The Transfer of Retinol from Serum Retinol-binding Protein to Cellular Retinol-binding Protein Is Mediated by a Membrane Receptor*

Manickavasagam SundaramDagger , Asipu Sivaprasadarao§, Monica M. DeSousa, and John B. C. Findlay

From the School of Biochemistry & Molecular Biology and § Department of Pharmacology, University of Leeds, Leeds LS2 9JT, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta -lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Higher eukaryotic organisms depend upon the dietary supply of vitamin A for a range of essential biological processes including vision, reproduction, growth, and development (1). Dietary vitamin A is first transported into the liver, where it forms a complex with a specific 21-kDa carrier protein, called retinol-binding protein (RBP).1 The retinol-RBP complex is then secreted into the plasma, where it associates with another protein, transthyretin forming the main transporting complex (2, 3). Within the cytoplasm of vitamin A-requiring cells, retinol is transported by cellular retinol-binding protein (CRBP), a 15-kDa protein that is structurally distinct from RBP (4). The mechanism by which the transfer of retinol from extracellular RBP to intracellular CRBP is effected is the subject of much debate. Accumulated evidence suggests that the initial step involves the interaction of RBP with a specific cell surface receptor (5-10). However, there have been conflicting reports concerning the mechanism by which the release of retinol to the intracellular phase occurs. Work with retinal pigment epithelial cells (11) and placental brush-border membranes (12) suggested that, following the binding of RBP to the receptor, retinol is delivered to the cell, and the resultant apo-RBP remains in the extracellular compartment. By contrast, with liver paranchymal (8) and F9 teratocarcinoma (10) cells, the RBP-receptor complex is believed to be internalized by endocytosis and subsequently degraded releasing the retinol. Whether the type of mechanism employed is cell type-specific remains to be established. However, in either case the mechanism by which retinol is subsequently transferred to CRBP is not clear. Others (13) argue that vitamin A, being in equilibrium with RBP and extremely hydrophobic, readily partitions into the lipid phase of the membrane. Retinol is then assumed to be transferred to the intracellular binding proteins, again by diffusion.

Further support for the existence of specific RBP receptors has been obtained from a number of more recent studies. First, purification of an RBP receptor has been reported from pigment epithelial cells (14) and placental membranes (15). Second, mutations introduced into RBP decrease or abolish the interaction of the protein with its receptor without affecting the retinol binding capacity of RBP (16).

Based on the knowledge of the three-dimensional structures of RBP (17) and CRBP (18) complexed with retinol, and the currently available information on RBP-receptor interactions, we have put forward a hypothesis according to which the RBP receptor directly mediates the transfer of retinol from extracellular RBP to intracellular CRBP (19). In this paper, using the recombinant binding proteins and human placental membranes, we provide experimental support for this hypothesis.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Streptavidin-agarose, 2-iminobiotin, human serum albumin, bovine beta -lactoglobulin, egg albumin, all-trans-retinol, p-chloromercuribenzenesulfonate (pCMBS) sodium salt, dibutyl phthalate, and transthyretin were purchased from Sigma. All-trans-[11,12-3H]retinol (40 Ci/mmol) was obtained from NEN Life Science Products Inc. Dinonyl phthalate was from BDH. Restriction and modification enzymes were purchased from Boehringer Mannheim or Promega. Pfu DNA polymerase was purchased from Stratagene. The GeneClean Kit was from Anachem. Bio-Beads SM-2 was from Bio-Rad. All other reagents and chemicals were of appropriate grade and obtained from Sigma, Pharmacia, or BDH. Escherichia coli XL1-Blue (Stratagene) and BL21 (DE3) (Novagen) were used for general cloning and expression purposes, respectively. pT7.7 was a gift from S. Tabor, Harvard University, Boston, MA. pASK60 was from Biometra. The human CRBP cDNA clone in the pSP64 vector was kindly provided by Dr. U. Eriksson (Ludwig Institute for Cancer Research, Stockholm, Sweden). Oligonucleotides were synthesized and supplied by Genosys.

