Fatty acid binding protein in locust and mammalian muscle. Comparison of structure,function and regulation

The flight muscle of adult desert locusts, Schistocerca gregaria, contains a fatty acid binding protein (FABP) that is homologous to mammalian M-FABP (cardiac FABP). In spite of the evolutionary distance between invertebrates and vertebrates, locust muscle FABP is similar to cardiac FABP in its amino acid sequence, structure, and binding behavior. While cardiac FABP is present already in the prenatal period, locust FABP is an adult specific protein; its expression is directly linked to metamorphosis. A correlation seems to exist between fatty acid oxidative capacity and FABP content in both locust and mammals. To accomplish the higher metabolic rate encountered during migratory flight, locust flight muscle cytosol contains more than three times as much FABP as that in mammalian heart. Increased fatty acid utilization by exercise or endurance training apparently induces FABP expression. Similarities and differences between vertebrate and invertebrate M-FABP are discussed in light of the proposed functions of muscle FABP as fatty acid transporter and cytoprotectant.     

Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding

Intracellular lipid-binding proteins have evolved from a common ancestral gene with the appearance of mitochondrial oxidation, to guarantee, for example, transport of fatty acids through the aqueous cytosol to their site of utilization. The mammalian forms of these lipid carriers are structurally well-characterized and have been categorized, on the basis of sequence similarities and several typical ligand-binding features, into four subfamilies. Only a single complex structure of an invertebrate fatty-acid-binding protein (FABP) has been reported to date, which reveals a unique ligand-binding arrangement yet unknown in vertebrate FABPs. In the present study, the structure of a second invertebrate FABP (locust muscle) complexed with a fatty acid has been determined on the basis of intermolecular NOE connectivities between the protein and the uniformly (13)C-enriched oleate ligand. The resulting ligand conformation, although resembling the closely related mammalian heart- and adipocyte-type FABPs, is characterized by certain binding features that differ significantly from the typical hairpin-turn ligand shapes of the latter forms. This is primarily due to an alanine-to-leucine substitution in locust FABPs that produces a steric hindrance for ligand binding. A comparison with an FABP from tobacco hornworm larvae furthermore demonstrates that certain amino acid substitutions that appear to be specific for invertebrates decidedly influence the binding arrangement inside the protein cavity. Hence, as a result of these evolutionary variations, invertebrate FABPs may display a much greater diversity in intracellular lipid binding than observed for the mammalian transport proteins, thus possibly providing new insights for the design of modified lipid carriers.     

Seasonal dynamics of flight muscle fatty acid binding protein and catabolic enzymes in a migratory shorebird

We developed an ELISA to measure heart-type fatty acid binding protein (H-FABP) in muscles of the western sandpiper (Calidris mauri), a long-distance migrant shorebird. H-FABP accounted for almost 11% of cytosolic protein in the heart. Pectoralis H-FABP levels were highest during migration (10%) and declined to 6% in tropically wintering female sandpipers. Premigratory birds increased body fat, but not pectoralis H-FABP, indicating that endurance flight training may be required to stimulate H-FABP expression. Juveniles making their first migration had lower pectoralis H-FABP than adults, further supporting a role for flight training. Aerobic capacity, measured by citrate synthase activity, and fatty acid oxidation capacity, measured by 3-hydroxyacyl-CoA-dehydrogenase and carnitine palmitoyl transferase activities, did not change during premigration but increased during migration by 6, 12, and 13%, respectively. The greater relative induction of H-FABP (+70%) with migration than of catabolic enzymes suggests that elevated H-FABP is related to the enhancement of uptake of fatty acids from the circulation. Citrate synthase, 3-hydroxyacyl-CoA-dehydrogenase, and carnitine palmitoyl transferase were positively correlated within individuals, suggesting coexpression, but enzyme activities were unrelated to H-FABP levels.     

Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an "open-cap" conformation or a "swivel-back" mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics.     

Solution structure of chicken liver basic fatty acid binding protein

Chicken liver basic fatty acid binding protein (Lb-FABP) belongs to the basic-type fatty acid binding proteins, a novel group of proteins isolated from liver of different non mammalian species whose structure is not known. The structure of Lb-FABP has been solved by ¹H NMR. The overall fold of Lb-FABP, common to the other proteins of the family, consists of ten antiparallel -strands organised in two nearly ortogonal -sheets with two alpha helices closing the protein cavity where small hydrophobic ligands can be bound. The binding specificity of the protein is not known, however, based on the high sequence and structural similarity with an orthologous protein, ileal lipid binding protein, it is suggested that bile acids may be the putative ligands.     

