Nomenclature of fatty acid-binding proteins

Summary A variety of designations is currently being used to refer to cellular fatty acid-binding proteins (FABPs). Besides from the use of other general names (e.g. Z protein), confusion mostly arises from the application of various abbreviations and symbols to denote the tissue(s) of origin and cellular localization (cytoplasm, plasma membrane) of a specific FABP. In order to minimize confusion a more unified and rational nomenclature is proposed, which is based on application of the formula X-FABPy. The prefix X is a capital letter indicating the tissue of greatest abundance, the suffix Y similarly denotes the (sub)cellular localization of the protein. The general and functional name fatty acid-binding protein (FABP) is preferred for the cellular proteins with the property to bind fatty acids, unless future research reveals that the binding of fatty acids is not the primary biological property or physiological role of (some of) these proteins.     

Acyl-CoA-binding protein (ACBP) and its relation to fatty acid-binding protein (FABP): an overview

Summary Acyl-CoA-binding protein is a 10 Kd protein which binds medium- and long-chain acyl-CoA esters with high affinity. The concentration in liver is 2–4 times the acyl-CoA concentration. ACBP has much greater affinity for acyl-CoA than FABP. FABP from bovine heart and liver is unable to compete with multilamellar liposomes, Lipidex and microsomal membrane in binding acyl-CoA esters, whereas ACBP effectively extracts acyl-CoA from all those sources. Previously published results on the effect of FABP on acyl-CoA metabolism need to be reevaluated due to possible contamination with ACBP. Recently it was discovered that ACBP is identical to a putative neurotransmitter diazepam binding inhibitor. The possibility therefore exists that ACBP has more than one function.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

Involvement of arginine in the binding of heme and fatty acids to fatty acid-binding protein from bovine liver

Fatty acid-binding protein from bovine liver but not from bovine heart binds hematin in a saturable manner with high affinity. This property is not confined to a particular isoform as both, pI 6.0- and pI 7.0 L-FABP, bind hematin similarly. In competition experiments hematin and oleic acid could replace each other demonstrating that they share at least parts of the same binding site. Common structural features, i.e. the presence of carboxylic groups and of hydrophobic carbon chains led to the hypothesis that both ligands interact similarly with L-FABP. This was supported by the decrease of binding affinity for either ligand upon modification with phenylglyoxal. Modification in the presence of fatty acid revealed the protection of one of the two arginines of L-FABP. By peptide mapping and Edman degradation Arg¹²² was identified as the counterpart of the fatty acids carboxylic group.     

Localization of mammary-derived growth inhibitor in capillary endothelial cells of the bovine mammary gland

Mammary-derived growth inhibitor (MDGI) has previously been localized in the mammary parencyma, dependent on the stage of differentiation of the mammary gland. Here, we have elucidated the distribution of MDGI in the mammary stroma by a combined immunohisto-and cytochemical analysis with antibodies raised against MDGI. Distinct staining of capillary endothelial cells has been revealed. Although its subcellular distribution resembles former observations in secretory epithelial cells, the expression of MDGI in capillary endothelial cells clearly precedes that in secretory epithelial cells. On the other hand, no endothelial MDGI staining has been detected in bovine heart, which contains a fatty acid-binding protein almost identical to MDGI. The localization of MDGI in the mammary capillary endothelium is discussed in terms of its possible involvement in the intracellular transport of hydrophobic ligands or in the regulation of endothelial cell proliferation.     

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).     

Fatty acid-binding protein and its relation to fatty acid oxidation

A relation between fatty acid oxidation capacity and cytosolic FABP content was found in heart and various muscles of the rat. Other tissues do not show such a relation, since they are involved in more or other pathways of fatty acid metabolism. At postnatal development FABP content and fatty acid oxidation capacity rise concomitantly in heart and quadriceps muscle in contrast to in liver and kidney. A dietary fat content of 40 en. % increased only the FABP content of liver and adipose tissue. Peroxisomal proliferators increased fatty acid oxidation in both liver and kidney, but only the FABP content of liver, and had no effect on heart and skeletal muscle. The FABP content of muscle did not show adaptation to various conditions. Only it increased in fast-twitch muscles upon chronic electrostimulation and endurance training.     

Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid

A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-³H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. ThefadL protein ofE. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of thefadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a K_d of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.Abbreviations BSA Bovine Serum Albumin - DAP m-Diazirinophenoxy - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis     

Cellular fatty acid-binding proteins: current concepts and future directions

Summary At least three different proteins are implicated in the cellular transport of fatty acid moieties: a plasmalemmal membrane and a cytoplasmic fatty acid-binding protein (FABP_PM and FABP_C, respectively) and cytoplasmic acyl-CoA binding protein (ACBP). Their putative main physiological significance is the assurance that long-chain fatty acids and derivatives, either in transit through membranes or present in intracellular compartments, are largely complexed to proteins. FABP_C distinguishes from the other proteins in that distinct types of FABP_C are found in remarkable abundance in the cytoplasmic compartment of a variety of tissues. Although their mechanism of action is not yet fully elucidated, current knowledge suggests that the function of this set of proteins reaches beyond simply aiding cytoplasmic solubilization of hydrophobic ligands, but that they can be assigned several regulatory roles in cellular lipid homeostasis.     

The fatty acid-binding heterocomplex FA-p34 formed by S100A8 and S100A9 is the major fatty acid carrier in neutrophils and translocates from the cytosol to the membrane upon stimulation

Since no data are available concerning fatty acid (FA) transport in neutrophils we studied the presence of possible FA carriers. The kFA-p34 complex, composed of S100A8 and S100A9, has been implicated in the intracellular transport of arachidonic acid and its precursors in human keratinocytes. Here, we show that FA-p34 is the major FA carrier in human neutrophils (nFA-p34). The complex is highly expressed in resting neutrophils (2.65% of cytosolic proteins) and translocates to the membrane fraction upon stimulation with opsonized zymosan. Comparison of purified nFA-p34 with kFA-p34 shows that both complexes are composed of nearly the same subunits and possess similar binding properties for oleic acid. Densitometrical analyses of 2D gels show that n and kFA-p34 contain twice as much S100A8 and S100A9 suggesting an estimated stoichiometry of (S100A8)2S100A9. A method is described allowing to distinguish n and kFA-p34 from S100A8/S100A9 homo- and heteromer complexes that are devoid of FA-binding properties. After solvent extraction, we find by GC analysis linoleic acid as major endogenous ligand of purified kFA-p34. Our results suggest that nFA-p34, might be involved in the shuttling of unsaturated FA between the cytosol and the plasma membrane of neutrophils.     

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.     

A comparison of heart and liver fatty acid-binding proteins: interactions with fatty acids and possible functional differences studied with fluorescent fatty acid analogues

Summary Fatty acid-binding proteins (FABP) are distinct but related gene products which are found in many mammalian cell types. They are generally present in high abundance, and are found in those tissues where free fatty acid (ffa) flux is high. The function(s) of FABP is unknown. Also not known is whether all FABP function similarly in their respective cell types, or whether different FABP have unique functions. The purpose of these studies was to assess whether different members of the FABP family exhibit different structural and functional properties. Two fluorescent analogues of ffa were used to compare the liver (L-FABP) and heart (H-FABP) binding proteins. The propionic acid derivative of diphenylhexatriene (PADPH) was used to examine the physical properties of the ffa binding site on L- and H-FABP, as well as the relative distribution of ffa between FABP and membranes. An anthroyloxy-derivative of palmitic acid, 2AP, was used to monitor the transfer kinetics of ffa from liver or heart FABP to acceptor membranes, using a resonance energy transfer assay. The results demonstrate that the ffa binding sites of both FABP are hydrophobic in nature, although the L-FABP site is more nonpolar than the H-FABP site. Equilibration of PADPH between L-FABP and phosphatidylcholine (PC) bilayers resulted in a molar partition preference of > 20: 1, L-FABP : PC. Similar studies with H-FABP resulted in a PADPH partition preference of only 3:1, H-FABP : PC. Finally, the transfer of 2AP from H-FABP to acceptor membranes was found to be 50-fold faster than transfer from L-FABP. These studies demonstrate that important structural and functional differences exist between different members of the FABP family, and therefore imply that the roles of different FABP may be unique.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - SUV Small Unilamellar Vesicle - PADPH 3-[p-(6-Phenyl)-1,3,5-Hexatrienyl]-phenylpropionic acid - 2AP 2-(9-Anthroyloxy)Palmitic acid - Q Quantum yield - _F Fluorescence lifetime     

Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium

Localization of a mammary-derived growth inhibitor (MDGI) in the bovine mammary gland was verified by light-and electron-microscopic methods. Expression of MDGI, which is known to inhibit the growth of mammary epithelial cell lines in vitro, was found to be highest in the late pregnant and in the lactating state. A combination of immunohistochemical and immunocytochemical methods with semi- and ultrathin resin sections revealed marked variations in MDGI staining. High MDGI levels were predominantly detectable in epithelial cells with large milk fat droplets. Distinct cell types that were almost free of label could be identified among bovine mammary epithelial cells that always exhibited high MDGI levels. Similar results were obtained when using a serum-free organ culture system in which MDGI was hormonally induced in cell types of comparable differentiation state. The specific occurrence of the growth inhibitor in developing alveoli and certain cell types points to the association between MDGI expression and functional differentiation in the normal mammary gland.     

Research of anin vitro model to study the expression of fatty acid-binding proteins in the small intestine

In order to find anin vitro model for studying the regulation of the biosynthesis of the cytoplasmic Fatty Acid-Binding Proteins (FABPc) expressed in the small intestine, Intestinal- and Liver-(I- and L-) FABPc expressions were tested by Northern blotting in 8 normal or cancerous intestinal cell lines from man, mouse and rat and in organ culture of mouse jejunal explants. Neither I- nor L-FABPc mRNA was detected in any cell strains tested except in the highly differentiated human enterocyte-like intestinal cell line Caco-2. In this line, Northern blot analysis revealed a single messenger of about 0.7 kb corresponding to the L-FABPc. A two-fold increase in mRNA L-FABPc occurred in differentiated Caco-2 cells treated for 7 days with 0.05 mM bezafibrate, a peroxisome-proliferating hypolipidemic drug. The lack of I-FABPc messengers in this strain led us to seek anotherin vitro model. I- and L-FABPc messengers were found using an organ culture of mouse jejunal explants. A clear rise in I- and, especially, L-FABPc mRNA levels occurred 6 and 24 hr after the addition of 0.05 mM bezafibrate in the culture medium. Our results demonstrate, to our knowledge for the first time, that: 1) organ culture of intestinal explants provides a useful model for studyingin vitro the simultaneous regulation of I- and L-FABPc expressions, 2) biosynthesis of L-FABPc may be exploredin vitro using the Caco-2 cell line, 3) fibrate peroxisome-proliferators exert a direct effect on I- and L-FABPc expression in the small intestine, 4) L-FABPc expression seems to be more sensitive to fibrate action than is I-FABPc expression.Abbreviations I-FABPc cytosolic Intestinal Fatty Acid-Binding Protein - L-FABPc cytosolic Liver Fatty Acid-Binding Protein - bp base pair - EDTA Ethylenediamine Tetraacetic Acid - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - HBSS Hanks Balanced Salt Solution     

Significance of cytoplasmic fatty acid-binding protein for the ischemic heart

Ischemia of the heart is accompanied by the tissue accumulation of long-chain fatty acids and their metabolic derivatives such as -hydroxy fatty acids and fatty acyl-CoA and acyl-L-carnitine esters. These substances might be detrimental for proper myocardial function. Previously, it has been suggested that intracellular lipid binding proteins like cytoplasmic fatty acid-binding protein (FABP) and acyl-CoA binding protein (ACBP) may bind these accumulating fatty acyl moieties to prevent their elevated levels from potentially harmful actions. In addition, the suggestion has been made that the abundantly present FABP may scavenge free radicals which are generated during reperfusion of the ischemic heart. However, these protective actions are challenged by the continuous physico-chemical partition of fatty acyl moieties between FABP and membrane structures and by the rapid release of FABP from ischemic and reperfused cardiac muscle. Careful evaluation of the available literature data reveals that at present no definite conclusion can be drawn about the potential protective effect of FABP on the ischemic and reperfused heart. Biochem123: 167–173, 1993)Abbreviations FABP Fatty Acid-Binding Protein - ACBP Acyl-CoA Binding Protein - MDGI Mammary-Derived Growth Inhibitor - CK Creatine Kinase - LDH Lactate Dehydrogenase     

