Cloning of human and rat ABCA5/Abca5 and detection of a human splice variant

We presently report the cloning of cDNA sequences encoding the novel rat ATP-binding cassette (ABC) transporter Abca5 and the orthologous human transporter, recently designated as ABCA5. Furthermore, the existence of a novel non-translated exon of the ABCA5 gene, previously assigned to an ABCA gene cluster in the chromosomal region 17q24.2-3, is demonstrated. Abca5 and ABCA5 cDNAs are predicted to give rise to proteins of 1642 amino acids which exhibit the typical domain arrangement of ABC full transporters and share 90% identity within the amino acid sequences. A cDNA representing an ABCA5 mRNA splice variant was cloned which would result in a truncated protein equivalent to an ABC half transporter. Northern blot analyses revealed expression of ABCA5 or Abca5 mRNA in several tissues, but particularly high Abca5 mRNA expression was observed in rat testis. Up-regulation of Abca5 mRNA expression during culture of primary rat hepatocytes suggests that hepatocyte cultures should provide a basis for investigation of Abca5 gene regulation and elucidation of Abca5 function.     

Three ATP-binding cassette transporter genes,Abca14, Abca15, and Abca16, form a cluster on mouse Chromosome 7F3

We have identified and cloned three mouse genes that belong to the ABCA subfamily of ATP-binding cassette (ABC) transporters. These three genes are arranged in a tandem head-to-tail cluster spanning about 300 kb on mouse Chromosome (Chr) 7F3. Phylogenetic analysis indicates that although the three genes are related to human and mouse ABCA3, they are not orthologs of any of the current list of 48 human ABC genes and were, therefore, named Abca14, Abca15, and Abca16. The coding region of each gene is split into 31 exons, has an open reading frame of more than 1600 amino acids, and encodes a full transporter molecule with two nucleotide-binding folds (NBF) and two transmembrane domains (TMD). All three genes are predominantly expressed in testis, which suggests that they may perform special functions in testicular development or spermatogenesis. Interestingly, the human genome contains only fragments (less than ten exons) of at least two different ABC genes in the syntenic region on Chromosome 16p12 that are scattered among other, unrelated genes and are not capable of coding functional ABC transporters.(Zhang-qun Chen and Tarmo Annilo) These authors contributed equally to this study.Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession numbers AY243470–AY243472.     

Molecular structure of a novel cholesterol-responsive A subclass ABC transporter,ABCA9

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.     

Genome-wide identification and evolution of ATP-binding cassette transporters in the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>: A case of functional divergence in a multigene family

Background  

In eukaryotes, ABC transporters that utilize the energy of ATP hydrolysis to expel cellular substrates into the environment are responsible for most of the efflux from cells. Many members of the superfamily of ABC transporters have been linked with resistance to multiple drugs or toxins. Owing to their medical and toxicological importance, members of the ABC superfamily have been studied in several model organisms and warrant examination in newly sequenced genomes.     

<Emphasis Type="Italic">AtMRP6</Emphasis>/<Emphasis Type="Italic">AtABCC6</Emphasis>, an ATP-Binding Cassette transporter gene expressed during early steps of seedling development and up-regulated by cadmium in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

ABC proteins constitute one of the largest families of transporters found in all living organisms. In Arabidopsis thaliana, 120 genes encoding ABC transporters have been identified. Here, the characterization of one member of the MRP subclass, AtMRP6, is described.     

Evolutionary dynamics of the ABCA chromosome 17q24 cluster genes in vertebrates

ABCA is a subfamily of ATP-binding-cassette (ABC) transporter genes. In this subfamily, it was found that five ABCA genes cluster in a head-to-tail pattern in the human and mouse genomes, but only one was found in fish. To understand better the evolution of this cluster of genes, we screened 11 vertebrate genome sequences and newly identified 28 ABCA cluster genes. Comparative genomic analysis reveals that the ABCA5 gene is relatively evolutionarily conserved. In contrast, the repertoires of the other ABCA genes in this cluster diverge tremendously among species, which is due mainly to postspeciation duplications. In addition, maximum likelihood analysis reveals that positive selection is acting on the paralogous genes ABCA6 and Abca8a, suggesting that these two genes have possibly acquired new functions after duplication. Because most eukaryotic ABC proteins integrate into the cytoplasmic membrane and transport a wide range of substrates across it, we conjecture that newly duplicated ABCA cluster genes are under diversifying selection for the ability to recognize a diverse array of substrates.     

