type I (CRBP-I) was assessed by Western blotting. Before supplementation with ROL, RE were lower in RCS rats. After ROL supplementation, esters increased and reached values that were similar in the two strains, but the increase, expressed relative to the initial value, was higher in RCS rats. The uptake of ROL and the level of CRBP-I were greater in RCS rats. Our results provide evidence of a functional retinol esterifying enzyme in cultured RCS RPE cells and suggest that CRBP-I could play a role in the uptake and esterification of ROL in the RPE cells.

1H and 15N assignments of rat holo cellular retinol-binding protein type I (CRBP-I)

1H and 15N assignments of rat holo cellular retinol-binding protein type I (CRBP-I)

1H and 15N assignments of rat holo cellular retinol-binding protein type I (CRBP-I)

1H and 15N assignments of rat holo cellular retinol-binding protein type I (CRBP-I)

Backbone relaxation data of rat holo cellular retinol-binding protein type I (CRBP-I)

Backbone relaxation data of rat holo cellular retinol-binding protein type I (CRBP-I)

Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques.

Recent developments in mass spectrometry have demonstrated the capability of this technique to transfer non-covalent protein complexes, involving low and high molecular weight ligands, from a condensed state to the gas phase. In this work, electrospray mass spectrometry with a quadrupole analyzer (ES-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) with its specific ligand, all-trans retinol (vitamin A), and with fatty acids. Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed determination of the molecular weight of apo-CRBP with good accuracy (<0.01%) and to identify a protein fraction ( approximately 20%) retaining the initial methionine. By adding saturating amounts of vitamin A, ES-MS studies on the protein in the holo-form under native conditions allowed detection of retinol bound within the cavity together with water molecules, as expected from its crystal structure. ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covalent interactions between CRBP and non-specific ligands such as fatty acids, bound to the protein as a result of expression in various strains of E. coli grown in different media. Since ES mass spectra do not elucidate which species interact with the protein, in order to investigate the ligands possibly retained in the active site of recombinant CRBP, liquid chromatography/ES-tandem mass spectrometry was used. In particular, this technique was applied to identify and quantify fatty acids bound to CRBP. Quantitative data indicated the presence of a few fatty acids at a total concentration lower than 2% of that of the protein. Similar findings were observed for the homolog rat cellular retinol-binding protein type-II, demonstrating the high degree of purity and homogeneity of apo-CRBP preparations derived from gene expression.

Characterization of a New Member of the Fatty Acid-binding Protein Family That Binds All-trans-retinol

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.

Defective expression of cellular retinol binding protein type I and retinoic acid receptors alpha2, beta2, and gamma2 in human breast cancer cells.

Because the retinoic acid (RA) signaling pathway regulates cell proliferation and differentiation, inactivation of genes integral to the pathway represents a potential mechanism of carcinogenesis. We have studied in human breast cancer cells (T47D, MCF-7, ZR75-1, MDA-MB-231, and BT20) the expression of a subset of retinoid signaling genes that are themselves transcriptionally up-regulated by RA, the cellular retinol binding protein type I (CRBPI) and the RA receptors (RARs) alpha2, beta2, and gamma2. We find that constitutive expression of these genes is low or undetectable, and that expression levels are seldom responsive to 24 h treatment with 1 microM all-trans or 9-cis RA (Northern blot analysis). This is in contrast to breast fibroblasts, which show RA-dependent expression of all four genes under the same conditions. Moreover, normal human breast epithelial cells express CRBPI and RARbeta2 at the mRNA level, suggesting that loss of expression of these genes is tied to malignant transformation. RARbeta2, but not CRBPI, was also expressed in RA-treated MTSV1-7 cells, an immortalized but nontumorigenic luminal epithelial cell line. Lack of CRBPI and RARbeta2 expression in cancer cells was not due to general impairment of RA signaling, as shown by RA activation of a RARE3-tk-CAT reporter in a subclone of MDA-MB-231 cells that did not express either CRBPI or RARbeta2. These results suggest that at least two independent defects in the expression of proteins that function in retinoid signaling may be involved in breast carcinogenesis.