Similar mechanisms of fatty acid transfer from human anal rodent fatty acid-binding proteins to membranes: Liver,intestine, heart muscle,and adipose tissue FABPs

The mammalian fatty acid-binding proteins (FABPs) are thought to be important for the transport and metabolism of fatty acids in numerous cell types. The transfer of FA from different members of the FABP family to membranes has been shown to occur by two distinct mechanisms, an aqueous diffusion-based mechanism and a collisional mechanism, wherein the FABP interacts directly with membrane acceptors. Much of the work that underlies this concept comes from efforts using rodent FABPs. Given the increasing awareness of links between FABPs and several chronic diseases in humans, it was important to establish the mechanisms of FA transfer for human FABPs. In the present studies, we examined the rate and mechanism of fatty acid transfer from four pairs of human and rodent (rat or mouse, as specified) FABPs: hLFABP and rLFABP, hIFABP and rIFABP, hHFABP and rHFABP, and hAFABP and mAFABP. In the case of human IFABP, both the Ala⁵⁴ and Thr⁵⁴ forms were examined. The results show clearly that for all FABPs examined, the mechanisms of ligand transfer observed for rodent proteins hold true for their human counterparts. Moreover, it appears that the Ala to Thr substitution at residue 54 of the human IFABP does not alter the fundamental mechanism of ligand transfer to membranes, but nevertheless causes a consistent decrease in the rate of transfer.     

Detection,tissue distribution and (sub)cellular localization of fatty acid-binding protein types

Summary This overview of recent work on FABP types is focussed on their detection and expression in various tissues, their cellular and subcellular distribution and their binding properties. Besides the 3 well-known liver, heart and intestinal types, new types as the adipose tissue, myelin and (rat) renal FABPs have been described. Recent observations suggest the occurrence of more tissue-specific types, e.g. in placenta and adrenals. Heart FABP is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. The cellular distribution of FABP types appears to be related to the function of the cells in liver, muscle and kidney. The presence of FABP in cellular organelles requires more evidence. The functional significance of the occurrence of more FABP types is unclear, in spite of the observed differences in their ligand-protein interaction.Abbreviations FABP(s) Fatty Acid-Binding Protein(s)     

Historic overview of studies on fatty acid-binding proteins

Summary Fatty acid-binding proteins (FABPs) were first identified in the cytosol of rat intestinal mucosa during studies on the regulation of intestinal fatty acid uptake. The subsequent finding of FABP activity in the cytosol of many other tissues initially was believed to reflect a single protein. However, the FABPs are now recognized as products of an ancient gene family comprised of at least 9 structurally related, soluble intracellular members, a number of which exhibit high-affinity binding of long-chain fatty acids. Despite recent insights into regulation and tissue-specific expression suggesting FABPs to subserve diverse roles, their precise biological functions remain to be elucidated.     

Expression of fatty acid-binding protein from bovine heart in Escherichia coli

Summary The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2. After induction with isopropyl--d-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E. coli amounting up to 5.7% of the soluble protein. cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay. The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing. cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins.     

Assay of the binding of fatty acids by proteins: evaluation of the Lipidex 1000 procedure

Summary Fatty acid (FA) binding by fatty acid-binding protein (FABP) is frequently Monitored with the so-called Lipidex 1000 assay, in which protein associated and non-protein bound FA are separated by selectively binding the latter to Lipidex 1000. Careful evaluation of this assay showed that the use of aqueous FA solutions resulted in a Marked decrease (60 to 70%) of FA concentration due to their aspecific binding to the surface of the test-tube used. In addition, solutions of rat heart FABP in the Molar range also showed a concentration decrease up to 80% due to protein binding to the surface of the test-tube. Introduction of detergents, Triton X-100 or Tween 20, limited the FA loss to less than 20% and totally eliminated FABP adsorption. Kinetic parameters for the binding of [1-¹⁴C]oleic acid by purified rat heart FABP, assayed in the presence of Triton X-100, were found to be similar to those assayed in the absence of detergent, when adequate corrections were Made for losses of FA and FABP due to surface adsorption. Use of Tween 20 resulted in a substantial increase of the dissociation constant. The addition of 100 M Triton X-100 to the assay medium considerably facilitates the determination of kinetic parameters of fatty acid-binding by proteins.