Methods

Construction of the CRBP Expression Vector-- The PstI fragment containing the human CRBP cDNA was first subcloned from the pSP64 vector into pKS Bluescript such that the cDNA was oriented in the 5' right-arrow 3' direction under the T7 promoter. The EcoRI and BamHI fragment from the resultant construct, pKS-CRBP, was then subcloned into the E. coli expression vector pT7.7. The resultant construct, pT7.7-CRBP (see Fig. 1 for a schematic diagram of the following steps) was subjected to deletion PCR (16) to remove the 5'-noncoding sequence of CRBP with concomitant fusion of the coding region (from second amino acid) to the initiation codon ATG in pT7.7, using the following primers: 5'-CCAGTCGACTTCACTGGGTAC-3' (sense) and 5-CATATGTATAATCTCCTTC-3' (antisense). The sense primer corresponds to nucleotides encoding +2 to +8 amino acids in the CRBP cDNA (20) while the antisense primer represents the ATG codon and 15 nucleotides upstream from it in the plasmid pT7.7. The PCR was performed in a 100-µl volume using 50 pmol of each phosphorylated primer, 10 ng of pT7.7-CRBP, 200 µM deoxynucleoside triphosphates (dNTPs), 10 µl of 10 × cloned Pfu DNA polymerase buffer (supplied by Stratagene), and 2.5 units of Pfu DNA polymerase. Thirty cycles, each consisting of 40 s at 94 °C, 1 min at 50 °C, and 10 min at 72 °C, with a final extension step at 72 °C for 10 min, were carried out. The PCR product was purified from agarose gels using the GeneClean kit, self-ligated at 16 °C overnight with 1 unit of T4 DNA ligase and used to transform E. coli XL-Blue cells. Clones were screened for loss of the EcoRI site by restriction enzyme analysis. Positive clones were subsequently sequenced to confirm the deletion and that no errors had occurred during PCR. The resulting plasmid is referred to as pT7.7-CRBP(del).


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Fig. 1.   Schematic representation of the construction of a vector for expression of the CRBP-Strep fusion protein. RBS, ribosome-binding site; ATG, translation initiation codon; STP, streptavidin recognition sequence; T7, T7 promoter. Positions of primers used for deletion-PCR are shown as horizontal arrows. For details see "Experimental Procedures."

To make the construct that allows the expression of CRBP as a fusion protein with streptavidin binding sequence (Ser-Ala-Trp-Arg-His-Pro-Glu-Phe-Gly-Gly) at its C terminus (21), the nucleotide sequence encoding this peptide (Strep-tag) was excised from the plasmid pASK60-Strep using BamHI and HindIII and subcloned into pT7.7-CRBP-(del). Deletion PCR was carried out (as described above) on the resultant construct to fuse the strep-tag to the last amino acid of CRBP, using the following primers: 5'-AGCGCTTGGCGTCACCCGC-3' (sense) and 5'-GTTCATAAGTTCTTCCACGTC-3' (antisense). The PCR product was self-ligated and used to transform E. coli XL blue cells. The desired clones were identified by the loss of the BamHI site and then confirmed by DNA sequencing. The construct was termed pCRBP-Strep.

Expression and Purification of the CRBP-Strep Fusion Protein-- E. coli strain BL21(DE3) was transformed with pCRBP-Strep and large scale expression carried out in a series of 20-ml cultures as follows. An overnight culture was set up in 5 ml of Luria broth supplemented with 50 µg/ml ampicillin (LB/amp medium) using a single colony from freshly transformed BL21 (DE3) cells. From this overnight culture, 80 µl were used to inoculate 4 ml of LB/amp medium. After growing the cells for 2 h at 37 °C and 200 rpm, 16 ml of LB/amp medium were added. Growth was continued until the cell density reached A600 of 0.3 at which point isopropyl-1-thio-beta -D-galactopyranoside was added to a final concentration of 0.5 mM and the induction carried out for 3 h at 30 °C. Induced cells were harvested by centrifugation at 5,000 × g for 10 min and resuspended in 10 ml of lysis buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl). The cells were then incubated with lysozyme at 0.5 mg/ml for 20 min at room temperature and sonicated (20 s bursts at 50% power) on ice, using an Ultrasonics Inc. sonicator. The cell debris was pelleted by centrifugation at 5,000 × g for 30 min at 4 °C and the supernatant applied to 0.5 ml of streptavidin-agarose, packed in a 2-ml column and equilibrated with 100 mM Tris-HCl, pH 8.0, 1 mM EDTA. After washing the resin with 5 ml of this equilibration buffer, the bound protein was eluted with 2.5 ml of elution buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM 2-iminobiotin). The fusion protein was dialyzed against distilled water at 4 °C, concentrated by freeze-drying and assayed for protein content by Lowry's method (22).

