Cellular Retinol-binding Protein Type Research Articles

Retinoic acid receptors and cellular retinoid binding proteins. III. Their differential transcript distribution during mouse nervous system development.

We have studied the transcript distribution of the retinoic acid receptors (RARs) and the cytoplasmic retinoid binding proteins during embryonic development of the mouse nervous system. Of the three retinoic acid receptors, only RAR-gamma was not expressed in developing neural structures. RAR-beta and RAR-alpha both showed rostral limits of expression in the medulla oblongata equivalent to their patterns of expression in the neuroepithelium of the early hindbrain neural tube. Within their expression domains in the spinal cord and brain, RAR-alpha was ubiquitously expressed, whereas RAR-beta transcripts showed very specific patterns of expression, suggesting that this receptor is involved in mediating retinoic acid-induced gene expression in relation to the development of specific neural structures or pathways. The cytoplasmic binding proteins, cellular retinoic acid binding proteins type I and II (CRABP I and CRABP II) and cellular retinol binding protein type I (CRBP I), were widely distributed in developing neural structures. Their differential spatiotemporal patterns of expression suggest that fine regional control of availability of retinoic acid (RA) to the nuclear receptors plays an important role in organization and differentiation of the nervous system. For instance, expression of CRABP I in the migrating cells that give rise to the olivary and pontine nuclei, which develop abnormally in conditions of retinoid excess, is consistent with observations from a variety of other systems indicating that CRABP I limits the access of RA to the nuclear receptors in normal physiological conditions. Similarly, expression of CRBP I in the choroid plexuses, which develop abnormally in conditions of vitamin A deficiency, is consistent with observations indicating that this binding protein mediates the synthesis of RA in tissues requiring high levels of RA for their normal developmental programme. RAR-beta and CRABP II, which are both RA-inducible, were coexpressed with CRBP I in the choroid plexus and in many other sites, perhaps reflecting the fact that all three genes are RA-inducible. The function of CRABP II is not well understood; its domains of expression showed overlaps with both CRABP I and CRBP I.

Expression of Cellular Retinoid-Binding Proteins During Normal and Abnormal Epidermal Differentiation

Retinoids have important roles in growth and differentiation of epidermal cells. We have analyzed the expression of two intracellular retinoid-binding proteins, the cellular retinol-binding protein type I and the cellular retinoic acid-binding protein type I, during normal and abnormal epidermal differentiation. Both proteins were found to be expressed in normal epidermis with increasing expression from basal layer towards superficial layers. In psoriatic lesions, a hyperproliferative condition of the skin, the epidermal expression of cellular retinol-binding protein I was induced, whereas expression of cellular retinoic acid-binding protein I was sharply down-regulated. This and other features of psoriatic lesions indicate that down-regulation of cellular retinoic acid-binding protein I expression might cause aberrant retinoid-regulated gene expression in skin. In basal and squamous cell carcinomas, cellular retinoic acid-binding protein I expression was down-regulated, whereas cellular retinol-binding protein I was expressed. Apart from epidermal cells, a mesenchymal, dendritic cell-type, strongly expressing cellular retinoic acid-binding protein I, was identified in the dermis. In several hyperproliferative conditions of the skin, including psoriasis, and squamous and basal cell carcinomas, this cell type was abundant. These results have implications for the role of retinoids in normal and abnormal epidermal differentiation and suggest that part of the phenotype of psoriasis is due to inappropriate metabolism of retinoic acid in skin.

Biosynthesis of all-trans-retinoic acid from retinal. Recognition of retinal bound to cellular retinol binding protein (type I) as substrate by a purified cytosolic dehydrogenase.

