Human Retinol-binding Protein Research Articles

Free energy simulations to study mutational effect of a conserved residue, Trp24, on stability of human serum retinol-binding protein

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a β-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP. Communicated by Ramaswamy H. Sarma

Isotope-dilution liquid chromatography-tandem mass spectrometry for quantification of human retinol binding protein 4 in serum

Serum retinol binding protein 4 levels play critical roles in the early diagnosis and therapeutic monitoring of diabetic kidney disease. In this paper, an liquid chromatography-tandem mass spectrometry approach for the absolute quantification of serum RBP4 with high sensitivity and specificity was presented. Following series of procedures including denaturation, reduction, alkylation and trypsin digestion, the signature peptides of RBP4 were separated on the HPLC system by gradient elution. Quantitative analysis was achieved by a triple quadrupole mass spectrometer under multiple reaction monitoring in the positive ion (ESI+) mode. The calibration range was from 6.25 to 125 mg/L (R2 > 0.993), the lower limit of quantification was 2.50 mg/L, and the lower limit of detection reached 0.0150 mg/L. In the study, the accuracy ranged from 94.6% to 107%, and the relative standard deviation of intra-assay and inter-assay imprecision was less than 5%. The method was demonstrated to realize sensitive and reliable absolute quantification of serum RBP4 conforming to guidelines for bioanalytical method validation.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

Investigation of galectin-3, lipocalin 2, retinol binding protein (RBP), small dense low-density lipoprotein (sdLDL) in patients with hirsutism.

IntroductionHirsutism is defined as excessive terminal hair in androgen-dependent areas in women. Adipose tissue is no longer regarded as a storage site for triglycerides or as a source of free fatty acids but is currently emerging as a key constituent of energy metabolism secreting numerous enzymes, cytokines, growth factors, and hormones.AimTo evaluate serum levels of galectin-3 (Gal3), lipocalin-2 (LCN2), retinol binding protein (RBP), and small dense low-density lipoprotein (sdLDL) in patients with hirsutism and patients co-presenting with polycystic ovary syndrome (PCOS) and hirsutism.Material and methodsThe study included 90 patients that were divided into three groups: (I) patients with idiopathic hirsutism (IH) (n = 30), (II) PCOS patients with hirsutism (H-PCOS) (n = 30), and (III) the control group (n = 30). Informed consent was obtained from each participant. Idiopathic hirsutism was scored using the modified Ferriman-Gallwey (mFG) scoring system and the diagnosis of PCOS was established based on the modified Rotterdam criteria. Human Gal3, LCN2, RBP, and sdLDL levels were analysed using a commercially available double-antigen sandwich enzyme-linked immunosorbent assay.ResultsNo significant difference was found between the three groups with regard to serum Gal3, LCN2, RBP, and sdLDL levels (p > 0.05). Median values were higher in the H-PCOS group than in the other two groups: body weight (median: 76.5 kg) (p < 0.001), waist circumference (median: 82.5 cm) (p = 0.001).ConclusionsNo significant correlation was found between the adipokines that play a role in the aetiology of numerous diseases and some mediators of the lipid metabolism and hirsutism.

In Silico Study on Retinoid-binding Modes in Human RBP and ApoD Lipocalins

Lipocalins are proteins with highly homologous structures but diverse sequences that are potential candidates for scaffold protein engineering with novel ligand-binding functions. Numerous crystal structures of lipocalin-ligand complexes have been identified and used in the study of their binding modes. On the other hand, crystallization studies cannot meet the increasing demand for novel lipocalin-ligand complexes in scaffold engineering, which requires rapid computational analyses of their binding modes in parallel. Human retinol-binding protein (RBP) and apolipoprotein D (apoD) are sequentially very distant proteins, but they show tight binding against retinoids, such as retinol and retinoic acid. In the present study, complexes of the two lipocalins with retinol and retinoic acid were modeled computationally by a molecular docking simulation, and their ligand-binding modes were analyzed at a molecular level. The models identified the crucial residues of lipocalins that interact with the ligands and revealed the similarities and differences in their retinoid-binding modes as well as in the specific interactions of the retinoid species within the same lipocalin. An analysis of the amino acid propensity of the retinoid-binding residues suggested that the evolutionary preference of the residues is restricted to the binding pocket rather than the entire protein. The distribution of charged residues around the terminus of retinoic acid showed a huge difference between RBP and ApoD, which might be a factor for the different binding affinities of lipocalins against retinoic acid. This in silico study is expected to be applied to scaffold protein engineering for novel retinoid-binding lipocalins.