Preparation of [3H]Retinol-RBP Complex-- The expression and purification of recombinant RBP (rRBP) was performed as described previously (23). Removal of retinol from rRBP and substitution with [3H]retinol was carried out as described by Chen and Heller (11). The specific activities of the final preparation varied from 3 to 5 Ci/mmol.

Preparation of Membranes-- Brush-border membrane vesicles were prepared from freshly obtained human placenta according to the method of Booth et al. (24). The membranes (30 mg of protein) were dialyzed exhaustively against deionized distilled water containing 0.2 mM phenylmethylsulfonyl fluoride at 4 °C, freeze-dried, and resuspended in 20 ml of ice-cold extraction buffer (10 mM sodium phosphate, pH 7.2, 10 mM EDTA, 0.5 M NaCl). After gentle homogenization with a Potter-Elvehjem homogenizer, the suspension was stirred for 2 h at 4 °C and then centrifuged at 105,000 × g for 1 h at 4 °C. The supernatant was dialyzed against distilled water containing 0.2 mM phenylmethylsulfonyl fluoride, freeze-dried, and resuspended in 30 mM sodium phosphate, pH 7.2, 0.2 M NaCl and assayed for retinol binding activity as below. The membrane pellet was resuspended in 15 ml of PBS containing 0.4% Triton X-100 and stored as aliquots at -20 °C.

Human red blood cell membranes were prepared according to the method of Dodge et al. (25). Packed red cells were washed four times with 10 volumes of 150 mM NaCl, 10 mM phosphate, pH 7.2, at 4 °C and the supernatant and white cell "buffy coat" removed by aspiration each time. The cells were lysed in 10 volumes of ice-cold 5 mM sodium phosphate, pH 8.0 (containing 0.1 mM phenylmethylsulfonyl fluoride), and centrifuged at 15,000 rpm for 20 min. The supernatant was carefully removed and the process repeated until the membranes were light pink (three times). These membranes were suspended in ice-cold extraction buffer and treated as above prior to the transport assay.

Binding of Retinol to CRBP-- The binding of all-trans-retinol to E. coli-derived apo-CRBP-Strep fusion protein was assayed by measuring the retinol-induced quenching of protein fluorescence as described by Cogan et al. (26). The titrations were carried out at 22 °C in PBS containing 1 mM beta -mercaptoethanol and 1 mM EDTA by adding small increments of freshly prepared ethanolic solutions of retinol to 2 ml of 1 µM CRBP. The excitation and emission wavelengths were 290 and 340 nm, respectively. A solution of N-acetyl-L-tryptophanamide having an absorbance at 290 nm equal to that of the protein was used as the control.

Assay for Transfer of [3H]Retinol from RBP to CRBP by Placental or Erythrocyte Membranes-- [3H]Retinol transfer from [3H]retinol-RBP complex to CRBP-Strep tag was assayed using placental or erythrocyte membranes washed as above. 20 µl of 10 × assay buffer (1% ovalbumin in 10 × PBS), 10 pmol of [3H]retinol-RBP (8-9 × 104 disintegrations/min) and 50 pmol of CRBP-Strep tag fusion protein were added to 100 µl of membranes (2 mg of protein/ml). The volume was made up to 200 µl with water. After incubation at 37 °C for 30 min, the sample was centrifuged at 13,000 rpm in a microcentrifuge for 10 min to pellet the membranes. The resulting supernatant was mixed with 50 µl of 500 mM Tris-HCl, 5 mM EDTA, pH 8.0, and applied to a column packed with 0.5 ml of streptavidin-agarose resin. After washing the resin with 5 ml of wash buffer (100 mM Tris-HCl, 1 mM EDTA, pH 8.0), the bound protein was recovered with 1.5 ml of elution buffer (100 mM Tris-HCl, 1 mM EDTA, 1 mM 2-iminobiotin, pH 8.0). The radioactivity in the eluate was measured in a Packard 1900TR liquid-scintillation counter. This gave a measure of [3H]retinol transferred to CRBP-Strep tag. Incubations in the absence of membranes were also carried out to estimate nonspecific transfer of [3H]retinol to CRBP. All assays were performed in duplicate.