An NAD-dependent rat liver cytosolic dehydrogenase accepted as substrate retinal generated in situ by microsomes from retinol bound to excess CRBP (cellular retinol binding protein, type I). This activity, which was not retained by anion-exchange chromatography at pH 9.15, was designated P1. P1 activity increased 2.5-fold, with no statistically significant change in its K or Hill coefficient, in liver cytosol from rats fed a retinoid-deficient diet. Orally dosed retinoic acid partially suppressed the increase. Activities chromatographically similar to hepatic P1 were observed in cytosols from rat kidney and testes. P1, purified from rat liver cytosol, had a pI of approximately 8.3, migrated as a tetramer (214 kDa) on a Sephadex G-200 column, and had a subunit molecular mass of 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With free retinal it catalyzed a maximum rate of retinoic acid synthesis of 265 nmol/min/mg of protein and exhibited allosteric kinetics with a K of 0.76 +/- 0.35 microM and a Hill coefficient of 1.5 +/- 0.13 (mean +/- S.D., n = 4). Substrate inhibition was noted with retinal concentrations greater than 6 microM. The purified enzyme not only recognized retinal generated by microsomes as substrate, but also recognized retinal bound to CRBP. The rates of retinoic acid synthesis from CRBP-retinal, with a series of increasing apoCRBP concentrations, exceeded the rates that would be supported by the free retinal present. The CRBP-retinal complex exhibited allosteric kinetics (K, 0.13 microM; Hill coefficient, 1.75; averages of duplicates) in the presence of excess apoCRBP (the ratio total CRBP/total retinal at each concentration of retinal was 2). This enzyme is likely to play a significant role in retinoic acid synthesis in vivo, because it participates in the synthesis of retinoic acid from a physiologically occurring form of retinol (holoCRBP), reflects retinoid status, and is distributed in extrahepatic tissues in addition to liver. These results also suggest a novel role for CRBP in retinoid metabolism, facilitating the conversion of retinal into retinoic acid.

Solubilization and partial characterization of lecithin-retinol acyltransferase from rat liver

Lecithin-retinol acyltransferase (LRAT) is a microsomal enzyme that can esterify retinol bound to either cellular retinol binding protein or cellular retinol binding protein type II, utilizing phosphatidylcholine as the acyl source. Here, we report the solubilization of rat liver microsomal LRAT by 1.0% Brij-35, and partial purification via Phenyl Sepharose CL-4B chromatography. Kinetic properties of the solubilized enzyme were determined for retinol bound to cellular retinol binding protein (Km of 2.4 μ M), retinol bound to cellular retinol binding protein type II (Km of 1.3 μ M) and dilauroylphosphatidylcholine (Km of 2.5 μ M). The solubilized enzyme was very selective for acyl donor in that phosphatidylethanolamine and phosphatidic acid were not utilized. With phosphatidylcholine as the acyl source, only the acyl group at the sn-I position was transferred to retinol. The reaction catalyzed by LRAT is reminiscent to that catalyzed by lecithin-cholesterol acyltransferase (LCAT); both enzymes transfer acyl groups from phosphatidylcholine to lipid alcohols to form esters. The catalytic mechanism of the two enzymes may also be similar. LCAT utilizes a serine for the phospholipase-like cleavage of phosphatidylcholine and one (or both) of two vicinal sulfhydryls as the intramolecular acceptor for the fatty acyl group, prior to transfer to cholesterol. Similar to LCAT, LRAT was inhibited by N-ethylmaleimide at concentrations as low as 15 μ M, and completely inhibited by 1.0 m M p-aminophenylarsineoxide, a reagent that requires vicinal sulfhydryls in order to form a stable covalent complex. But unexpectedly, LRAT retained over 50% of its activity after treatment with 20 m M diisopropylfluorophosphate, diethyl p-nitrophenyl phosphate, or m-aminophenyl boronic acid, reagents that have been shown to inhibit LCAT as well as other enzymes utilizing serine residues for hydrolysis. Solubilized LRAT was inhibited by 5.0 m M phenylmethylsulfonylfluoride, and this inhibition was reversible within seconds of dithiothreitol addition to the inactive enzyme. Similar results have been reported for the sulfhydryl-protease papain. These inhibitor studies suggest that the active residue in LRAT necessary for the phospholipase cleavage step is a cysteine rather than the serine employed by LCAT.

A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR.

