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.     

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     

NMR reveals molecular interactions and dynamics of fatty acid binding to albumin

Background

The molecular details of fatty acid (FA) interactions with albumin are fundamental to understanding transport in the plasma and cellular utilization of these key nutrients and building blocks of membranes.

Scope of review

This review focuses on the development and application of NMR methods to study FA binding to albumin [bovine (BSA) and human (HSA)]. The key strategy was to use ¹³C enrichment of a specific carbon in the FA as a non-perturbing probe to permit visualization of the small ligand complexed to the very large protein. NMR contributions to illuminating molecular interactions and FA dynamics are summarized from three decades of studies.

Major conclusions

Our early studies detected multiple binding sites that we hypothesized were distinguished because of the unique tertiary structure of the protein in close proximity to the FA labeled carbon in each site. Later crystallographic structures revealed the presence of polar and charged amino acid side chains near the carboxyl carbon of the FA and unique tertiary structures lining all of the FA binding pockets. In collaboration with the crystallography group, several FA sites in the crystalline state were matched with NMR resonances in the solution state. With the newest application of NMR, 2D NMR spectroscopy detected nine binding sites, and three were located in the crystal structure through displacement of drugs with identified sites.

General significance

NMR spectroscopy utilizing the FA as a probe allows characterization of site-specific interactions, molecular motions within binding sites, the order of filling and removal of FA from sites. This article is part of a Special Issue entitled Serum Albumin.     

Bifunctional lipid-transfer: fatty acid-binding proteins in plants

Summary A cytosolic protein, able to facilitate intermembrane movements of phospholipids in vitro, has been purified to homogeneity from sunflower seedlings. This protein, which has the properties of a lipid-transfer protein (UP), is also able to bind oleoyl-CoA, as shown by FPLC chromatography. This finding, in addition to previous observations suggesting that a lipid-transfer protein from spinach leaves can bind oleic acid and that oat seedlings contain a fatty acid-binding protein with similar features than lipid transfer proteins, provides a clear demonstration that plant cells contain bifunctional fatty acid/lipid transfer proteins. These proteins can play an active role in fatty acid metabolism which involves movements of oleyl-CoA between intracellular membranes.Abbreviations FABP Fatty Acid-Binding Proteins - UP Lipid-Transfer Protein - PC Phosphatidylcholine - PI Phosphatidylinositol - PE Phosphatidylethanolamine - pI Isoelectric point     

Isolation, characterization, and developmental expression of pig intestinal fatty acid-binding proteins

The goal of this study was to characterize and quantify intestinal fatty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centrifuged to obtain cytosol. Isolation of fatty acid-binding proteins from delipidated cytosol was achieved using molecular sieve, oleic acid affinity, and ion exchange chromatography. Fatty acid-binding protein isolation was monitored using a fatty-acid binding assay in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antisera to rat liver-fatty acid-binding protein cross reacted with an isolated intestinal fatty acid-binding protein of Mr = 13,000, whereas antisera to rat intestine-fatty acid-binding protein was not cross reactive with isolated pig intestinal proteins. These experiments identify a pig intestinal fatty acid-binding protein that exhibits strong immunochemical similarity to rat liver-fatty acid-binding protein. Cytosol prepared from intestinal mucosa of pigs at -4, 2, 4, 7, 15, 22, 28, and 35 d of age was assayed for fatty acid-binding protein activity. Preweaning fatty acid-binding protein activity in cytosol was maximal at 7 days of age when expressed as total jejunal fatty acid binding per kilogram bodyweight, intestinal or mucosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein activities declined to 28 days, but increased again by 35 days. Total soluble fatty acid-binding protein activity in pig intestine is regulated during postnatal development and this may account in part for the altered intestinal absorption of lipids observed in young pigs at weaning.     

Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in Escherichia coli. A comparative 13C NMR study

