Amino acid sequence and some ligand binding properties of fatty acid-binding protein from bovine brain

Summary A fatty acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified protein migrated as a single protein band in 15% polyacrylamide gel electrophoresis with an apparent molecular mass of 14.7 kDa. To ascertain that the purified protein was a FABP, it was submitted to fatty acid-binding tests. Oleic and palmitic acids bound to brain FABP but this was not the case for palmitoyl CoA. By Scatchard analysis the ligand binding values were: Kd = 0.28 µM, Bmax (mol/mol) = 0.6 for oleic acid and Kd = 0.8 µM, Bmax (mol/mol) = 2.1 for palmitic acid. The complete amino acid sequence of the brain FABP was determined and a microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP.This paper corresponds to a communication at the first international workshop on fatty acid binding proteins (Maastricht, the Netherlands, September 4–5, 1989).     

Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.     

Role of fatty acid-binding protein in cardiac fatty acid oxidation

Summary Although abundant in most biological tissues and chemically well characterized, the fatty acid-binding protein (FABP) was until recently in search of a function. Because of its strong affinity for long chain fatty acids and its cytoplasmic origin, this protein was repeatedly claimed in the literature to be the transcytoplasmic fatty acid carrier. However, techniques to visualize and quantify the movements of molecules in the cytoplasm are still in their infancy. Consequently the carrier function of FABP remains somewhat speculative. However, FABP binds not only fatty acids but also their CoA and carnitine derivatives, two typical molecules of mitochondrial origin. Moreover, it has been demonstrated and confirmed that FABP is not exclusively cytoplasmic, but also mitochondrial. A function for FABP in the mitochondrial metabolism of fatty acids plus CoA and carnitine derivatives would therefore be anticpated. Using spin-labelling techniques, we present here evidence that FABP is a powerful regulator of acylcarnitine flux entering the mitochondrial -oxidative system. In this perspective FABP appears to be an active link between the cytoplasm and the mitochondria, regulating the energy made available to the cell. This active participation of FABP is shown to be the consequence of its gradient-like distribution in the cardiac cell, and also of the coexistence of multispecies of this protein produced by self-aggregation.     

Tryptophan insertion mutagenesis of liver fatty acid-binding protein: L28W mutant provides important insights into ligand binding and physiological function

Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell.     

Fatty acid binding sites of rodent adipocyte and heart fatty acid binding proteins: characterization using fluorescent fatty acids

Murine adipocyte and rat heart fatty acid binding proteins (FABP) are closely related members of a family of cytosolic proteins which bind long-chain free fatty acids (ffa). The physical and chemical characteristics of the fatty acid binding sites of these proteins were studied using a series of fluorescent analogues of stearic acid (18:0) with an anthracene moiety covalently attached at seven different positions along the length of the hydrocarbon chain (AOffa). Previously, we used these probes to investigate the binding site of rat liver FABP (L-FABP) [Storch et al. (1989) J. Biol. Chem. 264, 8708-8713]. Here we extend those studies to adipocyte and heart FABP, two members of the FABP family which share a high degree of sequence homology with each other (62% identity) but which are less homologous with L-FABP (approximately 30%). The results show that the fluorescence emission spectra of AOffa bound to adipocyte FABP (A-FABP) are blue-shifted relative to heart FABP (H-FABP), indicating that AOffa bound to A-FABP are held in a more constrained configuration. For both proteins, constraint on the bound ffa probe is highest at the midportion of the acyl chain. Ffa are bound in a hydrophobic environment in both proteins. Excited-state lifetimes and fluorescence quantum yields suggest that the binding site of H-FABP is more hydrophobic than that of A-FABP. Nevertheless, acrylamide quenching experiments indicate that ffa bound to H-FABP are more accessible to the aqueous environment than are A-FABP-bound ffa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

Flavaspidic acid: a biochemical probe for the study of metabolic pathway in hepatocytes.

Displacement of fatty acids or their CoA esters from the cytosolic fatty acid-binding-protein (FABP) by β-flavaspidic acid-N-methyl-glucaminate (flavaspidic acid) shifted the flow of lactate from the lipogenic to the gluconeogenic pathway in hepatocytes isolated from livers of fed rats. Analyses of radiolabel in products of 1 hour incubations utilizing 40 mM DL-sodium lactate (U-¹⁴C sodium lactate or ³H₂O) as substrate showed fatty acid synthesis to be depressed in the presence of 5 mM flavaspidic acid to levels ranging to 28% of control. Glucose synthesis, on the other hand, rose to a level 809% of control, a rate approximately equivalent to the maximal rate of gluconeogenesis found in hepatocytes from diabetic rats. The production of ketone bodies was not influenced by flavaspidic acid whereas CO₂ production decreased reflecting the diversion of the flow of lactate from the lipogenic to the gluconeogenic pathway.These results are presented in support of the hypothesis that unbound long-chain acyl CoA esters act as physiological effectors of the adenine nucleotide translocation and through this action, integrate lipogenic and gluconeogenic activities. Flavaspidic acid thus serves as a useful probe for studies of the role of long chain acyl CoA esters in the intracellular regulation of intermediary metabolism and of the modulation exerted by the FABP.     

