Functional Characterization Reveals the Significance of Rare Coding Variations in Human Organic Anion Transporting Polypeptide 2B1 (SLCO2B1).

The organic anion transporting polypeptide 2B1 (OATP2B1), which is encoded by the SLCO2B1 gene, plays important roles in the absorption and disposition of its substrate drugs. Nonsynonymous variations of SLCO2B1 change its amino acid sequence and may alter its function. However, so far, very few genetic variants of SLCO2B1 have been functionally characterized. In the present study, first of all, 14 nonsynonymous single nucleotide variants (SNVs) of SLCO2B1 have been identified from the dbSNP database. Then, human embryonic kidney (HEK293) cells were employed as the expression system and functional studies were carried out for these 14 SNVs using substrates 4',5'-dibromofluorescein (DBF), estrone-3-sulfate (E3S), atorvastatin, and rosuvastatin. Our results showed that four nonsynonymous rare variants, namely, SLCO2B1 c.332G > A (p.R111Q), c.1184C > A (p.P395H), c.1624G > A (p.V542M), and c.1998C > A (p.F666L), have great effect on the function of OATP2B1. Surface biotinylation and immunoblot analysis indicated that the variant c.1184C > A (p.P395H) almost completely disrupted OATP2B1's expression on the plasma membrane. According to the three-dimensional structural model of OATP2B1 we developed, these four mutated residues are not located at the substrate binding region of OATP2B1. Their significant effect on the function of OATP2B1 could probably be attributed to jeopardizing OATP2B1's surface expression as exemplified by c.1184C > A (p.P395H), altering the transporter's overall structure and affecting its interactions with other proteins or the lipid bilayer. Taken together, our results demonstrated that rare coding variants could have a great impact on the function and expression of OATP2B1.

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis.

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.

[Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

A vesicular transporter that mediates aspartate and glutamate neurotransmission.

Aspartate, an excitatory amino acid, is known to be stored in synaptic vesicles and exocytosed from some neurons to perform aspartergic neurotransmission. Through in vitro reconstitution, we found that sialin, a lysosomal sialic acid exporter, is responsible for the vesicular storage of aspartate in hippocampal neurons and pinealocytes. Mutations found in Salla disease cause decreased aspartate transport activity without affecting sialic acid transport. Thus, sialin is a multifunctional transporter. It is possible that people with Salla disease lose the ability of aspartergic neurotransmission, and this could explain why Salla disease involves severe neurological defects.

Changes in the central component of the hypothalamus-pituitary-thyroid axis in a rabbit model of prolonged critical illness.

INTRODUCTION: Prolonged critically ill patients reveal low circulating thyroid hormone levels without a rise in thyroid stimulating hormone (TSH). This condition is labeled "low 3,5,3'-tri-iodothyronine (T3) syndrome" or "nonthyroidal illness syndrome (NTI)" or "euthyroid sick syndrome". Despite the low circulating and peripheral tissue thyroid hormone levels, thyrotropin releasing hormone (TRH) expression in the hypothalamus is reduced and it remains unclear which mechanism is responsible. We set out to study whether increased hypothalamic T3 availability could reflect local thyrotoxicosis and explain feedback inhibition-induced suppression of the TRH gene in the context of the low T3 syndrome in prolonged critical illness. METHODS: Healthy rabbits were compared with prolonged critically ill, parenterally fed animals. We visualized TRH mRNA in the hypothalamus by in situ-hybridization and measured mRNA levels for the type II iodothyronine diodinase (D2), the thyroid hormone transporters monocarboxylate transporter (MCT) 8, MCT10 and organic anion co-transporting polypeptide 1C1 (OATP1C1) and the thyroid hormone receptors alpha (TRalpha) and beta (TRbeta) in the hypothalamus. We also measured the activity of the D2 and type III iodothyronine deiodinase (D3) enzymes. RESULTS: In the hypothalamus of prolonged critically ill rabbits with low circulating T3 and TSH, we observed decreased TRH mRNA, increased D2 mRNA and increased MCT10 and OATP1C1 mRNA while MCT8 gene expression was unaltered as compared with healthy controls. This coincided with low hypothalamic thyroxine (T4) and low-normal T3 concentrations, without a change at the thyroid hormone receptor level. CONCLUSIONS: Although expression of D2 and of the thyroid hormone transporters MCT10 and OATP1C1 were increased in the hypothalamus of prolonged critical ill animals, hypothalamic T4 and T3 content or thyroid hormone receptor expression were not elevated. Hence, decreased TRH gene expression, and hereby low TSH and T3 during prolonged critical illness, is not exclusively brought about by hypothalamic thyrotoxicosis, and infer other TRH suppressing factors to play a role.

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.

Identification of a novel murine organic anion transporter like protein 1 (OATLP1) expressed in the kidney.

