Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host–parasite adaptation.     

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.     

The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1

We describe the 1.6 A crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease.The crystal structure reveals that EgFABP1 has the expected 10-stranded beta-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1.The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R em leader R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.     

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.     

Molecular cloning and characterization of a thioredoxin gene from Echinococcus granulosus.

The insert of a clone from a lambdagt11 Echinococcus granulosus (Platyhelminth, Cestoda) protoscolex cDNA library, showed an open reading frame whose deduced protein sequence presents a high homology with all described thioredoxins (TRX). The TRX active site (Trp-Cys-Gly-Pro-Cys) is completely conserved. With a monospecific antibody, selected from a total anti-protoscolex sera by the isolated clone, a 12 kDa polypeptide was immunoprecipitated from a protoscolex total protein extract. Furthermore, an antiserum raised against a recombinant EgTRX also recognizes a 12 kDa band in these extracts. The recombinant protein presents TRX activity, using the insulin reduction assay. Finally, a TRX activity was characterized in protoscolex extracts. In all organisms where TRXs were studied, they participate in a cascade of redox exchanges, contributing to the maintaining of cell homeostasis. Considering that the parasitic flatworm E. granulosus is probably submitted to an important oxidative stress due to host defences, EgTRX protein could be involved in the survival strategies of this parasite.     

Genomic typing of the Echinococcus granulosus isolates from the areas of Southern Urals

Nine larvocysts of Echinococcus granulosus isolated from nine patients and one cyst derived from a naturally infested cattle have been examined. Genomic typing was carried out in order to identify strains of E. granulosus. All DNA samples were shown to have the same genotype, E. granulosus G1.     

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.     

Tissue- and stage-specific expression of a fatty acid binding protein-like gene from amphioxus Branchiostoma belcheri

A cDNA clone encoding an amphioxus fatty acid binding protein-like (AmphiFABPL) protein was isolated from a gut cDNA library of Branchiostoma belcheri. It contained a 423 bp open reading frame corresponding to a deduced protein of 140 amino acids with a predicted molecular mass of approximately 15.9 kDa. Phylogenetic analysis showed that AmphiFABPL fell outside the vertebrate clade of fatty acid binding proteins (FABPs), being positioned at the base of the chordate lineage, and was almost equally homologous to various vertebrate FABPs, suggesting that it may be the archetype of vertebrate FABPs. Both northern blotting and in situ hybridization analyses demonstrated that AmphiFABPL was expressed in the hepatic caecum and hind-gut, and although at a much lower level, it was also present in the endostyle, ovary and testis. In addition, whole-mount in situ hybridization revealed that AmphiFABPL was initially expressed in the posterior two thirds of the primitive gut, including the mid-gut where the hepatic caecum will form later, in 2-day larvae. The expression pattern is closely similar to that of the L-FABP and I-FABP genes in vertebrates, supporting the hypothesis that the hepatic caecum in the amphioxus is homologous to the vertebrate liver.     

Polymorphisms in chicken extracellular fatty acid binding protein gene

In this study, we report the investigation of extracellular fatty acid binding protein gene (Ex-FABP) genetic polymorphism in a sample of 360 chicken individuals. The screening of the coding regions with their intron–exon boundaries and the proximal flanking regions was performed through a PCR-SSCP strategy. Following sequence analysis revealed 35 novel single nucleotide polymorphisms (SNPs) of chicken Ex-FABP gene. Among the 35 SNPs, twenty-five were found in the introns. And the remaining seven and three SNPs were in the coding region and the 5′UTR, respectively. Two SNPs in the coding region caused two missense mutants and the other five did not result in any amino acid changes. The nature and the distribution of Ex-FABP mutations in three chicken breeds were analyzed. Variations detected here might have an impact on Ex-FABP activity and function and underpin the development of gene markers for chicken fatty deposition and metabolism. The polymorphism, generated by C4715T mutation in exon5, was significantly associated with thickness of subcutaneous fat plus skin in cocks. Subcutaneous fat plus skin of cocks was more thick in TT genotype than in CC genotype (P < 0.05). The Ex-FABP gene could be a candidate locus or linked to a QTL that significantly affects fatty deposition and metabolism in chicken.     

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.     

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.     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

Nucleotide sequence of cDNA clones coding for a brain-type fatty acid binding protein and its tissue-specific expression in adult zebrafish (Danio rerio)

We have determined the nucleotide sequence for two cDNA clones coding for a fatty acid binding protein (FABP) from zebrafish (Danio rerio). Comparison of the sequence with GenBank entries revealed extensive amino acid identity between this zebrafish FABP and brain FABPs (B-FABP) from other species. The zebrafish B-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases BglII or EcoRI suggesting that a single copy of the B-FABP gene is present in the zebrafish genome. Northern blot analysis demonstrated that the zebrafish B-FABP mRNA is approximately 850 nucleotides in length. In situ hybridization revealed that the B-FABP mRNA was expressed in the periventricular gray zone of the optic tectum of the adult zebrafish brain.     

Nucleotide sequence of a cDNA clone coding for an intestinal-type fatty acid binding protein and its tissue-specific expression in zebrafish (Danio rerio)

We have cloned a cDNA from zebrafish (Danio rerio) that contains an open-reading frame of 132 amino acids coding for a fatty acid binding protein (FABP) of approximately 15 kDa. Multiple sequence alignment revealed extensive amino acid identity between this zebrafish FABP and intestinal-like FABPs (I-FABP) from other species. The zebrafish I-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases PstI Bg/II or EcoRI suggesting that a single copy of the I-FABP gene is present in the zebrafish genome. An oligonucleotide probe complementary to the zebrafish I-FABP mRNA hybridized to an mRNA of approximately 800 bases in Northern blot analysis. In situ hybridization revealed that the I-FABP mRNA was expressed exclusively in the intestine of the adult zebrafish.     

Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats

BACKGROUND/AIMS: Cytoplasmic liver fatty acid binding protein (L-FABP) has been suggested to be associated with cellular mitotic activity but the changes in L-FABP mRNA and protein levels during liver regeneration following partial hepatectomy (PHx) are not clear. METHODS: In the present study, we determined the time course of L-FABP mRNA expression and L-FABP levels following 70% PHx using Northern and Western blot, respectively. To elucidate one of the roles for L-FABP in PHx, [3H]-palmitic acid clearance in hepatocytes isolated from 24 h post-PHx and control animals was assessed. RESULTS: L-FABP mRNA increased at 30 min, peaked at approximately 1 h (163 +/- 17%; mean +/- SE, n = 5), and returned to control levels 6 h post-PHx. L-FABP level also increased at 1 h but peaked at 24-h (219 +/- 41%; mean +/- SE, n = 5). Hepatocyte [3H]-palmitic acid clearance increased by 29% at 24-h post-PHx, suggesting an increased intracellular transport (or binding) function by L-FABP. Pre-treatment with dexamethasone statistically reduced L-FABP levels (29%) and suppressed the regenerative process (mitotic activity). CONCLUSIONS: L-FABP mRNA increased sharply in response to PHx but the increase was short lived, while L-FABP level increased at a later stage. Both L-FABP content and fatty acid uptake increased significantly during liver regeneration induced by PHx in rats. It is likely that L-FABP is one of the factors responsible for hepatic regeneration.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.