Basic Fatty Acid Binding Protein Research Articles

Polycyclic Aromatic Hydrocarbons Alter the Hepatic Expression of Genes Involved in Sanderling (Calidris alba) Pre-migratory Fueling.

Polycyclic aromatic hydrocarbons (PAHs) impaired pre-migratory fueling in 49 orally dosed Sanderling (Calidris alba). In the present study, 8 genes related to fat deposition and PAH exposure were measured in liver subsamples from these same shorebirds. At the highest dose (1260 µg total PAH [tPAH]/kg body wt/day), PAH exposure decreased liver basic fatty acid binding protein 1 (Lbfabp) and hepatic lipase (Lipc) expression. The present study reveals candidate molecular-level pathways for observed avian pre-migratory refueling impairment. Environ Toxicol Chem 2021;40:1983-1991. © 2021 SETAC.

A comparative study of 3 alternative avian toxicity testing methods: Effects on hepatic gene expression in the chicken embryo.

There is growing interest in developing alternative methods to screen and prioritize chemical hazards, although few studies have compared responses across different methods. The objective of the present study was to compare 3 alternative liver methods derived from white Leghorn chicken (Gallus gallus domesticus): primary hepatocyte culture, liver slices, and liver from in ovo injected embryos. We examined hepatic gene expression changes after exposure to 3 chemicals (17β-trenbolone [17βT], 17β-estradiol [E2], and 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) using a custom quantitative polymerase chain reaction (qPCR) array with 7 genes (vitellogenin [VTG], apolipoprotein [Apo], cytochrome P450 1A4 [CYP1A4], liver basic fatty acid binding protein [LBFABP], 3β hydroxysteroid dehydrogenase [HSD3β1], stearoyl coenzyme A desaturase [SCD], and estrogen sulfotransferase [SULT1E1]). Gene expression across the 3 methods was examined using hierarchical clustering. Up-regulation of CYP1A4 in response to TCDD was consistent across all methods, and the magnitude was higher in hepatocytes (>150-fold) compared with slices (>31-fold) and in ovo liver (>27-fold). In hepatocytes, SCD and VTG up-regulation in response to 17βT and E2 was >4-fold and 16-fold, respectively. The rank order of cases with significant changes in gene expression among the 3 methods was: hepatocytes (22) > in ovo liver (11) > liver slices (6). Hierarchical clustering grouped liver slices and in ovo liver as more similar, whereas hepatocytes were grouped separately from in ovo liver. More introspective comparisons are needed to understand how and why alternative methods differ and to aid in their integration into toxicity testing. Environ Toxicol Chem 2019;38:2546-2555. © 2019 SETAC.

An acyl-CoA-binding protein (FcACBP) and a fatty acid binding protein (FcFABP) respond to microbial infection in Chinese white shrimp, Fenneropenaeus chinensis

Acyl-CoA-binding protein (ACBP) and fatty acid-binding protein (FABP) are involved in lipid metabolism. ACBP plays a key role in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, vesicular trafficking, and gene regulation. In our study, a 536 bp cDNA of ACBP ( FcACBP) was cloned and identified as a widely distributed gene in the Chinese white shrimp, Fenneropenaeus chinensis. Its expression in intestine was upregulated in response to white spot syndrome virus (WSSV) or Vibrio anguillarum infection. The expression patterns were confirmed by Western blot analysis. FABPs, members of the lipid-binding protein superfamily, play an important role in lipid metabolism and also participate in vertebrate innate immunity. A cDNA of FABP ( FcFABP) cloned from the hepatopancreas of the shrimp was 715 bp in size and encoded a 14 kDa protein. FcFABP appeared to be a basic fatty acid binding protein with a predicted isoelectric point of 9.16. It showed sequence similarity to both vertebrate and invertebrate FABPs. Phylogenetic analysis showed that FcFABP, together with LvFABP, were clustered into one group. FcFABP was detected mainly in the hepatopancreas and expression level increased after a challenge with WSSV. FcFABP was down-regulated by V. anguillarum challenge. The protein also had bacterial binding activity. These two lipid metabolism related proteins may play important roles in shrimp innate immunity.