Increasing the length and hydrophobicity of the C-terminal sequence of transthyretin strengthens its binding affinity to retinol binding protein.

Transthyretin (TTR) is a transporter for thyroid hormone (TH) and retinol, the latter via binding with retinol binding protein (RBP). Both the N‐terminal and C‐terminal regions of the TTR subunit are located in close proximity to the central binding channel for ligands. During the evolution of vertebrates, these regions changed in length and hydropathy. The changes in the N‐terminal sequence were demonstrated to affect the binding affinities for THs and RBP. Here, the effects of changes in the C‐terminal sequence were determined. Three chimeric TTRs, namely pigC/huTTR (human TTR with the C‐terminal sequence changed to that of Sus scrofa TTR), xenoN/pigC/huTTR (human TTR with the N‐terminal and C‐terminal sequences changed to those of Xenopus laevis and S. scrofa, respectively), and pigC/crocTTR (Crocodylus porosus TTR with the C‐terminal sequence changed to that of S. scrofa TTR), were constructed and their binding affinities for human RBP were determined at low TTR/RBP molar ratio using chemiluminescence immunoblotting. The binding dissociation constant (K d) values of pigC/huTTR, xenoN/pigC/huTTR and pigC/crocTTR were 3.20 ± 0.35, 1.53 ± 0.38 and 0.31 ± 0.04 μm, respectively, and the K d values of human and C. porosus TTR were 4.92 ± 0.68 and 1.42 ± 0.45 μm, respectively. These results demonstrate chimeric TTRs bound RBP with a higher strength than wild‐type TTRs, and the changes in the C‐terminal sequence of TTR had a positive effect on its binding affinity for RBP. In addition, changes to the N‐terminal and C‐terminal sequences showed comparable effects on the binding affinity.

Retinol as a cofactor for PKCδ-mediated impairment of insulin sensitivity in a mouse model of diet-induced obesity.

We previously defined that the mitochondria-localized PKCδ signaling complex stimulates the conversion of pyruvate to acetyl-coenzyme A by the pyruvate dehydrogenase complex. We demonstrated in vitro and ex vivo that retinol supplementation enhances ATP synthesis in the presence of the PKCδ signalosome. Here, we tested in vivo if a persistent oversupply of retinol would further impair glucose metabolism in a mouse model of diet-induced insulin resistance. We crossed mice overexpressing human retinol-binding protein (hRBP) under the muscle creatine kinase (MCK) promoter (MCKhRBP) with the PKCδ(-/-) strain to generate mice with a different status of the PKCδ signalosome and retinoid levels. Mice with a functional PKCδ signalosome and elevated retinoid levels (PKCδ(+/+)hRBP) developed the most advanced stage of insulin resistance. In contrast, elevation of retinoid levels in mice with inactive PKCδ did not affect remarkably their metabolism, resulting in phenotypic similarity between PKCδ(-/-)hRBP and PKCδ(-/-) mice. Therefore, in addition to the well-defined role of PKCδ in the etiology of metabolic syndrome, we present a novel PKCδ signaling pathway that requires retinol as a metabolic cofactor and is involved in the regulation of fuel utilization in mitochondria. The distinct role in whole-body energy homeostasis establishes the PKCδ signalosome as a promising target for therapeutic intervention in metabolic disorders.

Wards in the keyway: amino acids with anomalous pK as in calycins

As a follow-up to our recent analysis of the electrostatics of bovine β-lactoglobulin (Eberini et al. in Amino Acids 42:2019-2030, 2011), we investigated whether the occurrence in the native structure of calycins-the superfamily to which β-lactoglobulin belongs-of amino acids with anomalous pK (a)s is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK (a) of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the 'normalization' of anomalous pK (a)s may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.