Localization of projectin in locust flight muscle

Projectin is a giant filamentous protein of arthropod striated muscle. By using immunofluorescence microscopy, projectin was shown to span between the I band and the A band in locust (Locusta migratoria) flight muscle sarcomeres. The N- and C-terminal regions of projectin molecules were localized in the I band and A band, respectively. This observation explains the controversial reports of previous studies that projectin is localized either in the I band or in the A band of locust flight muscle sarcomeres. It is also observed that the N-terminal region of projectin is located in the I band of locust leg muscle sarcomeres.     

Fatty-acid-binding protein in locust flight muscle. Developmental changes of expression, concentration and intracellular distribution.

Fatty-acid-binding protein (FABP) from the flight muscle of the locust, Schistocerca gregaria, is similar to mammalian heart FABP in its primary structure and biochemical characteristics. We have studied developmental changes using enzyme-linked immunosorbent assays, RNA hybridization and electron microscopy of immunogold-labeled sections. Locust muscle FABP is the most abundant soluble muscle protein in fully developed adult locusts, comprising 18% of the total cytosolic protein. At the beginning of the adult stage, however, no FABP is detectable. Its concentration rises during the following 10 days, after which it reaches its maximal value. FABP mRNA is present shortly after adult ecdysis; its concentration increases for 10 days, before it diminishes and reaches a constant, low level, probably needed to maintain the established FABP level. The protein is abundant in cytosol and nuclei, but virtually absent in mitochondria.     

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.     

The muscle fatty acid binding protein of spadefoot toad (Scaphiopus couchii)

Fatty acid binding protein was purified from skeletal muscle of the spadefoot toad (Scaphiopus couchii), an estivating species. While estivating, this animal relies on the fatty acid oxidation for energy. Hence we were interested in the behaviour of fatty acid binding protein under conditions of elevated urea (up to 200 mM) and potassium chloride such as exist during estivation. Also we examined whether there were interactions between glycolytic intermediates and the binding ability of the protein. The amount of bound fatty acid (a fluorescence assay using cis-parinarate) was not affected (P < 0.05) by glucose, fructose 6-phosphate or phosphoenolpyruvate at physiological concentrations. By contrast, glucose 6-phosphate increased the amount of bound cis-parinarate but the apparent dissociation constant was not different from the control. Fructose 1,6-bisphosphate but not fructose 2,6-phosphate decreased cis-parinarate binding by 40%, commensurate with doubling the apparent dissociation constant (1.15-2.62 microM). Urea, guanidinium and trimethylamine N-oxide at 200 mM increased cis-parinarate binding 60% over controls. Urea (1 M) and KCl (200 mM) did not affect cis-parinarate binding compared to controls. The interaction of this fatty acid transporter with fructose 1,6-bisphosphate is discussed in terms of reciprocal interaction with phosphofructokinase since fatty acid is also an inhibitor of phosphofructokinase.     

Involvement of cyclic nucleotides in locust flight muscle metabolism

1. Flight had no significant effect on the levels of c-AMP of c-GMP in the flight muscles of Locusta migratoria.2. Injections of 0.01 or 0.1 corpus cardiacum equivalents into the abdominal cavity did not elicit any effect on cyclic nucleotide levels either.3. Injection of A23187 resulted in a decrease of the c-AMP level, but not the c-GMP level.4. Marked increase of the c-AMP level was found to occur in the flight muscles after injection of octopamine, whereas c-GMP level was not influenced.5. It is concluded that octopamine acts on locust flight muscles through c-AMP mediated activation of glycogen phosphorylase.     