Sucrose Density Gradient Centrifugation-- The sucrose density gradient centrifugation assay for retinol binding activity in the EDTA extract of the membranes (see above) was carried out as described by Sani (27).

Gel Filtration Assay for Retinol Binding Activity-- Unwashed placental membranes (5 mg of protein/ml) were resuspended in 1% Nonidet P-40 and after incubation on ice for 20 min, centrifuged at 105,000 × g for 1 h. The supernatant was treated with Bio-Beads SM-2 to remove excess detergent (28). The detergent-free extract containing 2.5 mg of protein was incubated at 4 °C in the dark for 4 h with 50 pmol of [3H]retinol in the absence or presence of 10 nmol of unlabeled retinol. The mixture was diluted with an equal volume of 6 M urea, dialyzed against 3 M urea in 20 mM sodium phosphate, 20 mM mercaptoethanol, 100 mM NaCl, pH 7.5 (equilibration buffer), and loaded onto a Sephadex G-75 column (75 × 2 cm) equilibrated with the same buffer. Fractions (1.5 ml) collected were counted for radioactivity.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression, Purification, and Characterization of CRBP-Strep Fusion Protein-- Levin et al. (29) have reported the over-expression of rat CRBP in E. coli and showed that the expressed protein was similar to the native protein in terms of its ability to bind retinol. Here, we expressed the human protein as a fusion construct with the 10-amino acid long streptavidin-binding sequence (Strep tag) (21) added to its C terminus. The fusion protein, referred to as CRBP-Strep, could be readily purified from the crude extracts of the cultures of E. coli BL21(DE3) transformed with pCRBP-Strep in a single affinity chromatographic step using streptavidin-agarose resin (Fig. 2a). The purified protein migrated on SDS-polyacrylamide gel electrophoresis as a single band of apparent molecular mass ~16 kDa, the size expected for the fusion protein (Fig. 2b). The CRBP-Strep fusion protein accounted for approximately 50% of the total E. coli protein.


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Fig. 2.   Purification of CRBP by streptavidin chromatography. a, the lysate, derived from 20 ml of E. coli culture expressing the CRBP-Strep fusion protein was applied to a column containing 0.5 ml of streptavidin-agarose resin. Arrow indicates the position at which the elution buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM 2-iminobiotin) was applied to the column. b, SDS-polyacrylamide gel electrophoresis profile of the purified protein (lane 2) run alongside marker proteins (Dalton Markers VII L, Sigma) of known molecular weight (lane 1).

To ensure that the addition of Strep tag does not affect its ability to bind retinol, we determined the affinity of the expressed fusion protein for retinol by monitoring the quenching of protein (tryptophan) fluorescence upon ligand binding (Fig. 3). The apparent dissociation constant (Kd), determined from the quenching data, was 20 ± 2 nM, in good agreement with the value (26 ± 20 nM) reported by Levin et al. (29) for rat CRBP. We also measured the UV-visible spectrum (240-380 nm) of the fusion protein and found no absorbance peaks other than that at 280 nm, suggesting the absence of any bound UV-absorbing ligand. The data suggest that the expressed tagged protein is similar to native CRBP in terms of its retinol binding ability and therefore should be suitable for the retinol transfer studies.


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Fig. 3.   Fluorescence titration of the E. coli-derived CRBP-Strep fusion protein with all-trans-retinol. Protein fluorescence was monitored following the addition of various amounts of all-trans-retinol, using excitation and emission wavelengths of 290 and 330 nm, respectively. The data points were fit to a monoexponential decay equation using nonlinear regression analysis.

Transfer of Retinol from RBP to CRBP Is Mediated by the Placental RBP Receptor-- To examine the hypothesis that the membrane-bound receptor might mediate the transfer of retinol from extracellular RBP to cytoplasmic CRBP, we have developed a simple assay using human placental membranes as the source of the receptor and the E. coli-expressed recombinant CRBP-Strep fusion protein. Membranes prepared from the placental brush-border tend to form sealed outside-out vesicles, thereby preventing access to the cytoplasmic surface of the membranes of exogenously added CRBP-Strep. The cytoplasmic phase of these membranes might also contain some endogeneous bound CRBP, thus reducing the number of binding sites available for the exogenously added CRBP-Strep. For these reasons, membranes prepared using the conventional protocols may be unsuitable for investigating the transfer of retinol to CRBP. We, therefore, treated the membranes with EDTA to remove any CRBP and added a small amount of Triton X-100 to prevent formation of sealed vesicles.