Enterocytes in the small intestinal mucosa contain abundant quantities of two homologous cytosolic proteins known as intestinal and liver fatty acid-binding proteins (I- and L-FABP, respectively). To elucidate structure-function relationships for these proteins, the interactions between 13C-enriched palmitate and oleate and Escherichia coli-expressed rat I- and L-FABP were systematically compared using 13C NMR spectroscopy. NMR spectra of samples containing fatty acids (FA) and I-FABP at different molar ratios (all at pH 7.2 and 37 degrees C) exhibited a single carboxyl resonance corresponding to FA bound to I-FABP (181.4 ppm, peak I) and an additional carboxyl resonance corresponding to unbound FA in a bilayer phase (179.6 ppm). Peak I reached a maximum intensity corresponding to 1 mol of bound FA/mol of I-FABP under all sample conditions examined. NMR spectra for samples containing FA and L-FABP also exhibited a single carboxyl resonance corresponding to FA bound to L-FABP but at a different chemical shift value (182.2 ppm, peak L). Its maximum intensity varied depending on the physical state of the unbound FA (liquid crystalline or crystalline), the FA used (palmitate or oleate), and the sample pH. In the presence of a liquid crystalline (bilayer) phase, up to 1 (oleate) or 2 (palmitate) mol of FA were bound/mol of L-FABP, but in the presence of a crystalline phase (1:1 acid-soap), up to 3 mol of palmitate were bound/mol of L-FABP (all at pH 7.2). Peak I exhibited little or no ionization shift over a wide pH range (pH 3.0-11.0), and its chemical shift was unaffected by the ionization of Lys and His residues. Hence, the carboxylate group of FA bound to I-FABP was solvent inaccessible and most likely involved in an ion-pair electrostatic interaction with the delta-guanidinium moiety of an Arg residue. In contrast, peak L exhibited an ionization shift and an estimated apparent pKa value similar to that obtained for monomeric FA in water, suggesting that the carboxylate groups of FA bound to L-FABP were solvent accessible and located at or near the protein solvent interface. With decreasing pH, FA dissociated from L-FABP but not I-FABP, as monitored by NMR peak intensities. Concurrently, a large decrease in circular dichroism molar ellipticity was observed with L-FABP but not I-FABP. In conclusion, I-FABP and L-FABP are distinct with regards to their FA-binding stoichiometries, binding mechanisms, and sensitivity to pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions

The structure ofE. coli-derived rat intestinal fatty acid-binding protein has recently been refined to 1.2 Å without bound fatty acid and to 2.0 Å and 1.75 Å with bound hexadecanoate (palmitate) and 9Z-octadecenoate (oleate), respectively. The structure ofE. coli-derived human muscle fatty acid-binding protein has also been solved to 2.1 Å with a C16 bacterial fatty acid. Both proteins contain 10 anti-parallel -strands in a+1, +1, +1... motif. The strands are arranged in two -pleated sheets that are orthogonally oriented. In each case, the fatty acid is enclosed by the -sheets and is bound to the proteins by feeble forces. These feeble forces consist of (i) a hydrogen bonding network between the fatty acid's carboxylate group, ordered solvent, and side chains of polar/ionizable amino acid residues; (ii) van der Waals contacts between the methylene chain of the fatty acid and the side chain atoms of hydrophobic and aromatic residues; (iii) van der Waals interactions between the methyl and the component methenyls of the phenyl side chain of a Phe which serves as an adjustable terminal sensor situated over a surface opening or portal connecting interior and exterior solvent; and (iv) van der Waals contacts between methylenes of the alkyl chain and oxygens of ordered waters that have been located inside the binding cavity. These waters are positioned over one face of the ligand and are held in place by hydrogen bonding with one another and with the side chains of protein's polar and ionizable residues. Binding of the fatty acid ligand is associated with minimal adjustments of the positions of main chain or side chain atoms. However, acquisition of ligand is associated with removal of ordered interior solvent suggesting that the free energy of dehydration of the binding site may be as important for the energy of the binding reaction as the free energy of stabilization of the fatty acid: protein complex.     

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.     

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     

1H, 13C and 15N assignments and chemical shift-derived secondary structure of intestinal fatty acid-binding protein

Summary Sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments have been established for rat intestinal fatty acid-binding protein complexed with palmitate (15.4 kDa) at pH 7.2 and 37°C. The resonance assignment strategy involved the concerted use of seven 3D triple-resonance expriments (CC-TOCSY, HCCH-TOCSY, HNCO, HNCA, ¹⁵N-TOCSY-HMQC, HCACO and HCA(CO)N). A central feature of this strategy was the concurrent assignment of both backbone and side-chain aliphatic atoms, which was critical for overcoming ambiguities in the assignment process. The CC-TOCSY experiment provided the unambiguous links between the side-chain spin systems observed in HCCH-TOCSY and the backbone correlations observed in the other experiments. Assignments were established for 124 of the 131 residues, although 6 of the 124 had missing amide ¹H resonances, presumably due to rapid exchange with solvent under these experimental conditions. The assignment database was used to determine the solution secondary structure of the complex, based on chemical shift indices for the ¹H, ¹³C, ¹³C and ¹³CO atoms. Overall, the secondary structure agreed well with that determined by X-ray crystallography [Sacchettini et al. (1989) J. Mol. Biol., 208, 327–339], although minor differences were observed at the edges of secondary structure elements.     