The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.

Identification and kinetic characterization of HtDTC, the mitochondrial dicarboxylate-tricarboxylate carrier of Jerusalem artichoke tubers.

Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant to cold and freezing. The aim of this study was to perform a kinetic characterization of the mitochondrial dicarboxylate-tricarboxylate carrier (HtDTC) and to assess a possible involvement of this carrier in the cold tolerance of tubers. The HtDTC was purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite and Matrex Gel Orange A. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31.6 kDa. A polyclonal antibody raised against the tobacco DTC cross-reacted with the purified protein on Western blot analysis. In gel trypsin, digestion of the purified HtDTC yielded peptides that exhibited strong amino acid sequence similarity to previously identified plant DTCs. Furthermore, using degenerate primers, a portion of the Htdtc cDNA was amplified and sequenced; this cDNA encoded for a protein with high sequence similarity to known plant homolog DTCs. When reconstituted in liposomes loaded with dicarboxylate (2-oxoglutarate, malate, malonate, succinate, and maleate) or tricarboxylate anions (citrate, trans-aconitate, and isocitrate), the purified HtDTC transported all these anions in exchange with external [14C]2-oxoglutarate. A kinetic characterization of HtDTC was performed: (a) the half-saturation constant Km and the Vmax at 25 degrees C of the 2-oxoglutarate/2-oxoglutarate exchange by reconstituted HtDTC were found to be 360 microM and 10.9 micromol/(min mg protein), respectively; (b) the activation energy Ea of the succinate/2-oxoglutarate exchange by the reconstituted HtDTC was found to be 50.7 kJ/mol constant between -5 and 35 degrees C. Similarly, the activation energy Ea of succinate respiration of isolated Jerusalem artichoke mitochondria, measured between -2 and 35 degrees C, was shown to be constant (65.3 kJ/mol). The physiological relevance of kinetic properties and temperature dependence of transport activities of HtDTC is discussed with respect to the cold tolerance ability of Jerusalem artichoke tubers.

Identification of a novel murine organic anion transporter like protein 1 (OATLP1) expressed in the kidney

The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.

Biology of a novel organic solute and steroid transporter, OSTalpha-OSTbeta.

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.

Novel sialic acid transporter of Haemophilus influenzae.

Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.

Functional characterization of SLCO1B1 (OATP-C) variants, SLCO1B1*5, SLCO1B1*15 and SLCO1B1*15+C1007G, by using transient expression systems of HeLa and HEK293 cells.

OBJECTIVES: SLCO1B1*5 and SLCO1B1*15 have been reported to reduce the clearance of pravastatin in healthy volunteers. However, there remains controversy in the effects of SLCO1B1*5 on the activity of OATP1B1 in vitro. In addition, the effect of SLCO1B1*15 on the function of OATP1B1 has not been studied using cDNA-expression systems. Object of the present study was to study the influence of SLCO1B1*5, *15 and *15+C1007G, a novel haplotype found in a patient with pravastatin-induced myopathy, on the functional properties of OATP1B1 by transient expression systems of HEK293 and HeLa cells using endogenous conjugates and statins as substrates. METHODS: Transporting assays for endogenous substrates were performed using tritium labeled estradiol-17beta-D-glucuronide and estrone-3-sulfate. Quantitation of pravastatin, atorvastatin, cerivastatin and simvastatin were carried out using HPLC tandem mass spectrometry. RESULTS: The transporting activities of cells expressing SLCO1B1*5, *15 and *15+C1007G decreased significantly but those of SLCO1B1*1b, *1a+C1007G and *1b+C1007G were not altered for all of the substrates tested except for simvastatin. Kinetic analysis of pravastatin and atorvastatin showed that Km values were not altered but Vmax values decreased significantly in cells expressing SLCO1B1*5, *15 and *15+C1007G. Immunocytochemical study showed that SLCO1B1*5, *15 and *15+C1007G proteins are localized not only at the plasma membrane but also in the intracellular space. CONCLUSIONS: These findings suggest that 521T>C, existing commonly in SLCO1B1*5, *15 and *15+C1007G, is the key single nucleotide polymorphism (SNP) that determines the functional properties of SLCO1B1*5, *15 and *15+C1007G allelic proteins and that decreased activities of these variant proteins are mainly caused by a sorting error produced by this SNP.

Isolation and characterization of a digoxin transporter and its rat homologue expressed in the kidney.