To assay the transfer of [3H]retinol from RBP to CRBP, we incubated RBP, complexed with [3H]retinol, and apo-CRBP-Strep with washed membranes under a variety of conditions. Following incubation, the membranes were removed by centrifugation and CRBP-Strep isolated from the supernatant by affinity chromatography with streptavidin-resin. Under the conditions used for chromatography, [3H]retinol associated with RBP was eluted in the flow-through, whereas that transferred to CRBP-Strep tag was retained by the resin. The resin-bound CRBP-Strep was subsequently released with iminobiotin and assayed for radioactivity.

The results (Table I) clearly show that the transfer of [3H]retinol from RBP to CRBP was about 3-fold greater in the presence of membranes than in their absence. To eliminate the possibility that the process is mediated by the lipid rather than the protein fraction of the membranes, the effect of heat treatment of the membranes on the transfer activity was examined. Heating the membranes at 90 °C for 10 min, prior to the transfer assay abolished the transfer of [3H]retinol from RBP to CRBP. The transfer activity reached equilibrium within 30 min.

                              
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Table I
Effect of placental membranes on [3H]retinol uptake by CRBP
The uptake of [3H]retinol by apo-CRBP (50 pmol) from the [3H]retinol-RBP complex (10 pmol) was studied in the absence and presence of placental or erythrocyte membranes (200 µg of protein). The incubations were carried out at 37 °C for 30 min in a volume of 200 µl. Prior to incubation, the placental membranes were treated with PBS alone or with 5 mM pCMBS, or heated at 90 °C for 10 min. Under each condition, [3H]retinol uptake by CRBP was determined as described under "Experimental Procedures." Values represent mean ± S.D. (n = 2).

To demonstrate that the membrane-mediated transfer is dependent on the RBP receptor, we treated the membranes with 5 mM pCMBS, a compound which has previously been shown to inhibit the ability of RBP to interact with its receptor (12). Unreacted pCMBS was removed by washing the membranes prior to the binding assay, to rule out the possibility that any inhibition observed could be the result of pCMBS interaction with the binding proteins (RBP and CRBP). As can be seen from the data in Table I, this treatment almost completely abolished the membrane-mediated transfer of [3H]retinol from RBP to CRBP, suggesting that the receptor is an obligatory part of the transport mechanism.

Finally, the experiment was repeated with washed erythrocyte ghosts. This membrane system is not thought to contain the RBP receptor (11). The results show a transfer rate not significantly different from that obtained with heat-treated placental membranes, offering further support for a receptor-mediated transfer mechanism.

beta -Lactoglobulin and Serum Albumin Cannot Substitute for RBP in the Transfer Process-- The specificity of the transfer activity of placental membranes with respect to RBP was assessed by examining the ability of a homologue, beta -lactoglobulin, and serum albumin to inhibit the transfer of [3H]retinol from the [3H]retinol-RBP complex to CRBP-Strep. These two proteins resemble RBP and CRBP in terms of their ability to bind retinol with high affinity but neither competes with RBP for binding to its receptor (6). It was found that neither of these proteins, in their retinol-bound form, inhibited the transfer process (Table II). The fact that beta -lactoglobulin, despite its high degree of structural similarity to RBP (30), failed to inhibit the transfer process again indicates that the receptor is highly specific for RBP.

                              
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Table II
Specificity of retinol transfer activity of placental membranes
Placental membranes (200 µg of protein) were incubated with 10 pmol of [3H]retinol-RBP and 50 pmol of apo-CRBP for 30 min at 37 °C in the presence of different retinol-binding proteins (2 µM). [3H]Retinol taken up by CRBP was then assayed as detailed under "Experimental Procedures." None represents the total uptake determined in the absence of any competing protein. Values are the mean ± S.D. (n = 2).

Fig. 4 shows that the retinol transfer activity of the membranes is inhibited by unlabeled RBP in a concentration-dependent manner, approximately 50% inhibition occurring at a 15-fold molar excess of the unlabeled protein. This indicates that the transfer activity is mediated by a limited number of receptor molecules.