An in vitro reversible interconversion of rat liver fatty acid binding protein having different isoelectric points by virtue of the fatty acid content.

Rat liver fatty acid binding protein (FABP) was purified to homogeneity by procedures including Sephadex G-100 and DEAE-cellulose column chromatographies. FABP was resolved into two major peaks, A and B, by the first DEAE-cellulose column chromatography. Each of these two fractions exhibited apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight of 14,000 Da and amino acid analysis of these fractions has revealed that they are virtually identical or closely resemble each other. However, their fatty acid content was significantly different and heterogeneity was clearly demonstrated in the patterns of isoelectric focusing. In this communication, a single isoform (pI 5.0) from peak B FABP was further purified by successive DEAE-cellulose column chromatography and used as the final preparation. When the final FABP was partly freed of fatty acids by a mild delipidation technique using Lipidex 1,000, the pI shifted upward from 5.0 to 7.0. However, the pI of the delipidated FABP returned to its original pI of 5.0 after recombining fatty acids. These in vitro manipulations of bound fatty acid content made clear its possible cause of the microheterogeneity of FABP.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.     

Characterization of crystalline rat liver fatty acid binding protein produced in Escherichia coli

The principal absorptive cell of the rat small intestinal epithelium contains two homologous cytosolic proteins that bind long chain fatty acids. These are known as intestinal and liver fatty acid binding proteins (FABP). While their precise physiological roles have not been defined, they are believed to represent a multifunctional cytosolic transport system that is involved in the trafficking of exogenous lipids to sites of metabolic processing. 13C NMR studies have revealed differences in their fatty acid binding stoichiometries, binding mechanisms, and the ionization properties of bound fatty acids. To understand the functional differences, liver FABP has been crystallized for eventual comparison with the known crystal structure of intestinal FABP. The lattice type is trigonal with unit cell dimensions of a = b = 84.1 A and c = 44.2 A. The space group as determined by examination of the Patterson symmetry is either P3(1)21 or P3(2)21.     

Spectroscopic investigations on the binding site of bovine hepatic fatty acid binding protein. Evidence for the existence of a single binding site for two fatty acid molecules

The hydrophobic region of the binding site of a bovine fatty acid binding protein (pI 7.0-FABP) has been characterized using fluorescence and circular dichroism (CD) spectroscopy. Blue-shifts of fluorescence emission maxima and increased lifetimes of naphthylamine dyes, anthroyloxy-fatty acids, pyrene nonanoic acid and trans-parinaric acid indicated a hydrophobic interaction with FABP. The fluorescence quenching of various anthroyloxy-fatty acids by iodide and acrylamide showed lower accessibility to the fluorophore linked to the carbon adjacent to the carbonyl group and towards the methyl end of the fatty acid. Binding stoichiometries were different for fatty acids and their bulky fluorescent analogues. trans-Parinaric acid when bound to FABP showed a complex induced CD-spectrum, which is explained by a close proximity of two ligands in the same binding site. Fluorescent derivatives of phosphatidylcholine with trans-parinaric acid and cholesteryl trans-parinarate did not bind to FABP. Thus, the binding site appears to be constructed for high affinity binding of long chain fatty acids.     

Inhibitory effect of fatty acids on the binding of androgen receptor and R1881

Unsaturated long chain fatty acids are known to inhibit the binding between estrogen and estrogen receptor, or progesterone and progesterone receptor in rat uterus. The effects of long chain fatty acids on the binding between androgen receptor of castrated rat prostate and 3H-R1881 were studied. The binding was not affected by saturated fatty acids such as palmitic acid (16:0) or stearic acid (18:0). But unsaturated fatty acids such as oleic acid (18:1), arachidonic acid (20:4) and docosahexaenoic acid (22:6) inhibited the binding between androgen receptor and 3H-R1881. The inhibitory effect of arachidonic acid was dose dependent. Scatchard analysis showed that the addition of arachidonic acid markedly decrease the number of binding sites of androgen receptor. But the dissociation constant was not affected. The inhibitory effect of arachidonic was not a competitive one.     