The vitamin A derivative retinoic acid exerts its effects on transcription through two distinct classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). We provide evidence that expression of the gene for cellular retinol-binding protein type II (CRBPII), a key protein in the intestinal absorption of vitamin A, is dramatically up-regulated by retinoic acid in the presence of RXR but not RAR. This regulation is conferred through a specific cis element in the CRBPII promoter that contains five nearly perfect tandem repeats of the sequence AGGTCA spaced by a single nucleotide. The discovery of this new RX response element provides a means for distinguishing between the two retinoid receptor systems and suggests that an RXR-mediated pathway exists for modulating vitamin A metabolism.

Regulation of cellular retinol binding protein type II by 1,25-dihydroxyvitamin D3

Previously we purified and sequenced an 18-kDa chick duodenal protein that was modulated by 1,25-dihydroxyvitamin D3. The N-terminus of this protein has striking sequence homology to cellular retinol binding protein type II (CRBP II). Furthermore, this purified chick protein binds retinol. Antibodies have now been generated to the chick protein and used for immunoblot analysis to demonstrate that the chick protein has molecular weight, tissue distribution, and subcellular localization similar to rat CRBP II. These antibodies also cross-reacted with rat CRBP II. Antibodies to rat CRBP II cross-react with the chick protein. Northern analysis using a cDNA probe for rat CRBP II showed a single 860 base pair mRNA in both chick and rat intestinal RNA preparations. These results demonstrate that the 1,25-dihydroxyvitamin D3 modulated protein in chick embryonic organ culture is chick CRBP II. Pulse-chase experiments in chick embryonic duodenal organ culture strongly suggest that 1,25-dihydroxyvitamin D3 markedly decreases the synthesis of CRBP II, while not changing the degradation rate. The concentration of 1,25-dihydroxyvitamin D3 required for the decrease in CRBP II synthesis is approximately that required to stimulate calcium uptake into embryonic chick duodenal organ cultures.

Levels of cellular retinol-binding proteins in the small intestine of rats during pregnancy and lactation.

The movement and metabolism of vitamin A is dependent on a number of specific carrier proteins. The small intestine contains both cellular retinol-binding protein (type two) (CRBP(II], restricted to the villus-associated enterocytes, and cellular retinol-binding protein (CRBP), present primarily in supporting mesenchymal cells. The content of these proteins in the small intestine of prepartum and postpartum Sprague-Dawley rats was determined by radioimmunoassay. Levels of CRBP(II), but not CRBP, changed dramatically during this period. Total content of CRBP(II) in the small intestine rose precipitously in late pregnancy and continued to rise throughout lactation to a peak at day 21 postpartum more than 300% greater than in nulliparous, nonpregnant controls. In contrast, total small intestinal weight and CRBP content increased only approximately 100% from late pregnancy to day 21 of lactation. CRBP(II) concentration in the proximal and middle segments of small intestine (expressed on a g wet tissue, mg protein, or mg DNA basis) remained at control levels through day 17 of pregnancy, increased 50-100% in late pregnancy, then rose markedly at parturition to levels two- to threefold greater than controls. CRBP(II) concentration was then maintained at a relatively constant elevated level during the remainder of lactation, but decreased markedly after weaning, approaching control levels within 1 week. The concentrations of CRBP(II) in enterocytes isolated from the proximal two-thirds of the small intestine from rats on day 20 of pregnancy and days 1 and 16 of lactation, expressed on a mg DNA basis, were similar and approximately 60% greater than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, Primary Structure Characterization, and Cellular Distribution of Two Forms of Cellular Retinol-binding Protein, Type II from Adult Rat Small Intestine