Effect of the N‐terminal sequence on the binding affinity of transthyretin for human retinol‐binding protein

During vertebrate evolution, the N-terminal region of transthyretin (TTR) subunit has undergone a change in both length and hydropathy. This was previously shown to change the binding affinity for thyroid hormones (THs). However, it was not known whether this change affects other functions of TTR. In the present study, the effect of these changes on the binding of TTR to retinol-binding protein (RBP) was determined. Two wild-type TTRs from human and Crocodylus porosus, and three chimeric TTRs, including a human chimeric TTR in which its N-terminal sequence was changed to that of C. porosus TTR (croc/huTTR) and two C. porosus chimeric TTRs (hu/crocTTR in which its N-terminal sequence was changed to that of human TTR and xeno/crocTTR in which its N-terminal sequence was changed to that of Xenopus laevis TTR), were analyzed for their binding to human RBP by native-PAGE followed by immunoblotting and a chemilluminescence assay. The K(d) of human TTR was 30.41 ± 2.03 μm, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 μm. The binding affinities increased in croc/huTTR (K(d) = 23.57 ± 3.54 μm) and xeno/crocTTR (K(d) = 0.61 ± 0.16 μm) in which their N-termini were longer and more hydrophobic, but decreased in hu/crocTTR (K(d) = 5.03 ± 0.68 μm) in which its N-terminal region was shorter and less hydrophobic. These results suggest an influence of the N-terminal primary structure of TTR on its function as a co-carrier for retinol with RBP.

Quantitative Mass Spectrometry Evaluation of Human Retinol Binding Protein 4 and Related Variants

BackgroundRetinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker.MethodsRBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants.ResultsThe linear range of the assay was 7.81–500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 – 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA = 1.05× ELISA – 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%.ConclusionThe novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker.

Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris

Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2×10−7M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.

Discrete Molecular Dynamics as a Tool to Test Soluble Protein Models

Discrete molecular dynamics (DMD) has been applied in many areas of protein folding and aggregation because relatively long time scales can be simulated. Coarse-graining and implicit solvent models have been implemented to maximize its efficiency. However, there has been no rigorous evaluation with DMD on experimentally known structures. Here, we ran DMD with four-bead peptide model on X-ray structure of a human serum retinol-binding protein and NMR structure of a regulator of G-Protein signaling, and compared the results to those of simulations by NAMD (all atom molecular dynamics with explicit water). DMD showed larger root mean squared deviation (RMSD) from the starting conformation compared to NAMD though tertiary structures were mostly maintained. We developed a new implicit solvent model for DMD based on Miyazawa-Jernigan interaction pair potential that has advantage of unambiguous implementation to address dependencies in implicit solvent models. Results obtained with the Miyazawa-Jernigan implicit solvent model were comparable to those obtained with a previously used implicit solvent model based on Kyte-Doolittle hydropathy scale. We ran DMD and NAMD on incorrectly folded models. Structures of the incorrectly folded models were very poorly preserved during the DMD simulations. Both methods were able to distinguish between the correct and incorrect structures based on differences in the magnitudes of the RMSD from the starting conformation. Hence, we suggest that DMD may be useful as a tool to test soluble protein models.

Two physiological forms of human retinol binding protein.

Journal Article Two Physiological Forms of Human Retinol Binding Protein Get access Nutrition Reviews, Volume 30, Issue 4, April 1972, Pages 90–92, https://doi.org/10.1111/j.1753-4887.1972.tb03997.x Published: 01 April 1972

Retinol binding protein is a major carrier of progesterone in vitro and in human follicular fluid

OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands.DESIGN: Experimental study.MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF.RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex.CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells. OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands. DESIGN: Experimental study. MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF. RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex. CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells.

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase beta/alpha barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution.

An Essential Ligand-binding Domain in the Membrane Receptor for Retinol-binding Protein Revealed by Large-scale Mutagenesis and a Human Polymorphism

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.

Interaction of diethylstilbestrol and ioxynil with transthyretin in chicken serum

The association of suspected endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), ioxynil and pentachlorophenol (PCP), with chicken serum proteins was investigated in relation to thyroid system disruption. All of these chemicals strongly inhibited l-[ 125I]thyroxine ([ 125I]T 4) binding to purified transthyretin (TTR) whereas PCP was less potent inhibitor than DES and ioxynil of [ 125I]T 4 binding to diluted whole chicken serum. This result suggested that PCP interacted with serum proteins other than TTR in whole chicken serum. Following the incubation of chicken serum with each chemical (final concentrations 0.25–1.0 µM), serum proteins were fractionated by gel filtration chromatography (Cellulofine GCL-1000) and affinity chromatography (human retinol-binding protein coupled to Sepharose 4B). Although all chemicals were detected in the gel filtration chromatography 50–100 kDa fractions, DES and ioxynil, but not PCP, were co-eluted with TTR during affinity chromatography. Our results indicated that a significant proportion of DES and ioxynil, but a low proportion of PCP, interacted with TTR in whole chicken serum.