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Fig. 4.   Effect of unlabeled RBP on [3H]retinol transfer from [3H]retinol-RBP complex CRBP. The indicated amounts of unlabeled RBP were included in the retinol transfer assay reactions containing 10 pmol of [3H]retinol-RBP, 50 pmol of apo-CRBP, and 200 µg of placental membranes. After 30 min incubation at 37 °C, [3H]retinol transferred to CRBP was estimated as described under "Experimental Procedures."

Apo-, but Not the Holo-form of CRBP Can Receive Retinol from RBP-- The efficiency of apo-CRBP to act as the recipient of retinol delivered via the RBP receptor system was examined by testing the ability of the defatted forms of human serum albumin and bovine beta -lactoglobulin to substitute for CRBP. Since these native proteins possess no tags to aid their purification, a different approach was used. This involved measuring the loss of [3H]retinol from the RBP complex isolated from the reaction mixture using a transthyretin affinity resin. The data (Table III) show that both human serum albumin and bovine beta -lactoglobulin are much less efficient than apo-CRBP in terms of their ability to accept [3H]retinol delivered from RBP even though they have similar affinities for the ligand. The radioactivity transferred with these proteins probably arises from free equilibration. Table III also demonstrates that the holo-form of CRBP is unable to induce retinol release from RBP. Interestingly, this was also true of the membrane in the absence of any protein acceptor.

                              
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Table III
Specificity of [3H]retinol uptake by CRBP
[3H]Retinol-RBP (10 pmol) was incubated at 37 °C for 30 min with placental membranes (200 µg of protein) in the absence and presence of the indicated proteins at 2 µM concentration. Both beta -lactoglobulin and serum albumin used in the assay were defatted. After centrifugation at 13,000 rpm in a Microfuge for 10 min, 40 µl of 250 mM Tris-HCl, pH 7.4, 0.25 M NaCl was added to the supernatant and applied to a 0.5-ml column of TTR-affinity resin. After washing the resin with 50 mM Tris-HCl, 0.5 M NaCl, pH 7.4, [3H]retinol-RBP, retained by transthyretin, was eluted with deionized water and the radioactivity present in the eluate was measured. The radioactivity transferred to CRBP was calculated by subtracting the test values from controls, lacking membranes, which were carried out in parallel. Values represent mean ± S.D. (n = 2).

Extracts of Placental Membranes Contain a 15-kDa Retinol-binding Protein-- Fig. 5 shows that when the EDTA extract of placental membranes was incubated with [3H]retinol and then subjected to sucrose density gradient centrifugation, a single radioactive peak at 2 S was observed. This peak was not present in the incubations carried out in the presence of a 200-fold molar excess unlabeled retinol. To determine the molecular weight of the protein, detergent-solubilized placental membrane proteins were treated with [3H]retinol and subjected to gel filtration using Sephadex G-75. Gel filtration was performed in the presence of 3 M urea to prevent any aggregation of the protein. A radioactive peak was observed at the 15 kDa position (Fig. 6). The peak was absent when the incubations were carried out in the presence of excess unlabeled retinol. These studies suggest normal association of apo-CRBP with native placental membranes. The data, however, do not exclude the possibility that a protein distinct from, but similar to, CRBP might be present in the extract.


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Fig. 5.   Sucrose density gradient profile of the EDTA extract of placental membranes. 0.3 ml of the EDTA extracts (0.6 mg of protein) were incubated with 36 pmol of [3H]retinol in the absence (open circle ) and presence (bullet ) of 5 nmol of unlabeled retinol for 4 h in the dark at 4 °C. The unbound retinol was removed with dextran-coated charcoal and the protein subjected to sucrose density gradient (5-20%) centrifugation. Positions of bovine serum albumin (1) and ovalbumin (2), used as external standards, are shown by arrows.


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Fig. 6.   Gel filtration of Nonidet P-40 extract of placental membranes. Gel filtration of the placental membrane proteins treated with 50 pmol of [3H]retinol alone (open circle ) or [3H]retinol plus 10 nmol of unlabeled retinol (bullet ) was performed as described under "Experimental Procedures." The column was calibrated using standard molecular weight marker proteins. Positions of the marker proteins, bovine serum albumin (1), ovalbumin (2), and beta -lactalbumin (3) are shown by arrows.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This paper reports an investigation into the process by which retinol from the extracellular RBP is transported to intracellular CRBP across the plasma membrane (human placental brush-borders). Previous studies have demonstrated that these membranes contain receptors that specifically interact with the circulating RBP and induce the release of retinol from the binding protein (11, 12). Here, we examined the hypothesis that the retinol so released is transferred to apo-CRBP by a membrane dependent mechanism. To do so, a new assay system involving the isolation of affinity-tagged recombinant CRBP has been developed.