Cellular retinol-binding protein type II (CRBP(II)) is a major protein in the small intestine, accounting for more than 1% of the soluble protein recovered from rat jejunal mucosa. Two forms of the protein, called CRBP(II)A and CRBP(II)B, were purified from rat small intestine using a three-column procedure. The two forms were present in equal abundance. The primary structures of CRBP(II)A and CRBP(II)B were determined using a combination of techniques including amino acid composition and sequence analyses, and fast atom bombardment and gas chromatography-electron impact mass spectrometry. The primary structures of both proteins were found to be identical, but they differed in their NH2-terminal processing. CRBP(II)B was acetylated at its NH2 terminus, while CRBP(II)A was not. The results also confirmed the amino acid sequence of CRBP(II)A that was deduced from the cDNA sequence by Li et al. (Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5770-5783). Antibodies capable of distinguishing between the two forms of CRBP(II) were used for immunohistochemical studies which indicated that the organ and cellular distributions of the two forms were identical. The 50% acetylation observed here in vivo fits the pattern predicted by recent in vitro studies which described the effect of NH2-terminal sequence on cotranslational NH2-terminal processing of cytosolic proteins (Boissel, J. P., Kasper, T. J., and Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449). Our results provide a basis for investigating the possibility of different roles of CRBP(II)A and CRBP(II)B within cells, as well as the importance of acetylation of the amino terminus for these biological functions.

Cellular retinol-binding protein (type two) is abundant in human small intestine.

Human small intestine was found to contain a retinol-binding protein similar to the gut-specific cellular retinol-binding protein, type two [CRBP (II)], described in the rat. This newly detected human protein was immunochemically distinct from human cellular retinol binding protein previously described but immunochemically similar to rat CRBP (II). The partially purified protein bound retinol and exhibited fluorescence excitation and emission spectra distinct from those spectra for retinol bound to pure human CRBP but similar to the spectra for retinol bound to rat CRBP (II). Human CRBP (II) could be localized to the villus-associated enterocytes by immunohistochemistry, using antiserum against rat CRBP (II). The protein was abundant representing 0.4% of the total soluble protein in a jejunum mucosal extract. This protein may play an important role in the absorption and necessary intestinal metabolism of vitamin A.

Specificity of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin

We have reported previously that cellular retinol-binding protein (CRBP) is able to transfer retinol to specific binding sites in nuclei and chromatin. In this report, we have examined the specificity of the interaction of the protein moiety of retinol-CRBP (R-CRBP) with chromatin and nuclei in the transfer process. We first determined the ability of apo-CRBP, apo-serum retinol-binding protein (RBP), and apo β-lactoglobulin (BLG), all capable of retinol binding, to compete with R-CRBP in the transfer of retinol to chromatin and nuclei. Apo-CRBP was an effective competitor but apo-RBP and apo-BLG showed no competitive ability. On the other hand, cellular retinol-binding protein type II (CRBP(II)), whose amino acid sequence shows a considerable similarity to CRBP, did compete for the transfer of retinol from the R-CRBP complex, but less effectively than CRBP. These results demonstrate that the interaction of the protein moiety of the R-CRBP complex with nuclei and chromatin is quite specific.

Cell-specific immunohistochemical localization of a cellular retinol-binding protein (type two) in the small intestine of rat.

One of us recently has reported the purification of a new retinol-binding protein that is distinctly different from the well-known cellular retinol-binding protein, CRBP. This protein, which we propose to name cellular retinol-binding protein type II [CRBP(II)], was found almost exclusively in the small intestine of the adult rat at levels 1000 times greater than that of CRBP. Here we have determined the cellular location of these two proteins in the small intestine of the rat. By using an immunohistochemical technique, the absorptive cells of the small intestine, from the duodenum to the ileum, were strongly stained when antiserum against CRBP(II) was used. More intense staining was observed in absorptive cells near the tips of the villi than in those located at the base of the villi. However, the proliferative cells in the crypts of Lieberkühn were stained only lightly if at all. In contrast to absorptive cells, goblet cells in the villi did not stain. When tissue sections containing the gastroduodenal junction were examined, no staining was observed in the gastric epithelium, while the epithelium of the most proximal portion of the duodenum showed very strong staining. In tissue sections containing the ileocecal junction, staining terminated abruptly at the end of the distal ileum. No staining was observed in the epithelium of the colon. In contrast, the cellular location of CRBP in the small intestine was quite different from the cellular location of CRBP(II). The epithelial cells of the small intestine showed no staining when affinity-purified anti-CRBP was used. Staining was observed for connective tissue cells in the lamina propria and in cells located within the gut-associated lymphoid tissue. The cell-specific localization pattern determined for these two proteins suggests that CRBP(II), rather than CRBP, is the protein that plays a role in the absorption of retinol.