Immunoassay of urinary retinol binding protein as a putative renal marker in cats

The presence of low molecular weight retinol binding protein (RBP) in urine reflects tubular damage. Therefore, RBP has been used as a renal marker in humans and dogs. Using an anti-human RBP antibody (Ab), this study first demonstrates feline urinary RBP by Western blot analysis and then evaluates its potential as a renal marker in cats by enzyme-linked immunosorbent assay (ELISA). Urine was taken by cystocentesis, centrifuged and stored at −80 °C until analysis. Urinary RBP levels were compared in clinically healthy cats (H), chronic renal failure patients (CRF) and cats with hyperthyroidism (HT). The detection of a band at the same position as the human RBP standard with Western blot analysis, indicated that RBP was present in the urine of CRF and HT patients but minimally present in H cats. The data obtained with ELISA were in accordance with these observations. RBP levels were expressed as RBP:creatinine (RBP:c) ratios following normalisation with urinary creatinine. The functional assay sensitivity was 1.37 μg/l RBP. Parallelism between the trend lines of the human RBP standard curve and the curves obtained from sequentially diluted urine samples indicated that feline RBP was recovered. The mean intra-assay coefficient of variance was 7% and the standardised agreement index revealed satisfactory day-to-day repeatability. The RBP:c ratio in all H cats (n=10) was below the assay sensitivity. The groups of CRF and HT patients had increased mean RBP:c ratios of 1.6±0.5×10−2 μg/mg (mean±SEM, n=10) and 1.4±0.4×10−2 μg/mg (n=13), respectively. Both groups showed a large variation in the relative RBP concentrations of individual cats. In conclusion, RBP is demonstrated for the first time in urine from most CRF and HT patients and the validated ELISA allows its evaluation as a putative renal marker in cats.

Biochemical basis for retinol deficiency induced by the I41N and G75D mutations in human plasma retinol-binding protein

Retinol-binding protein (RBP) is the retinol-specific carrier protein present in plasma, where it circulates almost entirely bound to thyroxine-binding transthyretin (TTR). Recently, depressed plasma retinol and RBP levels in carriers of the I41N and G75D RBP point mutations have been reported. We show here that although recombinant human N41 and D75 RBPs can form complexes with retinol and TTR in vitro, the retinol-mutated RBP complexes are significantly less stable than human normal holo-RBP, as revealed by the markedly facilitated retinol release by mutated holo-RBPs to phospholipid membranes, in accordance with the location of mutated residues inside the RBP retinol-binding cavity. Taken together, the data are consistent with the I41N and G75D point mutations being the cause of an altered interaction of retinol with RBP, resulting in a remarkably reduced stability of the retinol–RBP complex, which in turn can lead to the lowering of plasma retinol and RBP levels.

Accumulation of retinol in the liver after prolonged hyporetinolemia in the vitamin A-sufficient rat

We assessed the effects of prolonged reduction of plasma retinol concentrations (hyporetinolemia) on the distribution of tissue vitamin A (VA) and of its active compounds using a model of continuous recombinant human interleukin-6 (rhIL-6) infusion via osmotic minipumps in VA-sufficient male rats. Plasma retinol and retinol-binding protein (RBP) concentrations remained decreased and lower in rhIL-6-treated rats compared with controls from 7.5 h throughout 7 days of infusion (P < 0.001). This reduction was accompanied by a 68% increase in hepatic retinol concentration by 7 days (P < 0.05). Hepatic and renal retinyl palmitate and retinoic acid concentrations did not change, and renal megalin content remained unchanged; hepatic RBP concentrations were 41% lower in rhIL-6-treated rats compared with controls (P < 0.05). These results indicate that instead of being lost, retinol accumulated in the liver during inflammation and that hyporetinolemia was attributable to a decrease in the availability of hepatic RBP. A plausible consequence of the effect of rhIL-6-induced hyporetinolemia is that by 7 days tissues that are dependent on plasma retinol may become deprived of VA. These results have important implications in understanding the mechanism by which measles infection induces hyporetinolemia and VA deficiency of extrahepatic tissues.