The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation

ATP-binding cassette (ABC) transporters form a large superfamily of transporters that bind and hydrolyze ATP to transport various molecules across limiting membranes or into vesicles. The ABCA subfamily members are thought to transport lipid materials. ABCA12 is a keratinocyte transmembrane lipid transporter protein associated with the transport of lipids via lamellar granules. ABCA12 is considered to transport lipids including ceramides to form extracellular lipid layers in the stratum corneum of the epidermis, which is essential for skin barrier function. ABCA12 mutations are known to underlie the three major types of autosomal recessive congenital ichthyoses: harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma. ABCA12 mutations result in defective lipid transport via lamellar granules in the keratinocytes, leading to ichthyosis phenotypes from malformation of the stratum corneum lipid barrier. Studies on ABCA12-deficient bioengineered models have revealed that lipid transport by ABCA12 is required for keratinocyte differentiation and epidermal morphogenesis. Defective lipid transport due to loss of ABCA12 function leads to the accumulation of intracellular lipids, including glucosylceramides and gangliosides, in the epidermal keratinocytes. The accumulation of gangliosides seems to result in the apoptosis of Abca12^−/− keratinocytes. It was reported that AKT activation occurs in Abca12^−/− granular-layer keratinocytes, which suggests that AKT activation serves to prevent the cell death of Abca12^−/− keratinocytes. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for α-keto acid binding

Background  

The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H⁺ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds α-keto acids in vitro.     

ABC transporters as cancer drivers: Potential functions in cancer development

Background

ABC transporters have attracted considerable attention for their function as drug transporters in a broad range of tumours and are therefore considered as major players in cancer chemoresistance. However, less attention has been focused on their potential role as active players in cancer development and progression.

Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

Background  

The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) on human retinal pigment epithelial cells is investigated.     

ABC transporter FtsABCD of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> mediates uptake of ferric ferrichrome

Background  

The Streptococcus pyogenes or Group A Streptococcus (GAS) genome encodes three ABC transporters, namely, FtsABCD, MtsABC, and HtsABC, which share homology with iron transporters. MtsABC and HtsABC are believed to take up ferric (Fe³⁺) and manganese ions and heme, respectively, while the specificity of FtsABCD is unknown.     

脊椎动物ABCA基因亚家族研究进展

ABC（ATP-binding cassette）基因家族编码膜蛋白,其成员负责多种物质的跨膜运输。基于氨基酸序列的同源性,人的48个ABC成员被分为7个亚家族：ABCA~ABCG。与其他亚家族相比,ABCA基因编码的蛋白具有独特的拓扑结构,并且其家族成员在两栖动物和哺乳动物分化之后各发生过一次大的扩展（expanding）。基因结构分析发现这两次扩展均是通过基因倍增实现的,这些倍增的产物在啮齿目和食肉目中得到保留,而在灵长目中却有一半变成假基因或被删除。ABCA成员主要负责不同组织器官脂类和胆固醇的跨膜运输,部分成员的突变与疾病相关。     

Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling

Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1^−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1^+/+ and Abca1^−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1^+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1^−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.Lipid rafts are cholesterol-enriched membrane microdomains, thought to be present in all cells, that concentrate and organize cell-surface signal transduction events in several signaling cascades, including those of the Toll-like receptors (TLRs) (1). The selectivity of rafts for particular proteins, and, consequently, the signal strength of pathways initiating from ligated raft-resident receptors, are thought to derive in large part from the high cholesterol content of raft microdomains (2–4). In vitro, altering raft cholesterol of living cells downward or upward with chemical tools (e.g. cyclodextrins) leads to parallel changes in raft protein abundance (3, 4). The relevance of cholesterol-driven alterations in the raft proteome to disease is suggested by reports that hypercholesterolemia cholesterol-loads macrophage rafts and amplifies their responsiveness to lipopolysaccharide (LPS) (3, 4). Proteomic strategies have recently been applied to raft isolates from a variety of cell types, aiming to better understand the identity of proteins tonically present in rafts, as well as proteins dynamically recruited to rafts upon cell stimulation (2, 5–8). To date, however, most reports have used cell lines of uncertain physiological relevance. In addition, although raft cholesterol levels are regulated in vivo by intracellular cholesterol trafficking (1), no reports to date have sought to define how the raft proteome is physiologically regulated by cholesterol trafficking proteins.ATP binding cassette (ABC) A1, a member of the ABC transporter superfamily, plays a key role in regulating levels of cholesterol in macrophages and other cells via promoting efflux of cellular cholesterol to extracellular acceptors, in particular lipid-free apolipoprotein (apo) A-I (9). The importance of ABCA1¹ to human health is clearly illustrated by Tangier disease, a rare ABCA1 mutation syndrome typified by severe HDL deficiency, widespread macrophage foam cells, and premature atherosclerosis (10). In addition, the large number of common ABCA1 polymorphisms that have been associated with human cardiovascular disease (10) suggest a broad-spanning impact of ABCA1 on human health. It remains somewhat controversial whether ABCA1-effluxed cholesterol derives from raft or extra-raft membranes (11). Nonetheless, both human Tangier disease cells and ABCA1-null murine macrophages have been shown to have greatly expanded lipid rafts that contain increased cholesterol and increased TLR4 (12, 13). These changes are associated with enhanced responsiveness to LPS that can be reversed by cholesterol depletion (13–15). Collectively, these findings indicate that ABCA1 may regulate the raft proteome and innate immune response through control of raft cholesterol. However, no proteomic analysis of rafts from ABCA1-deficient cells has been reported to date.Herein, we report a proteomic analysis of raft isolates from naive and LPS-stimulated Abca1^+/+ and Abca1^−/− primary murine macrophages. Unexpectedly, we found that ABCA1 deletion and LPS stimulation induced many similar changes in the raft proteome. Stomatin-like protein 2 (SLP-2), a lesser known member of the stomatin-prohibitin-flotillin-HflK/C (SPFH) family of membrane scaffolding proteins, was unique among SPFH proteins in being robustly up-regulated in rafts of unstimulated Abca1^−/− cells compared with Abca1^+/+ counterparts. We found that rafts of SLP-2 knockdown cells were abnormal, displaying increased binding of cholera toxin subunit B—a probe for the raft-specific ganglioside GM1—but markedly decreased protein, including flotillins-1 and -2, and CD14. Whereas SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux, it reduced macrophage responsiveness to LPS and multiple additional TLR ligands. Taken together, we report that ABCA1 regulates the macrophage raft proteome and identify SLP-2 as a novel ABCA1-dependent regulator of raft composition that controls the innate immune response.     

Gestational age‐dependent gene expression profiling of ATP‐binding cassette transporters in the healthy human placenta

The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan^® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.     

ABC transporters B1, C1 and G2 differentially regulate neuroregeneration in mice

Background

ATP-binding cassette (ABC) transporters are essential regulators of organismic homeostasis, and are particularly important in protecting the body from potentially harmful exogenous substances. Recently, an increasing number of in vitro observations have indicated a functional role of ABC transporters in the differentiation and maintenance of stem cells. Therefore, we sought to determine brain-related phenotypic changes in animals lacking the expression of distinct ABC transporters (ABCB1, ABCG2 or ABCC1).

Methodology and Principal Findings

Analyzing adult neurogenesis in ABC transporter-deficient animals in vivo and neuronal stem/progenitor cells in vitro resulted in complex findings. In vivo, the differentiation of neuronal progenitors was hindered in ABC transporter-deficient mice (ABCB1^0/0) as evidenced by lowered numbers of doublecortin⁺ (−36%) and calretinin⁺ (−37%) cells. In vitro, we confirmed that this finding is not connected to the functional loss of single neural stem/progenitor cells (NSPCs). Furthermore, assessment of activity, exploratory behavior, and anxiety levels revealed behavioral alterations in ABCB1^0/0 and ABCC1^0/0 mice, whereas ABCG2^0/0 mice were mostly unaffected.

Conclusion and Significance

Our data show that single ABC transporter-deficiency does not necessarily impair neuronal progenitor homeostasis on the single NSPC level, as suggested by previous studies. However, loss of distinct ABC transporters impacts global brain homeostasis with far ranging consequences, leading to impaired neurogenic functions in vivo and even to distinct behavioral phenotypes. In addition to the known role of ABC transporters in proteopathies such as Parkinson''s disease and Alzheimer''s disease, our data highlight the importance of understanding the general function of ABC transporters for the brain''s homeostasis and the regeneration potential.