Using this assay, we demonstrate that the transfer of [3H]retinol from RBP to CRBP is greatly facilitated by the presence of placental membranes (Table I) and is unaffected by other retinol-binding proteins such as serum albumin and beta -lactoglobulin (Tables II and III). It is further suggested that the transfer of retinol occurs via the agency of specific RBP receptors, since this transfer is substantially reduced in receptor-deficient erythrocyte membranes, on denaturation of placental membrane proteins, on treatment with pCMBS which abolishes RBP-receptor interaction (Table I), and in competition with unlabeled RBP (Fig. 4). Since little specific retinol transfer was observed with erythrocyte membranes or when placental membranes were heat-treated, the transfer process was not mediated solely or substantially by membrane lipids. This is further supported by other experiments (Table III) which indicate limited release of retinol via the membrane in the absence of a retinol acceptor protein. Finally, since the studies were carried out on isolated membranes, the data also suggest that the transfer of retinol across the placental brush-border is unlikely to involve energy-dependent endocytosis.

These studies also show that the transfer of retinol occurred only when apo-CRBP was used as the acceptor protein; the defatted forms of neither serum albumin nor beta -lactoglobulin, both of which can bind retinol with high affinity, were able to substitute for apo-CRBP (Table III). This implies that a specific membrane-binding site for apo-CRBP might exist on the cytoplasmic phase of the membrane. Consistent with the hypothesis put forward before (19), the holo-form of CRBP is ineffective in inducing the release of retinol from the receptor-bound RBP. The demonstration that a 15-kDa protein with retinol-binding property is associated with native placental membranes (Figs. 5 and 6) suggests that under physiological conditions, apo-CRBP might receive retinol via the RBP receptor at the membrane level. The association of CRBP with the plasma membranes has been previously reported by other workers (31, 32).

Taken together, the evidence could be interpreted to suggest that the RBP receptor protein induces the release of retinol from circulating RBP and facilitates its transfer to the intracellular CRBP in a membrane-dependent manner. However, the mechanism by which the receptor mediates this transfer is not clear. X-ray crystallographic studies revealed that in RBP, retinol is situated in the binding pocket with its beta -ionone ring deep inside and the terminal hydroxyl near the open end (17). By contrast, in CRBP the orientation of the ligand in the binding pocket is opposite to that in RBP (18). More recently, mutagenesis study has shown that RBP interacts with the receptor with the loops that make up the entrance/exit site of retinol, thereby perhaps facilitating direct release of ligand to the transfer mechanism. This is followed by the loss of, or reduction in, the affinity of the receptor for RBP (6, 33). The exact process by which the receptor facilitates the subsequent transfer of retinol to CRBP, however, has yet to be elucidated. One possibility is that the receptor, either on its own, or with associated but as yet unidentified protein(s), might form the transmembrane machinery through which retinol is transported into the binding pocket of CRBP, with the hydroxyl group leading the way. Such a mechanism would place the retinol in CRBP in the correct orientation. This would require CRBP to interact directly with the cytoplasmic side of the transport mechanism with the loops near the entrance to its ligand binding pocket. Final proof for such a mechanism awaits reconstitution experiments using the purified receptor (15).

Whether the transport system proposed here is unique to retinol or may represent one member of a new large family of related processes designed for efficient handling of hydrophobic materials or substances required in only small quantities by eukaryotic cells, remains to be established. Certainly, there are suggestions that some fatty acids may utilize a related mechanism.

    FOOTNOTES

* This work was supported by a grant from the Biotechnology and Biological Sciences Research Council, United Kingdom (to J. B. C. F. and A. S.), and the Royal Society (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Physiology, University of Alberta, Edmonton, Canada.

To whom correspondence should be addressed. Tel.: 44-0113-2334326; Fax: 44-0113-2334331; E-mail: a.sivaprasadarao{at}leeds.ac.uk.

1 The abbreviations used are: RBP, retinol-binding protein; CRBP, cellular retinol-binding protein; pCMBS, p-chloromercuribenzenesulfonate; PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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