Characterization of critical factors influencing gene expression of two types of fatty acid-binding proteins (L-FABP and Lb-FABP) in the liver of birds

In avian species, two types of intracellular lipid-binding proteins are abundant in the liver, the liver fatty acid-binding protein (L-FABP) and the liver basic fatty acid-binding protein (Lb-FABP). Both FABPs are capable of forming complexes with free fatty acids and bile acids, but the functional distinction between L-FABP and Lb-FABP in avian liver is not fully understood. To gain insights into the functional distinction between L-FABP and Lb-FABP, we investigated the expression of both genes in relation to the pre- and post-hatching development, diurnal cycle and feeding state in the livers of chicken ( Gallus gallus) and Japanese quail ( Coturnix japonica). In chickens, the Lb-FABP mRNA was expressed only in the liver, while the L-FABP was expressed in both liver and intestinal tissues. Only small amounts of the L-FABP and Lb-FABP mRNAs were detected in the liver during chicken embryogenesis, but at the onset of hatching a dramatic increase in mRNA expression was observed for both genes, suggesting that the expression of the L-FABP and Lb-FABP genes is synchronized at developmental stages. Remarkably, the diurnal expression pattern differed between the two genes under a 16L:8D condition in sexually mature quail: L-FABP gene expression transiently increased at the end of the light cycle, whereas Lb-FABP gene expression peaked during the early part of the light cycle and gradually decreased as the dark period approached. We attempted to identify the factors regulating the diurnal gene expression pattern, and found that feeding stimulation was a critical factor inducing Lb-FABP gene expression irrespective of light condition. On the other hand, feeding stimulation only slightly stimulated expression of the L-FABP gene, and was not always its primary determinant. These results suggest that L-FABP and Lb-FABP play different roles in metabolic process during the postprandial state.

Proteomic study of the effects of complex environmental stresses in the livers of goldfish (Carassius auratus) that inhabit Gaobeidian Lake in Beijing, China

Recent advances in proteomics have provided an excellent opportunity to understand biological adaptation under complex environmental stress at the protein level. Gaobeidian Lake, located in Beijing, China, is characterized by complex environmental stresses by serving as both the effluent of a wastewater treatment plant and a coolant of a nearby thermal power plant. Liver is the primary organ of energy metabolism and xenobiotic detoxification. To further our understanding of how organisms that live in Gaobeidian Lake acclimatize themselves to these complex environmental stresses, hepatic protein expression patterns were examined in goldfish Carassius auratus that inhabit the lake. Huairou Reservoir, a drinking water source, was used as a reference site. Twenty four protein spots, which were differently expressed in the two sites, were further digested with trypsin and analyzed by matrix-assisted laser desorption/ionization (MALDI) tandem time of flight mass spectrometry (TOF/TOF). The expression of several energy metabolism and oxidative stress proteins, such as glutathione peroxidase (GPx), ferritin H3, and liver basic fatty acid-binding protein (Lb-FABP) were found to be altered in this stressful environment. In addition to the up-regulation of GPx translation, both the mRNA levels and enzymatic activity of GPx protein were elevated in goldfish living in Gaobeidian Lake. The expression of both peroxisome proliferator activated receptor (PPAR), one of the most important metabolism and stress regulation genes as well as cytochrome P450 1A1 (CYP1A1), a detoxification gene, was also detected by real-time PCR at the two sites. Increased expression levels of both PPAR-beta and CYP1A1 (P < 0.1) were observed in Gaobeidian Lake. Our study provides an integrative view of the expression levels of hepatic proteins and genes in goldfish under complex environmental stress that live in Gaobeidian Lake. Our results showed that anthropogenic environmental stresses in Gaobeidian Lake activated the regulation gene of lipid metabolism PPAR, elevated the lipid metabolism levels, and activated the anti-oxidative adaptation mechanism of organisms in the lake.

