Structural and biochemical characterization of the lungfish (Lepidosiren paradoxa) liver basic fatty acid binding protein

Only one fatty acid-binding protein (FABP) from the liver of the lungfish (Lepidosiren paradoxa) was isolated and characterized. The sequence comparison of lungfish FABP with that of the known members of the liver FABP (L-FABP) and liver basic FABP (Lb-FABP) subfamilies indicates that it is more closely related to chicken, iguana, frog, axolotl, catfish, and shark Lb-FABPs than to mammalian and axolotl L-FABPs. Lungfish liver expression of this single Lb-FABP contrasts with the other fish studied so far which coexpress an Lb-FABP with heart-adipocyte and/or intestinal FABP types. The lungfish liver FABP expression pattern resembles that of tetrapods, which only expresses liver type FABPs. Lungfish Lb-FABP is one of the two FABPs reported to have a disulfide bridge. The molecular modeling of lungfish Lb-FABP predicts that nine of the conserved residues of Lb-FABPs are oriented toward the binding cavity, thus suggesting they are related to the protein binding characteristics.     

Structural and biochemical characterization of toad liver fatty acid-binding protein

Two paralogous groups of fatty acid-binding proteins (FABPs) have been described in vertebrate liver: liver FABP (L-FABP) type, extensively characterized in mammals, and liver basic FABP (Lb-FABP) found in fish, amphibians, reptiles, and birds. We describe here the toad Lb-FABP complete amino acid sequence, its X-ray structure to 2.5 A resolution, ligand-binding properties, and mechanism of fatty acid transfer to phospholipid membranes. Alignment of the amino acid sequence of toad Lb-FABP with known L-FABPs and Lb-FABPs shows that it is more closely related to the other Lb-FABPs. Toad Lb-FABP conserves the 12 characteristic residues present in all Lb-FABPs and absent in L-FABPs and presents the canonical fold characteristic of all the members of this protein family. Eight out of the 12 conserved residues point to the lipid-binding cavity of the molecule. In contrast, most of the 25 L-FABP conserved residues are in clusters on the surface of the molecule. The helix-turn-helix motif shows both a negative and positive electrostatic potential surface as in rat L-FABP, and in contrast with the other FABP types. The mechanism of anthroyloxy-labeled fatty acids transfer from Lb-FABP to phospholipid membranes occurs by a diffusion-mediated process, as previously shown for L-FABP, but the rate of transfer is 1 order of magnitude faster. Toad Lb-FABP can bind two cis-parinaric acid molecules but only one trans-parinaric acid molecule while L-FABP binds two molecules of both parinaric acid isomers. Although toad Lb-FABP shares with L-FABP a broad ligand-binding specificity, the relative affinity is different.     

鸡L-FABP基因全长cDNA克隆表达及与杂种鸡脂肪沉积的关系

肝脏脂肪酸结合蛋白(L-FABP)与脂肪运输及沉积关系密切。文章以8周龄丝羽乌鸡(CC)、农大褐蛋鸡(DD)及其正反杂交组合鸡(CD和DC)为试验材料,利用mRNA差异显示技术,在肝脏组织中获得一条阳性差异表达片段。通过片段回收、测序及序列比对发现,该差异表达片段为鸡L-FABP基因的全长cDNA编码序列(NCBI登录号:AY321365)。Northern杂交和半定量RT-PCR结果显示,该基因在正反杂交组合CD及DC的肝脏组织中表达量均明显高于亲本CC和DD,与杂种鸡的高腹脂及较大肌间脂宽表型趋势相同。鉴于L-FABP基因的高表达可能导致杂种鸡的脂肪沉积高于亲本,有必要针对鸡L-FABP基因进行深入的功能研究。     

Evolutionary diversification of the avian fatty acid-binding proteins

Phylogenetic analysis of avian and other vertebrate fatty acid binding proteins (FABPs) supported the hypothesis that several gene duplications within this family occurred prior to the most recent common ancestor (MRCA) of tetrapods and bony fishes. The chicken genome encodes two liver-expressed FABPs: (1) L-FABP or FABP1; and (2) Lb-FABP. We propose that the latter be designated FABP10, because in our phylogenetic analysis it clustered with zebrafish FABP10. Bioinformatic analysis of across-tissue gene expression patterns in the chicken showed some congruence with phylogenetic relationships. On the basis of expression, chicken FABP genes seemed to form two major groups: (1) a cluster of genes many of which showed predominant expression in the digestive system (FABP1, FABP2, FABP6, FABP10, RBP1, and CRABP1); and (2) a cluster of genes most of which had predominant expression in tissues other than those of the digestive system, including muscle and the central nervous system (FABP3, FABP4, FABP5, FABP7, and PMP2). Since these clusters corresponded to major clusters in the phylogenetic tree as well, it seems a plausible hypothesis that the earliest duplication in the vertebrate FABP family led to the divergence of a gut-specialized gene from a gene expressed mainly in nervous and muscular systems. Data on gene expression in livers of two lines of chickens selected for high growth and low growth showed differences between FABP1 and FABP10 expressions in the liver, supporting the hypothesis of functional divergence between the two chicken liver-expressed FABPs related to food intake.     

