Cloning and Tissue Expression of Chicken Heart Fatty Acid-Binding Protein and Intestine Fatty Acid-Binding Protein Genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

Cloning and tissue expression of chicken heart fatty acid-binding protein and intestine fatty acid-binding protein genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

Identification of a human heart FABP pseudogene located on chromosome 13

The fatty acid-binding proteins (FABPs) constitute a conserved group of cytosolic low molecular mass proteins, which consists of several types: liver, heart, myelin, epidermal, adipocyte, brain, intestinal and ileal type. The FABP gene structure is well conserved during evolution and exhibits a four-exon/three-intron structure. In the past, multiple hybridizing fragments were detected upon Southern blot analysis using heart FABP (H-FABP) cDNA as a probe. The origin of these fragments was not clear. We screened a human genomic library and isolated an intronless gene (FABP3-ps) with 85% similarity to the human H-FABP cDNA and high similarity (76 and 79%) to the H-FABP cDNAs of mouse and bovine, respectively. By means of fluorescence in situ hybridization this processed pseudogene could be assigned chromosome 13q13-q14, whereas the gene for human H-FABP (FABP3) resides on chromosome 1p32-p33. No expression of the processed pseudogene could be detected in skeletal muscle or fetal brain.     

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.     

The multigene family of fatty acid-binding proteins (FABPs): Function, structure and polymorphism

Fatty acid-binding proteins (FABPs) are members of the superfamily of lipid-binding proteins (LBP). So far 9 different FABPs, with tissue-specific distribution, have been identified: L (liver), I (intestinal), H (muscle and heart), A (adipocyte), E (epidermal), Il (ileal), B (brain), M (myelin) and T (testis). The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport. The structure of all FABPs is similar - the basic motif characterizing these proteins is beta-barrel, and a single ligand (e.g. a fatty acid, cholesterol, or retinoid) is bound in its internal water-filled cavity. Despite the wide variance in the protein sequence, the gene structure is identical. The FABP genes consist of 4 exons and 3 introns and a few of them are located in the same chromosomal region. For example, A-FABP, E-FABP and M-FABP create a gene cluster. Because of their physiological properties some FABP genes were tested in order to identify mutations altering lipid metabolism. Furthermore, the porcine A-FABP and H-FABP were studied as candidate genes with major effect on fatness traits.     

Tissue-specific Functions in the Fatty Acid-binding Protein Family

The intracellular fatty acid-binding proteins (FABPs) are abundantly expressed in almost all tissues. They exhibit high affinity binding of a single long-chain fatty acid, with the exception of liver FABP, which binds two fatty acids or other hydrophobic molecules. FABPs have highly similar tertiary structures consisting of a 10-stranded antiparallel β-barrel and an N-terminal helix-turn-helix motif. Research emerging in the last decade has suggested that FABPs have tissue-specific functions that reflect tissue-specific aspects of lipid and fatty acid metabolism. Proposed roles for FABPs include assimilation of dietary lipids in the intestine, targeting of liver lipids to catabolic and anabolic pathways, regulation of lipid storage and lipid-mediated gene expression in adipose tissue and macrophages, fatty acid targeting to β-oxidation pathways in muscle, and maintenance of phospholipid membranes in neural tissues. The regulation of these diverse processes is accompanied by the expression of different and sometimes multiple FABPs in these tissues and may be driven by protein-protein and protein-membrane interactions.     

Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes

Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes.     

Evolutionary genomics of the Fox genes: Origin of gene families and the ancestry of gene clusters

Over the past decade genomic approaches have begun to revolutionise the study of animal diversity. In particular, genome sequencing programmes have spread beyond the traditional model species to encompass an increasing diversity of animals from many different phyla, as well as unicellular eukaryotes that are closely related to the animals. Whole genome sequences allow researchers to establish, with reasonable confidence, the full complement of any particular family of genes in a genome. Comparison of gene complements from appropriate genomes can reveal the evolutionary history of gene families, indicating when both gene diversification and gene loss have occurred. More than that, however, assembled genomes allow the genomic environment in which individual genes are found to be analysed and compared between species. This can reveal how gene diversification occurred. Here, we focus on the Fox genes, drawing from multiple animal genomes to develop an evolutionary framework explaining the timing and mechanism of origin of the diversity of animal Fox genes. Ancient linkages between genes are a prominent feature of the Fox genes, depicting a history of gene clusters, some of which may be relevant to understanding Fox gene function.     

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.     