Basic liver-type fatty acid binding protein in rainbow trout Oncorhynchus mykiss: sequence and gene expression in tissue

Basic liver-type fatty acid binding protein in rainbow trout Oncorhynchus mykiss: sequence and gene expression in tissue

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid‐binding protein bound to cholic and oleic acid

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent.

Determinants of protein stability and folding: Comparative analysis of beta‐lactoglobulins and liver basic fatty acid binding protein

A new energy decomposition approach, aimed at identifying residues playing a folding key role, has been applied here to three homologous proteins, belonging to the calycin superfamily, namely bovine and porcine beta-lactoglobulins and Liver basic fatty acid binding protein, sharing the same beta-barrel fold and different degree of sequence identities. All-atom, explicit solvent molecular dynamics simulations around the native conformation were used to generate, for each of the three proteins, energy maps which were further simplified through eigenvalue decomposition. Analysis of the components of the eigenvector associated with the lowest eigenvalue singled out those residues (hot sites) behaving as strongly interacting and possible nucleation centers. The results fit well with experimental folding data and, especially, with the analysis of side chain-side chain interaction conservation.

Chicken Liver Bile Acid-Binding Protein Is in a Compact Partly Folded State at Acidic pH. Its Relevance to the Interaction with Lipid Membranes

Chicken liver bile acid-binding protein (formerly known as chicken liver basic fatty acid-binding protein) binds to anionic lipid membranes acquiring a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B., and Montich, G. (2003) Biochim. Biophys. Acta 1611, 98-106]. To understand the mechanisms of its interactions with membranes, we have investigated the presence of partly folded states in solution. Using fluorescence spectroscopy of the single Trp residue, circular dichroism in the far- and near-UV, Fourier transform infrared spectroscopy, and size-exclusion chromatography, we found that L-BABP was partly unfolded at pH 2.5 and low ionic strength, retaining some of its secondary structure. Addition of 0.1 M NaCl at pH 2.5 or decreasing the pH to 1.5 produced a more compact partly folded state, with a partial increase of secondary structure and none of tertiary structure. Fluorescence emission spectra of this state indicate that the Trp residue is within an environment of low polarity, similar to the native state. This environment is not produced by the insertion of the Trp into soluble aggregates as revealed by size-exclusion chromatography, fluorescence anisotropy, and infrared spectroscopy. The presence of partly folded states under acidic conditions in solution suggests the possibility that membrane binding of L-BABP occurs via this state.

Interactions of chicken liver basic fatty acid-binding protein with lipid membranes

The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with large unilamellar vesicles (LUVs) of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) were studied by binding assays, Fourier transform infrared (FT-IR) spectroscopy, monolayers at air–water interface, and low-angle X-ray diffraction. Lb-FABP binds to POPG LUVs at low ionic strength but not at 0.1 M NaCl. The infrared (IR) spectra of the POPG membrane-bound protein showed a decrease of the band corresponding to β-structures as compared to the protein in solution. In addition, a cooperative decrease of the β-edge band above 70 °C in solution was also evident, while the transition was less cooperative and took place at lower temperature for the POPG membrane-bound protein. Low- and wide-angle X-ray diffraction experiments with lipid multilayers indicate that binding of the protein produces a rearrangement of the membrane structure, increasing the interlamellar spacing and decreasing the compactness of the lipids.

Crystallization and preliminary X-ray study of two liver basic fatty acid-binding proteins.

The fatty acid-binding proteins (FABPs) are a very well known protein family which includes the liver basic FABPs (Lb-FABPs), a subgroup so far characterized in several vertebrates but not in mammals. The most important difference recognized between the proteins in this subgroup and the better known mammalian liver FABPs (L-FABPs) is the stoichiometry of ligand binding: two fatty acid molecules in L-FABPs compared with one in Lb-FABPs. The only Lb-FABP with a known three-dimensional structure is that of chicken Lb-FABP, but the details of ligand binding are still unresolved as the crystals of the protein are grown at an acidic pH and the protein has been shown to lose its ligand under these conditions. The two proteins whose crystallizations are reported here are the second and third members of this subfamily to be crystallized. The crystals of axolotl Lb-FABP belong to either space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 65.38, c = 60.90 A, and diffract to a resolution of 2.0 A on a conventional source at room temperature. The crystals of toad Lb-FABP belong to either space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 48.14, c = 135.23 A, and diffract to 2.5 A resolution under the same conditions. It is expected that the solution of these two structures will help to clarify the structural differences between Lb-FABPs and L-FABPs and will possibly explain the different binding stoichiometries observed in these otherwise so similar protein subfamilies.