The regulation of glycolysis in perfused locust flight muscle

Concentrations of glycolytic intermediates, amino acids and possible regulator substances were measured in extracts from locust thoracic muscles perfused under different conditions. The conversion of [(14)C]glucose into intermediates and CO(2) by muscle preparations was also followed. When muscles perfused with glucose were made anaerobic changes in metabolite concentrations occurred that could be accounted for by an activation of phosphofructokinase and pyruvate kinase. When butyrate and glucose were present in the perfusion medium the rate of glycolytic flux was lower than with glucose alone, and the aldolase reaction appeared to be inhibited. When butyrate alone was supplied to the muscle the concentrations of most glycolytic intermediates were similar to those found when glucose was supplied. Iodoacetate caused changes in concentrations of intermediates that appeared to result from inhibition of glyceraldehyde 3-phosphate dehydrogenase. Fluoroacetate-poisoned muscles showed a high citrate concentration, but no obvious site of inhibition by citrate was apparent in the glycolytic pathway. Mechanisms for control of glycolysis in locust flight muscle are discussed and related to the known properties of isolated enzymes. It is proposed that trehalase, hexokinase, phosphofructokinase, aldolase, and pyruvate kinase may be control enzymes in this tissue.     

Binding properties of Echinococcus granulosus fatty acid binding protein.

EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.     

Liver fatty acid binding protein expression enhances branched-chain fatty acid metabolism

Although liver fatty acid binding protein (L-FABP) is known to enhance uptake and esterification of straight-chain fatty acids such as palmitic acid and oleic acid, its effects on oxidation and further metabolism of branched-chain fatty acids such as phytanic acid are not completely understood. The present data demonstrate for the first time that expression of L-FABP enhanced initial rate and average maximal oxidation of [2,3-3H] phytanic acid 3.5- and 1.5-fold, respectively. This enhancement was not due to increased [2,3-3H] phytanic acid uptake, which was only slightly stimulated (20%) in L-FABP expressing cells after 30 min. Similarly, L-FABP also enhanced the average maximal oxidation of [9,10-3H] palmitic acid 2.2-fold after incubation for 30 min. However, the stimulation of L-FABP on palmitic acid oxidation nearly paralleled its 3.3-fold enhancement of uptake. To determine effects of metabolism on fatty acid uptake, a non-metabolizable fluorescent saturated fatty acid, BODIPY-C16, was examined by laser scanning confocal microscopy (LSCM). L-FABP expression enhanced uptake of BODIPY-C16 1.7-fold demonstrating that L-FABP enhanced saturated fatty acid uptake independent of metabolism. Finally, L-FABP expression did not significantly alter [2,3-3H] phytanic acid esterification, but increased [9,10-3H] palmitic acid esterification 4.5-fold, primarily into phospholipids (3.7-fold) and neutral lipids (9-fold). In summary, L-FABP expression enhanced branched-chain phytanic acid oxidation much more than either its uptake or esterification. These data demonstrate a potential role for L-FABP in the peroxisomal oxidation of branched-chain fatty acids in intact cells.     

Three-dimensional structure of recombinant human muscle fatty acid-binding protein.

The three-dimensional structure of recombinant human muscle fatty acid-binding protein with a bound fatty acid has been solved and refined with x-ray diffraction data to 2.1 A resolution. The refined model has a crystallographic R factor of 19.5% for data between 9.0 and 2.1 A (7243 unique reflections) and root-mean-square deviations in bond length and bond angle of 0.013 A and 2.7 degrees. The protein contains 10 antiparallel beta-strands and two short alpha-helices which are arranged into two approximately orthogonal beta-sheets. Difference electron density maps and a multiple isomorphous derivative electron density map showed the presence of a single bound molecule of a long chain fatty acid within the interior core of the protein. The hydrocarbon tail of the fatty acid was found to be in a "U-shaped" conformation. Seven ordered water molecules were also identified within the interior of the protein in a pocket on the pseudo-si face of the fatty acid's bent hydrocarbon tail. The methylene tail of the fatty acid forms van der Waals interactions with atoms from 13 residues and three ordered waters. The carboxylate of the fatty acid is located in the interior of the protein where it forms hydrogen bonds with the side chains of Tyr128 and Arg126 and two ordered water molecules. A comparison of the three-dimensional structure of human muscle fatty acid-binding protein and rat intestinal fatty acid-binding protein shows strong similarity. Both proteins bind a single fatty acid within their interior cores, but the bound fatty acids are very different in their conformations and interactions. These findings suggest that the intestinal and muscle fatty acid-binding proteins have evolved distinct binding sites in order to satisfy different requirements within the tissues where they are expressed.