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

Cloning and Tissue Expression of Chicken Heart Fatty Acid-Binding Protein and Intestine Fatty Acid-Binding Protein Genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

Cloning and tissue expression of chicken heart fatty acid-binding protein and intestine fatty acid-binding protein genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

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.     

Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Altered feeding differentially regulates circadian rhythms and energy metabolism in liver and muscle of rats

Energy metabolism follows a diurnal pattern responding to the light/dark cycle and food availability. This study investigated the impact of restricting feeding to the daylight hours and feeding a high fat diet on circadian clock (bmal1, dbp, tef and e4bp4) and metabolic (pepck, fas, ucp3, pdk4) gene expression and markers of energy metabolism in muscle and liver of rats. The results show that in chow-fed rats switched to daylight feeding, the peak diurnal expression of genes in liver was shifted by 6–12 h while expression of these genes in muscle remained in a similar phase to rats feeding ad libitum. High fat feeding during the daylight hours had limited effect on clock gene expression in liver or muscle but shifted the peak expression of metabolic genes (pepck, fas) in liver by 6–12 h. The differential effects of daylight feeding on gene and protein expression in muscle and liver were accompanied by an 8% reduction in whole body energy expenditure, a 20–30% increased glycogen content during the light phase in muscle of day-fed rats and increased adipose tissue deposition per gram food consumed. These data demonstrate that a mismatch of feeding and light/dark cycle disrupts tissue metabolism in muscle with significant consequences for whole body energy homeostasis.     

Isolation, characterization and binding properties of two rat liver fatty acid-binding protein isoforms

Mammalian liver has only one fatty acid-binding protein (L-FABP) while the liver of non-mammalian vertebrates expresses a liver basic FABP (Lb-FABP) in addition to other members of the FABP family. We explore the possibility that L-FABP isoforms accomplish, in the liver of mammals, the metabolic functions corresponding to the different FABPs present in the liver of non-mammalian vertebrates. We have isolated isoforms I and II which have a different residue 105, Asn in the former and Asp in the latter. We made a conformational comparison of the apo-isoforms by intrinsic fluorescence emission and fourth-derivative spectroscopy, native-state proteolysis and unfolding curves. Ligand affinity was studied by measuring cis-parinaric acid displacement by different ligands. They have differences in their molecular conformation, including the environment of the binding site. Isoform II has probably a more open conformation than isoform I, thus allowing the binding of a greater variety of ligands. The affinity of isoform II for lysophospholipids, prostaglandins, retinoids, bilirubin and bile salts is greater than that of isoform I. These characteristics of rat L-FABP isoforms I and II suggest that they may accomplish different functions as happens with those of the different FABP types in non-mammalian species.     

Effects of linoleic and gamma-linolenic acids (efamol evening primrose oil) on fatty acid-binding proteins of rat liver

Summary We have studied the effects of Efamol evening primrose oil (EPO) on fatty acid-binding proteins (L-FABP) of rat liver. EPO contains 72% cis-linoleic acid and 9% cis-gamma linolenic acid. EPO has been clinically used for treatment of a number of diseases in humans and animals. EPO is also known to lower cholesterol level in humans and animals. Feeding of an EPO supplemented diet to rats (n = 9) for 2 months decreases the oleate binding capacity of purified L-FABP of rat liver whereas the palmitate binding activity was increased by 38%. However, EPO feeding did not alter the L-FABP concentrations significantly as measured by using the fluorescence fatty acid probe, dansylamino undecanoic acid. Endogenous fatty acid analysis of L-FABPs revealed significant qualititative and quantitative changes in fatty acid pattern after EPO feeding. EPO feeding decreased the endogenous palmitate level by 53% and oleate level by 64% in L-FABPs and also EPO feeding decreased the total endogenous fatty acid content from 62 nanomole per mg of protein to 42 nanomole per mg of L-FABP (n = 3).     

Crystal structure of chicken liver basic fatty acid-binding protein complexed with cholic acid

Two paralogous groups of liver fatty acid-binding proteins (FABPs) have been described: the mammalian type liver FABPs and the basic type (Lb-FABPs) characterized in several vertebrates but not in mammals. The two groups have similar sequences and share a highly conserved three-dimensional structure, but their specificity and stoichiometry of binding are different. The crystal structure of chicken Lb-FABP complexed with cholic acid and that of the apoprotein refined to 2.0 A resolution are presented in this paper. The two forms of the protein crystallize in different space groups, and significant changes are observed between the two conformations. The holoprotein binds two molecules of cholate in the interior cavity, and the contacts observed between the two ligands can help to explain the reason for this stoichiometry of binding. Most of the amino acids involved in ligand binding are conserved in other members of the Lb-FABP family. Since the amino acid sequence of the Lb-FABPs is more similar to that of the bile acid-binding proteins than to that of the L-FABPs, the possibility that the Lb-FABPs might be more appropriately called liver bile acid-binding proteins (L-BABPs) is suggested.     

