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.     

An ATP-binding cassette gene (ABCG5) from the ABCG (White) gene subfamily maps to human chromosome 2p21 in the region of the Sitosterolemia locus.

We characterized a new human ATP-binding cassette (ABC) transporter gene that is highly expressed in the liver. The gene, ABCG5, contains 13 exons and encodes a 651 amino acid protein. The predicted protein is closely related to the Drosophila white gene and a human gene, ABCG1, which is induced by cholesterol. This subfamily of genes all have a single ATP-binding domain at the N-terminus and a single C-terminal set of transmembrane segments. ABCG5 maps to human chromosome 2p21, between the markers D2S117 and D2S119. The abundant expression of this gene in the liver suggests that the protein product has an important role in transport of specific molecule(s) into or out of this tissue.     

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s)

The purpose of the present study was to clarify the expression, transport properties and regulation of ATP-binding cassette G2 (ABCG2) transporter at the rat blood-brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2-transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY-prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide-linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain-to-blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR-BBB13), astrocyte (TR-AST4) and pericyte (TR-PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR-BBB13 cells with conditioned medium of TR-AST4 cells, the Ko143 (an ABCG2-specific inhibitor)-sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR-PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.     

ABCG2 Dysfunction Increases Serum Uric Acid by Decreased Intestinal Urate Excretion

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.     

Generation of an ABCG2 transgenic line - A tool to study ABCG2 expression in mice

The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.     

cGMP transport by vesicles from human and mouse erythrocytes

cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.     

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.     

人肾高亲和力钠离子依赖性二羧酸转运蛋白基因的克隆和生物信息学分析

为探讨人高亲和力钠离子依赖性二羧酸转运蛋白 (humanhigh affinitysodium dependentdicar boxylatetransporter,hSDCT2orhNaDC3 )基因在人体内的生理功能及其与疾病的关系 ,借助生物信息学成功地从人肾中克隆了hSDCT2基因 (GenBank接收号 :AY0 72 810 ) .首先将大鼠SDCT2cDNA与人EST数据库进行同源性比较 ,获得具有高度同源性EST片段并用DNAstar软件将它们拼接成EST重叠群 .在重叠群上设计PCR引物从人肾总RNA中用RT PCR扩增出hSDCT2基因并测序 ,然后用软件对其结构特性、组织分布及基因定位进行分析 .序列测定结果显示 ,hSDCT2开放阅读框为180 9bp ,共编码 6 0 2个氨基酸 .蛋白同源性分析表明 ,其氨基酸序列与大鼠及小鼠SDCT2分别有85 %和 87%相同 .二级结构分析显示 ,该蛋白有 12个跨膜螺旋区 .Northern分析显示 ,该基因可在肾、肝、脑、胎盘等多种组织中表达 ,并定位于 2 0号染色体的q12～q13 1     

Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain

Using in situ hybridization for the mouse brain, we analyzed developmental changes in gene expression for the ATP-binding cassette (ABC) transporter subfamilies ABCA1-4 and 7, and ABCG1, 2, 4, 5 and 8. In the embryonic brains, ABCA1 and A7 were highly expressed in the ventricular (or germinal) zone, whereas ABCA2, A3 and G4 were enriched in the mantle (or differentiating) zone. At the postnatal stages, ABCA1 was detected in both the gray and white matter and in the choroid plexus. On the other hand, ABCA2, A3 and A7 were distributed in the gray matter. In addition, marked up-regulation of ABCA2 occurred in the white matter at 14 days-of-age when various myelin protein genes are known to be up-regulated. In marked contrast, ABCA4 was selective to the choroid plexus throughout development. ABCG1 was expressed in both the gray and white matters, whereas ABCG4 was confined to the gray matter. ABCG2 was diffusely and weakly detected throughout the brain at all stages examined. Immunohistochemistry of ABCG2 showed its preferential expression on the luminal membrane of brain capillaries. Expression signals for ABCG5 and G8 were barely detected at any stages. The distinct spatio-temporal expressions of individual ABCA and G transporters may reflect their distinct cellular expressions in the developing and adult brains, presumably, to regulate and maintain lipid homeostasis in the brain.     

Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.     

ABCG4: a novel human white family ABC-transporter expressed in the brain and eye

White family transporters are a group of ATP-binding cassette (ABC) proteins that show sequence homology to the Drosophila white gene product. The Drosophila protein is a subunit of heterodimeric transporters of precursors for eye-pigment synthesis. A novel, human member of this family (ABCG4) has been identified. Northern blotting shows that ABCG4 is expressed specifically in the brain and the eye.     

Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS)

Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28--> q29.     

The 70 kDa peroxisomal membrane protein: an ATP-binding cassette transporter protein involved in peroxisome biogenesis

The 70 kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. cDNAs for human and rat PMP70 have been cloned and sequenced and the gene mapped to the human chromosome 1p21-22. The predicted amino acid sequence showed homology to members of the ATP-binding cassette transporter family. In humans, mutations in the PMP70 gene have been found in a subset of patients with Zellweger syndrome, a lethal inborn error of peroxisome biogenesis. These results suggest that PMP70 functions in transporting molecules or possibility peptides across the peroxisomal membrane and has an important role in peroxisome assembly.     

SHRSP/Izm and WKY/NCrlCrlj rats having a missense mutation in Abcg5 deposited plant sterols in the body, but did not change their biliary secretion and lymphatic absorption-comparison with Jcl:Wistar and WKY/Izm rats

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.     

Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. We previously reported the cloning of C4ST cDNA from mouse brain. We here report the cloning and expression of human C4ST cDNA. The cDNA was isolated from a human fetal brain cDNA library by hybridization with a DNA probe prepared from rat poly(A)(+) RNA used for the cloning of mouse C4ST cDNA. The cDNA comprises a single open reading frame that predicts a Type II transmembrane protein composed of 352 amino acids. The protein has an amino acid sequence homology of 96% with mouse C4ST. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that transfers sulfate to both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis indicated that human C4ST mRNAs (6.0 and 1.9 kb) are expressed ubiquitously in various adult human tissues. Dot blot analysis has shown that human C4ST is strongly expressed in colorectal adenocarcinoma and peripheral blood leukocytes, whereas strong expression of human chondroitin 6-sulfotransferase (C6ST) is observed in aorta and testis. These observations suggest that the expression of C4ST and C6ST may be controlled differently in human tissues. The C4ST gene was localized to chromosome 12q23.2-q23.3 by fluorescence in situ hybridization.     

Deletion of the transmembrane transporter ABCG1 results in progressive pulmonary lipidosis

We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1(-/-) mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3- and 8-month-old Abcg1(-/-) mice. These data suggest that the lungs of young Abcg1(-/-) mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1(-/-), but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1(-/-)mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.     

SHRSP/Izm and WKY/NCrlCrlj Rats Having a Missense Mutation in Abcg5 Deposited Plant Sterols in the Body,but Did Not Change Their Biliary Secretion and Lymphatic Absorption—Comparison with Jcl:Wistar and WKY/Izm Rats

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.