Ablation of the liver fatty acid binding protein gene decreases fatty acyl CoA binding capacity and alters fatty acyl CoA pool distribution in mouse liver

Although liver fatty acid binding protein (L-FABP) is known to bind not only long chain fatty acid (LCFA) but also long chain fatty acyl CoA (LCFA-CoA), the physiological significance of LCFA-CoA binding has been questioned and remains to be resolved. To address this issue, the effect of L-FABP gene ablation on liver cytosolic LCFA-CoA binding, LCFA-CoA pool size, LCFA-CoA esterification, and potential compensation by other intracellular LCFA-CoA binding proteins was examined. L-FABP gene ablation resulted not only in loss of L-FABP but also in concomitant upregulation of two other intracellular LCFA-CoA binding proteins, acyl CoA binding protein (ACBP) and sterol carrier protein-2 (SCP-2), by 45 and 80%, respectively. Nevertheless, the soluble fraction from livers of L-FABP (-/-) mice bound 95% less radioactive oleoyl-CoA than wild-type L-FABP (+/+) mice. The intracellular LCFA-CoA binding protein fraction (Fraction III) from wild-type L-FABP (+/+) mice, isolated by gel permeation chromatography of liver soluble proteins, exhibited one high-affinity binding and several low-affinity binding sites for cis-parinaroyl-CoA, a naturally occurring fluorescent LCFA-CoA. In contrast, high-affinity LCFA-CoA binding was absent from Fraction III of L-FABP (-/-) mice. While L-FABP gene ablation did not alter liver LCFA-CoA pool size, LCFA-CoA acyl chains of L-FABP (-/-) mouse livers were enriched 2.1-fold in C16:1 and decreased 1.9-fold in C20:0 fatty acids. Finally, L-FABP gene ablation selectively increased the amount of LCFAs esterified into liver phospholipid > cholesteryl ester, while concomitantly decreasing the amount of fatty acids esterified into triglycerides by 40%. In summary, these data with L-FABP (-/-) mice demonstrated for the first time that L-FABP is a physiologically significant contributor to determining liver cytosolic LCFA-CoA binding capacity, LCFA-CoA acyl chain distribution, and esterified fatty acid distribution.     

Interactions of oleic acid with liver fatty acid binding protein: a carbon-13 NMR study

13C NMR spectroscopy was used to probe the structural interactions between carboxyl-13C-enriched oleic acid (18:1) and rat liver fatty acid binding protein (FABP) and the partitioning of 18:1 between FABP and unilamellar phosphatidylcholine (PC) vesicles. Spectra of systems containing 2-8 mol of 18:1/mol of FABP (but no PC) exhibited one carboxyl resonance (182.2 ppm) corresponding to FABP-bound 18:1. At pH values less than 8.0, an additional carboxyl resonance, corresponding to unbound 18:1 in a lamellar phase, was observed. Both resonances exhibited ionization shifts with estimated apparent pKa values of less than 5 (bound 18:1) and greater than 7 (unbound 18:1). The intensity of the resonance corresponding to FABP-bound 18:1 increased with increasing 18:1/FABP mole ratio and at 8/1 mole ratio indicated that at least 2 and 6 mol of 18:1/mol of FABP were FABP-bound at pH 7.4 and 8.6, respectively. NMR spectra of systems containing equal concentrations (w/v) of FABP and PC and from 1 to 4 mol of total fatty acid (FA)/mol of FABP exhibited two 18:1 carboxyl resonances (182.2 and 178.5 ppm, pH 7.4). The downfield resonance corresponded to FABP-bound 18:1 and the upfield resonance to PC vesicle bound 18:1. At 1/1 mole ratio (FA/FABP), the intensities of both resonances were approximately equal, but at 4/1 mole ratio the resonance for PC vesicle bound 18:1 was 3-fold more intense than that for FABP-bound 18:1. The following conclusions are reached: (i) The carboxyl groups of 18:1 bound to liver FABP experience only one type of binding environment (the aqueous milieu adjacent to the protein surface).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid binding proteins from heart

Heart contains a fatty acid binding protein (FABP) concentration comparable to liver, when it is determined with a fatty acid-binding assay. The low concentration detected with anti-liver FABP antibodies is related to the different chemical forms and physiochemical properties of liver and heart FABP. The ratio of fatty acid bound per purified protein molecule is one or lower. Rat heart mitochondria oxidize FABP-bound fatty acids. The FABP content of rat heart is dependent on sex and diurnal cycle, but is not influenced by starvation or clofibrate feeding. It is also not different in the newborn rat. FABP was obtained from human heart in a yield of 11%. It shows similar binding characteristics to palmitic, oleic and arachidonic acid. The functional significance of the specific heart FABP is discussed in relation to myocardial fatty acid metabolism in normal and pathological conditions.     

Relation of lung fatty acid binding protein to the biosynthesis of pulmonary phosphatidic acid and phosphatidylcholine

The activities of glycerophosphate and lysophosphatidylcholine (LPC) acyltransferases were determined using lung microsomes in the presence of lung fatty acid binding protein (FABP). The synthesis of phosphatidic acid (PA) was increased two- to fourfold in the presence of FABP as compared to albumin. Lung FABP did not increase the incorporation of palmitoyl CoA into phosphatidylcholine. The results indicate that FABP-bound fatty acyl CoA may be a preferred substrate for glycerophosphate acyltransferase.