Digoxin, which is one of the most commonly prescribed drugs for the treatment of heart failure, is mainly eliminated from the circulation by the kidney. P-glycoprotein is well characterized as a digoxin pump at the apical membrane of the nephron. However, little is known about the transport mechanism at the basolateral membrane. We have isolated an organic anion transporter (OATP4C1) from human kidney. Human OATP4C1 is the first member of the organic anion transporting polypeptide (OATP) family expressed in human kidney. The isolated cDNA encodes a polypeptide of 724 aa with 12 transmembrane domains. The genomic organization consists of 13 exons located on chromosome 5q21. Its rat counterpart, Oatp4c1, is also isolated from rat kidney. Human OATP4C1 transports cardiac glycosides (digoxin, K(m) = 7.8 microM and ouabain, K(m) = 0.38 microM), thyroid hormone (triiodothyronine, K(m) = 5.9 microM and thyroxine), cAMP, and methotrexate in a sodium-independent manner. Rat Oatp4c1 also transports digoxin (K(m) = 8.0 microM) and triiodothyronine (K(m) = 1.9 microM). Immunohistochemical analysis reveals that rat Oatp4c1 protein is localized at the basolateral membrane of the proximal tubule cell in the kidney. These data suggest that human OATP4C1/rat Oatp4c1 might be a first step of the transport pathway of digoxin and various compounds into urine in the kidney.

Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans.

We have cloned and functionally characterized an Na+-coupled citrate transporter from Caenorhabditis elegans (ceNAC-2). This transporter shows significant sequence homology to Drosophila Indy and the mammalian Na+-coupled citrate transporter NaCT (now known as NaC2). When heterologously expressed in a mammalian cell line or in Xenopus oocytes, the cloned ceNAC-2 mediates the Na+-coupled transport of various intermediates of the citric acid cycle. However, it transports the tricarboxylate citrate more efficiently than dicarboxylates such as succinate, a feature different from that of ceNAC-1 (formerly known as ceNaDC1) and ceNAC-3 (formerly known as ceNaDC2). The transport process is electrogenic, as evidenced from the substrate-induced inward currents in oocytes expressing the transporter under voltage-clamp conditions. Expression studies using a reporter-gene fusion method in transgenic C. elegans show that the gene is expressed in the intestinal tract, the organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Functional knockdown of the transporter by RNAi (RNA interference) not only leads to a significant increase in life span, but also causes a significant decrease in body size and fat content. The substrates of ceNAC-2 play a critical role in metabolic energy production and in the biosynthesis of cholesterol and fatty acids. The present studies suggest that the knockdown of these metabolic functions by RNAi is linked to an extension of life span and a decrease in fat content and body size.

CD36/fatty acid translocase in rats: distribution, isolation from hepatocytes, and comparison with the scavenger receptor SR-B1.

The new mAb UA009 recognizes an antigen expressed by microvascular endothelium, by lymphatic endothelium, and by some epithelia in a number of organs, including the small intestine, lactating mammary gland, kidney, lung, sebaceous glands, and circumvallate papillae of the tongue. This antigen is also expressed abundantly in the splenic red pulp and marginal zone and by monocytes, macrophages, and erythrocytes (but not by platelets). Among tissues that store or metabolize fatty acids, the antigen is expressed by adipocytes, cardiomyocytes, and red skeletal muscle. Importantly, it is expressed by steroidogenic cells in the adrenal gland, testis, and ovary, whereas in the liver it is expressed by hepatocytes in a pattern that is dependent on gender and genetic background. mAb UA009 immunoprecipitated a mol wt 85-kDa surface protein from detergent extracts of hepatocytes from Dark Agouti female rats. The N-terminal amino acid sequence of this protein was identical to fatty acid translocase (FAT), the rat cluster of differentiation 36 (CD36) ortholog. The mAb also reacted with COS-7 cells transfected with cDNA encoding FAT. cDNAs encoding a CD36/FAT-like polypeptide were prepared from both liver and heart RNA by RT-PCR. The nucleotide sequences obtained from these cDNAs (Dark Agouti rats) revealed identity and 99% similarity, respectively, with the published sequences of Cd36/Fat in rats of the Wistar and Sprague-Dawley strains. The absence of the UA009 antigen in CD36/FAT-deficient SHR/N rats confirmed the identity of the UA009 antigen and CD36/FAT. We suggest that CD36/FAT might function in the liver as a sex-regulated accessory molecule, either in reverse cholesterol transport and/or in fatty acid uptake.

Isolation and partial characterization of CD36 from skim milk.

CD36, a common milk fat globule membrane glycoprotein, was isolated from skim milk by methods similar to those previously utilized for the isolation of sulfhydryl oxidase. Two separate methods that were employed, gave similar purity as observed by electrophoresis. The first was based on differential centrifugation and size-exclusion chromatography, whereas the second combined ultrafiltration and affinity chromatography. After significant purification, the protein was identified by Western blotting and sequence analysis. Deglycosylation decreased the apparent molecular mass from approximately 85 to 57 kDa. These results suggested tissue-specific glycosylation. The purified fractions also exhibited low levels of sulfhydryl oxidase activity, the significance of which will require further study.