Interaction of chicken liver basic fatty acid-binding protein with fatty acids: a 13C NMR and fluorescence study.

Two different groups of liver fatty acid-binding proteins (L-FABPs) are known: the mammalian type and the basic type. Very few members of this second group of L-FABPs have been characterized and studied, whereas most of the past studies were concerned with the mammalian type. The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with 1-(13)C-enriched palmitic acid (PA) and oleic acid (OA) were investigated by (13)C NMR spectroscopy. Samples containing fatty acids (FA) and Lb-FABP at different molar ratios exhibited only a single carboxylate resonance corresponding to bound FA, and showed a binding stoichiometry of 1:1 both for PA and for OA. Fluorescence spectroscopy measurements yielded the same binding stoichiometry for the interaction with cis-parinaric acid [K(d) = 0.38(4) microM]. Competition studies between cis-parinaric acid and the natural ligands indicated a decreasing affinity of chicken Lb-FABP for PA, OA, and retinoic acid (RA). (13)C NMR proved that pH and ionic strength affect complex stability. The carboxyl signal intensity reversibly decreased upon lowering the pH up to 5. The pH dependence of the bound carboxyl chemical shift yielded an apparent pK(a) of 4.8. A decrease of the integrated intensity of the bound carboxylic signal in the NMR spectra was observed while increasing the chloride ion concentration up to 200 mM. This body of evidence indicates that the bound FA is completely ionized at pH 7.4, that its polar head is positioned in a solvent-accessible region, that a FA-protein strong ionic bond is not present, and that high ionic strength causes the release of the bound FA. The reported results show that, insofar as the number of bound ligands and its relative affinity for different FAs are concerned, chicken Lb-FABP is remarkably different from the mammalian liver FABPs, and, within its subfamily, that it is more similar to catfish Lb-FABP while it behaves quite differently from shark or axolotl Lb-FABPs.

Properties of a stationary phase based on immobilised chicken liver basic fatty acid-binding protein

The fatty acid-binding proteins (FABPs) are a class of low-molecular-mass proteins that bind fatty acids and are thought to be involved in their intracellular transport. FABPs have been isolated and studied from several tissues, but their precise function and mechanism of action are still not clear. Chicken liver (basic) fatty acid-binding protein (bFABP) was immobilised on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to bFABP. The retention and enantioselectivity of the new column for a large number of chiral drugs was investigated. The enantiomers of basic and neutral compounds were poorly retained and not resolved by the bFABP column. On the contrary the resolution of the enantiomers of some acidic compounds was obtained. Therefore the influence of the mobile phase pH and organic modifier on the chromatographic performance of acidic compounds was studied. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short-chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure–retention relationship correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results.

Crystallization of chicken liver (basic) fatty acid binding protein after purification in multicompartment electrolyzers with isoelectric membranes.

A preparation of chicken liver (basic) fatty acid binding protein was purified to homogeneity in multicompartment electrolyzers with isoelectric membranes. Large amounts of the isoelectric point (pI) 9.7 protein were collected into a compartment delimited by pI 8.8 and 11.0 membranes. The protein thus purified produced crystals which diffract to higher resolution than those obtained by purification via preparative isoelectric focusing (IEF) in soluble carrier ampholytes. In addition, a novel orthorhombic form with a different molecular packing was obtained. It is hypothesized that, when using conventional IEF, traces of carrier ampholytes could adhere to the protein, particularly in the hydrophobic ligand-binding pocket, rendering the interpretation of the electron density maps difficult. Multicompartment electrolyzers do not present this drawback, since they are based on insoluble buffering species.