Genomic insights into versatile lifestyle of three new bacterial candidate phyla

Metagenomic explorations of the Earth’s biosphere enable the discovery of previously unknown bacterial lineages of phylogenetic and ecological significance. Here, we retrieved 11 metagenomic-assembled genomes (MAGs) affiliated to three new monophyletic bacterial lineages from the seawater of the Yap Trench. Phylogenomic analysis revealed that each lineage is a new bacterial candidate phylum, subsequently named Candidatus Qinglongiota, Candidatus Heilongiota, and Candidatus Canglongiota. Metabolic reconstruction of genomes from the three phyla suggested that they adopt a versatile lifestyle, with the potential to utilize various types of sugars, proteins, and/or short-chain fatty acids through anaerobic pathways. This was further confirmed by a global distribution map of the three phyla, indicating a preference for oxygen-limited or particle-attached niches, such as anoxic sedimentary environments. Of note, Candidatus Canglongiota genomes harbor genes for the complete Wood- Ljungdahl pathway and sulfate reduction that are similar to those identified in some sulfate-reducing bacteria. Evolutionary analysis indicated that gene gain and loss events, and horizontal gene transfer (HGT) play important roles in shaping the genomic and metabolic features of the three new phyla. This study presents the genomic insight into the ecology, metabolism, and evolution of three new phyla, which broadens the phylum-level diversity within the domain Bacteria.

     

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity,Lifetime and Anisotropy

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions.     

Fatty acid-binding protein from human heart localized in native and denaturing two-dimensional gels

Summary A group of low molecular weight fatty acid-binding cytosolic proteins, FABP_c with high abundance in heart, liver, skeletal muscle, intestine and adipose tissue, are anticipated to play a role in long-chain fatty acid metabolism in these tissues. Recently, a FABP_c with M_T 15 kDa has been purified from human heart muscle and found to be present in levels 2–4% of cytosolic proteins of human heart myocytes. In the present study two-dimensional gel electrophoresis under native and denaturing conditions has been used to characterize FABP_c from human heart and this protein is found to be a major protein of human heart myocytes. The pI of FABP_c from human heart was found to be about 5.3 under native conditions and about 6.5 in the presence of 9 M urea.     

Fatty acid-binding proteins--insights from genetic manipulations

Fatty acid-binding proteins (FABPs) belong to the conserved multigene family of the intracellular lipid-binding proteins (iLBPs). These proteins are ubiquitously expressed in vertebrate tissues, with distinct expression patterns for the individual FABPs. Various functions have been proposed for these proteins, including the promotion of cellular uptake and transport of fatty acids, the targeting of fatty acids to specific metabolic pathways, and the participation in the regulation of gene expression and cell growth. Novel genetic tools that have become available in recent years, such as transgenic cell lines, animals, and knock-out mice, have provided the opportunity to test these concepts in physiological settings. Such studies have helped to define essential cellular functions of individual FABP-types or of combinations of several different FABPs. The deletion of particular FABP genes, however, has not led to gross phenotypical changes, most likely because of compensatory overexpression of other members of the iLBP gene family, or even of unrelated fatty acid transport proteins. This review summarizes the properties of the various FABPs expressed in mammalian tissues, and discusses the transgenic and ablation studies carried out to date in a functional context.     

The analysis of genomic structures in the L1 family of cell adhesion molecules provides no evidence for exon shuffling events after the separation of arthropod and chordate lineages

Members of the L1 family of neural cell adhesion molecules consist of multiple extracellular immunoglobulin and fibronectin type III domains that mediate the adhesive properties of this group of transmembrane proteins. In vertebrate genomes, these protein domains are separated by introns, and it has been suggested that L1-type genes might have been subject to exon-shuffling events during evolution. However, comparison of the human L1-CAM and the chicken neurofascin gene with the genomic structure of their Drosophila homologue, neuroglian, indicates that no major rearrangement of protein domains has taken place subsequent to the split of the arthropod and chordate phyla. The Drosophila neuroglian gene appears to have lost most of the introns that have been conserved in the human L1-CAM and the chicken neurofascin gene. Nevertheless, exon shuffling or the generation of new exons by mutational changes might have been responsible for the generation of additional, alternatively spliced exons in L1-type genes.     

The evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine

Ergothioneine (EGT) is a histidine betaine derivative that exhibits antioxidant action in humans. EGT is primarily synthesized by fungal species and a number of bacterial species. A five-gene cluster (egtA, egtB, egtC, egtD & egtE) responsible for EGT production in Mycobacteria smegmatis has recently been identified. The first fungal biosynthetic EGT gene (NcEgt-1) has also been identified in Neurospora crassa. NcEgt-1 contains domains similar to those found in M. smegmatis egtB and egtD. EGT is biomembrane impermeable. Here we inferred the evolutionary history of the EGT cluster in prokaryotes as well as examining the phyletic distribution of Egt-1 in the fungal kingdom. A genomic survey of 2509 prokaryotes showed that the five-gene EGT cluster is only found in the Actinobacteria. Our survey identified more than 400 diverse prokaryotes that contain genetically linked orthologs of egtB and egtD. Phylogenetic analyses of Egt proteins show a complex evolutionary history and multiple incidences of horizontal gene transfer. Our analysis also identified two independent incidences of a fusion event of egtB and egtD in bacterial species. A genomic survey of over 100 fungal genomes shows that Egt-1 is found in all fungal phyla, except species that belong to the Saccharomycotina subphylum. This analysis provides a comprehensive analysis of the distribution of the key genes involved in the synthesis of EGT in prokaryotes and fungi. Our phylogenetic inferences illuminate the complex evolutionary history of the genes involved in EGT synthesis in prokaryotes. The potential to synthesize EGT is a fungal trait except for species belonging to the Saccharomycotina subphylum.