In vivo NMR detection of diet-induced changes in adipose tissue composition

We introduce an in vivo spectroscopic method to assess the effects of diet on fatty acid composition of the predominant chemical constituent of adipocytes in mice. To do this, we make use of a nonlinear NMR signal that, unlike a standard NMR signal, is intrinsically insensitive to local magnetic field inhomogeneities and which naturally suppresses the large water signal from nonfatty tissues. Our method yields fat composition information from fat depots distributed over large sample volumes in a single experiment, without requiring the use of tedious shimming procedures, voxel selection, or water suppression. Our results suggest that this method can reveal clear differences in adipose tissue composition of mice fed a standard chow diet compared with mice fed a diet rich in polyunsaturated fatty acids. With further developments this method could be used to obtain information on human lipid composition noninvasively and to track changes in lipid composition induced by diet intervention, pharmaceutical drugs, and exercise.     

The analysis of fatty acid binding to protein using a modified equilibrium dialysis method: detailed analysis of chromophore-fatty acid-protein interactions

In this paper we extend our previous analysis of fatty acid-chromophore-protein interactions using a modified equilibrium dialysis method described previously. A more rigorous mathematical treatment is combined with a micro-dialysis method using a maximum volume of dialyzate of between 250 microliters and 400 microliters to examine the suitability of different chromophores (mepacrine, quinine, chloroquine, chlorpromazine, methylene blue, rhodamine 6G, 6-carboxyfluorescein) for studying the binding of fatty acid to protein. The macro- and micro-methods of dialysis are compared, and the binding of fatty acid to bovine serum albumin and beta-lactoglobulin discussed as examples of the method. Problems associated with propagated errors in the measurements and obtaining the number of binding sites and the binding constants from curve-fitting are also considered.     

Mobility of lipid in complexes of amylose–fatty acids by deuterium and C solid state NMR

Palmitic and lauric acid complexes with amylose were studied by solid state methods: ¹³C CP/MAS NMR, deuterium NMR, X-ray powder diffraction and differential scanning calorimetry (DSC). The crystalline amylose complexes were found to be in a V-type sixfold single chain helix. The melting points of the complexes were over 100°C, at least 40–50°C higher than the melting points of the free fatty acids. CP/MAS ¹³C NMR spectra revealed two resonance peaks at 33.6 and 32.4 ppm for the palmitic acid, which were assigned as free and complexed fatty acid, respectively. A single resonance peak at 32.4 ppm was found for the lauric acid and assigned to the complex. The chemical shift of 32.4 ppm for the complexed fatty acids suggests a combined trans and gauche conformation for the fatty acid chain in the complex. T₁ relaxation measurements on the two palmitic acid resonances show different behavior: a very slow relaxation for the 33.6 ppm and a much faster relaxation (1.2 s) for the 32.4 ppm resonances. The latter was similar to the relaxation of the single resonance of the lauric acid (1.1 s). Temperature dependent deuterium spectra of the amylose–lauric acid and amylose–palmitic acid complexes suggest a complete complexation for the amylose–lauric acid, whereas the amylose–palmitic acid complex is partially disassociated by the thermal treatment. Based on the overall data, a partially disordered model is proposed: an imperfect helix with the fatty acid partly inside and partly out, depending on crystallization conditions and the necessity of placing the carboxyl head outside the V-helix.     

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE⁺ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a sup-Estrain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel -strands organized into two orthogonally situated -sheets. The overall conformation of the protein resembles that of a clam — hence the term -clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg¹⁰⁶ and Gln¹¹⁵. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the -clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations pf several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism. Comparison of the C coordinates of apo- and holo-I-FABP with those of other proteins indicates it is a member of a superfamily that currently includes (i) 10 mammalian intracellular lipid binding proteins, (ii) the photoactive yellow protein from the purple photoautotrophic bacterium Ectothiorhodospira halophila and (iii) a group of extracellular lipid binding proteins from a diverse number of phyla that have a common barrel consisting of 8 anti-parallel -strands stacked in two nearly orthogonal sheets. In summary, E. coli-derived I-FABP not only represents a useful model for assessing the atomic details of fatty acid-protein interactions and the mechanisms which regulate acquisition and release of this type of ligand, but also structure/function relationships in other superfamily members.Abbreviations I-FABP Intestinal Fatty Acid Binding Protein - r.m.s root mean square     

Intracellular lipid binding proteins of the small intestine

The small intestine contains three distinct proteins belonging to the intracellular lipid binding protein family: the liver-type fatty acid binding protein (L-FABP), the intestinal fatty acid binding protein (I-FABP) and the ileal lipid binding protein (ilbp). The function of these proteins in the small intestine has remained enigmatic. Targeted gene disruption studies may shed insights into the physiological importance of these proteins. In the case of I-FABP, this approach has demonstrated that the complete elimination of this protein in murine intestine does not compromise dietary fat absorption in vivo but is associated with the development of insulin resistance.     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.