Presence of two new fatty acid binding proteins in catfish liver.

A basic fatty acid binding protein (FABP), closely related to that of chicken liver, was isolated and characterized from catfish (Rhamdia sapo) liver in a previous work. Results herein show the presence of another two FABPs in which partial amino acid sequences reveal great similarity with the corresponding sequences of other already known FABPs belonging to the heart type. The purification procedures for both proteins involve gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (as a last step). Because both FABP N-termini were blocked, they were submitted to in-gel tryptic digestion and the resulting peptides were separated by high performance liquid chromatography, and sequenced by Edman degradation. One of these proteins presented the highest identity percentage when compared with those of the human and bovine heart and bovine brain (81%), and the other when compared with those of chicken retina (75%) and mouse and bovine heart FABP (70%). The presence of several FABPs plus the fact that they belong to different types, as found in the Rhamdia sapo liver, is unusual in mammals, which express a characteristic liver-type member of this protein family.

The primary structure of a basic (pI 9.0) fatty acid-binding protein from liver of Gallus domesticus

The complete amino acid sequence of a basic (pI 9.0) fatty acid-binding protein purified from liver of Gallus domesticus was determined by automated Edman degradation of tryptic, CNBr/HFBA and Staphylococcus aureus protease peptides. The protein contains 125 amino acid residues which correspond to a molecular mass of 14094. The identification of the blocked N-terminus Ac-Ala required digestion of a SV-8 peptide with the acylamino acid-releasing enzyme prior to sequence analysis. Sequence comparison shows that chicken liver basic-FABP has a significant similarity to other proteins belonging to the superfamily of intracellular lipid molecule binding proteins. Moreover, these sequence data confirm that basic-FABP probably binds its substrate in a slightly different way when compared with other FABPs. Basic-FABP was submitted to the EMBL Data Library with an accession number of P80226

Conformational and binding properties of chicken liver basic fatty acid binding protein in solution

The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained KD = (1.0 +/- 0.1) x 10(-6) M in a 0.9: 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile-water, ethylene glycol-water, and dioxane-water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1:9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of alpha-helix.

Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 � resolution

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 A resolution by X-ray crystallography. Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built. This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32. The protein is structured as a compact 10-stranded-beta-barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule. The NH2-terminus portion of the molecule contains two short alpha-helices. The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods.

Chicken liver basic fatty acid-binding protein (p I = 9.0) Purification, crystallization and preliminary X-ray data

Chicken liver basic fatty acid-binding protein (p I = 9.0) has been purified with a high yield by a modification of a method originally applied to rat liver. The final product is highly homogeneous and can be used to grow crystals that belong to two different space groups. The crystals are either tetragonal, space group P4 22 12 with a = b = 60.2 Å and c = 138.1 Å or orthorhombic, space group P2 12 12 1 with a = 60.7 Å, b = 40.1 Å and c = 66.7 Å. The second form appears to be more suitable for X-ray diffraction studies, it diffracts to at least 2.8 Å resolution and it is believed to contain one protein molecule in the crystallographic asymmetric unit.

Purification and binding characteristics of a basic fatty acid binding protein from Avena sativa seedlings

A fatty acid binding protein has been purified from etiolated Avena sativa seedlings. The successive steps of purification comprised heat denaturation (50°C), (NH 4) 2SO 4 fractionation, gel filtration on Sephacryl S-200, CM-chromatography, and preparative isoelectric focusing. The homogeneity of the protein was ascertained by electrophoretic titration and SDS-polyacrylamide gel electrophoresis. The protein has an isoelectric point of 8.4 and an apparent molecular weight of 8700 ± 500. The purified protein is capable of binding long-chain fatty acids and their CoA-esters. A high affinity ( K d 3.5 · 10 −6 M) of oleic acid for the basic protein was found by independent measurements of association and dissociation kinetics.