Localization of liver fatty acid-binding protein and its mRNA in the liver and jejunum of rats: an immunohistochemical and in situ hybridization study

Summary The localization of liver fatty acid-binding protein (L-FABP) and its mRNA in the liver and jejunum was examined in normal and 3-day-fasted rats by means of immunohistochemistry using a specific antibody to L-FABP and in situ hybridization using a synthetic oligonucleotide complementary to L-FABP mRNA as probe. In the liver from normally fed rats, the signal for L-FABP mRNA in hepatocytes was distributed throughout the lobule, with higher intensity in the periportal than in the centrolobular region. After a 3-d fasting, the mRNA signal declined in intensity throughout the lobule, in accordance with the result of Northern blot analysis. Immunohistochemistry for L-FABP showed intralobular patterns of immunoreactivity similar to those of the mRNA signal in both fed and fasted animals. In the jejunum from fed rats, L-FABP-mRNA signal was abundant in the absorptive epithelial cells lining the lower two-thirds of villus and less abundant in the villus tip cells, while the intensity of L-FABP immunoreactivity remained high in the latter cells. Fasting brought about a downward shift of the mRNA signal to an area including the upper half of the crypt and the lower portions of villus, with decreased intensity in the rest of the villus. Immunohistochemistry also showed a downward extension of the immunoreactivity into the upper crypt area. The present results suggest that in situ hybridization is a useful tool to analyze regulations of the expression of L-FABP gene in the digestive organs in association with epithelial cell migration and dietary condition.     

Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies

Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that the parinaric acid microenvironment in the fatty acid-binding site of I-FABP was much less polar than that of L-FABP. Parinaric acids displayed similar rotational correlation time and limiting anisotropy when bound to I-FABP and to L-FABP. These results are consistent with a close proximity of bound fatty acids to the tyrosine and tryptophan residues and with immobilization of the polyene fatty acids in the fatty acid-binding site(s) of L-FABP and I-FABP. The two proteins differ in that only L-FABP has two fatty acid-binding sites and appears to undergo significant conformational change upon fatty acid binding.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Characterization of a BODIPY-labeled {fl}uorescent fatty acid analogue. Binding to fatty acid-binding proteins, intracellular localization, and metabolism

The BODIPY-labeled fatty acid analogues are a useful addition to the tools employed to study the cellular uptake and metabolism of lipids. In this study, we show that BODIPY FL C₁₆ binds to purified liver and intestinal fatty acid-binding proteins with high affinity at a site similar to that for the physiological fatty acid oleic acid. Further, in human intestinal Caco-2 cells BODIPY FL C₁₆ co-localizes extensively with mitochondria, endoplasmic reticulum/Golgi, and L-FABP. Virtually no esterification of BODIPY FL C₁₆ was observed under the experimental conditions employed. We conclude that BODIPY FL C₁₆ may be a useful tool for studying the distribution and function of FABPs in a cellular environment.     

Fatty acid-binding protein expression in the liver: its regulation and relationship to the zonation of fatty acid metabolism

Summary Liver fatty acid-binding protein (L-FABP) is expressed in a declining gradient between the portal and central zones of the liver acinus. This paper discusses the results of experimental studies which address the questions: (a) What factors regulate L-FABP expression in liver and produce its acinar gradient? (b) What is the relationship between the acinar gradient of L-FABP and acinar gradients in the transport and metabolism of long-chain fatty acids? Both high-fat diets and clofibrate-treatment increase L-FABP proportionally at both extremes of the liver acinus and the small intestine, with preservation of the L-FABP gradient in both tissues. Female rats differ from males, however, in showing a greater hepatic abundance of L-FABP which is expressed almost equally throughout the acinus. Dietary studies show that L-FABP is induced with increased fatty acid flux derived from dietary fat but not from de novo hepatic fatty acid synthesis. Studies of the synthesis and utilization of fatty acids by hepatocytes isolated from the periportal and pericentral zones of the liver acinus suggest that the acinar gradient of L-FABP is not associated with differences in the instrinsic capacity of zone 1 and zone 3 hepatocytes to utilize or synthesize fatty acids. In addition, studies of the acinar uptake pattern of a fluorescent fatty acid derivative by isolated perfused livers indicate that the acinar distribution of L-FABP does not determine the pattern of fatty acid uptake in the intact acinus. Rather, the acinar gradient of L-FABP is most likely to represent a response to physiological conditions existing in the intact acinus which may include gradients in the flux of fatty acids, fatty acid metabolites and hormones.Abbreviations ALT Alanine Aminotransferase - FABP Fatty Acid Binding Protein - I-FABP Intestinal-type Fatty Acid Binding Protein - L-FABP Liver-type Fatty Acid Binding Protein - 12-NBD-stearate 12-(N-methyl)-N-(7-nitrobenzo-2-oxa-1, 3,-diazol-4-yl)amino)-octadecanoic acid