Molecular determinants of specificity for synthetic nucleoside analogs in the concentrative nucleoside transporter, CNT2

Members of the concentrative nucleoside transporter (CNT) family (SLC28) mediate the transport of naturally-occurring nucleosides, and nucleoside analog drugs across the plasma membrane of epithelial cells. Each of the three CNT family members has a distinct specificity for naturally occurring nucleosides, and residues that contribute to the specificity of each transporter have been identified. In contrast, the molecular determinants of specificity for synthetic nucleoside analogs are not known. In this study, we take advantage of the large species difference that exists between human and rat CNT2 (hCNT2 and rCNT2) in their ability to transport the nucleoside analog drug cladribine, 2CdA, (rCNT2 > > > hCNT2) to identify the critical domains and amino acid residues that contribute to the observed difference in specificity between CNT2 orthologs. Using chimeric proteins of human and rat CNT2, we determined that the C-terminal half of CNT2 contained the determinants of 2CdA selectivity. We replaced key residues in the C terminus of hCNT2 with the equivalent residue in rCNT2. One residue in the C-terminal portion of CNT2 was found to significantly contribute to 2CdA selectivity: hCNT2-S354A. This mutant caused an increase of 5-6-fold over hCNT2. The 2-chloro pharmacophore, rather than the 2'-deoxyribose was responsible for the reduced 2CdA uptake by hCNT2. Our data are consistent with a model in which an increased capability for hydrogen bonding in critical amino acids that reside in the C terminus of rCNT2 contributes to its enhanced selectivity for 2CdA.     

Cloning, genomic organization and chromosomal localization of the gene encoding the murine sodium-dependent, purine-selective, concentrative nucleoside transporter (CNT2)

A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides. The cDNA was used to screen a murine (strain 129SvJ/6) genomic library in pBeloBAC11 to identify a clone containing the mCNT2 gene. A PCR strategy was used to identify and sequence the intron-exon boundaries and to determine the approximate sizes of the introns. The mCNT2 gene spans approximately 13.7 kb and is encoded by 15 exons. The gene was mapped to mouse chromosome 2e3 by fluorescence in situ hybridization.     

Characterization of equilibrative and concentrative Na+-dependent (cif) nucleoside transport in acute promyelocytic leukemia NB4 cells

Nucleoside transport processes can be classified by the transport mechanism, e = equilibrative and c = concentrative, by the sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), s = sensitive and i = insensitive, and also by permeant selectivity. To characterize nucleoside transport in acute promyelocytic NB4 cells, nucleoside transport was resolved into different components by selective elimination of transport processes with NBMPR and with Na⁺-deficient media. Initial transport rates were estimated from time course experiments. For adenosine, uridine, and formycin B, equilibrative transport accounted for approximately 60% of their uptake, with ei and es transport contributing almost equally, and Na⁺-dependent transport accounting for the remaining 40% of the total uptake. Thymidine uptake was mediated exclusively by equilibrative systems with ei and es systems each contributing 50% to total uptake. Adenosine accumulated above equilibrative concentrations, suggesting that a concentrative transport process was active and/or that metabolism led to adenosine's accumulation. Formycin B, a nonmetabolizable analog, also accumulated in the cells, supporting the concentrative potential of the Na⁺-dependent transporter. Kinetic analyses also provided evidence for three distinct high affinity transport mechanisms. NBMPR binding assays indicated the presence of two high affinity (K_m 0.10 and 0.35 nM) binding sites. In conclusion, NB4 cells express ei and es transport, as well as a large ci transport component, which appears to correspond to cif (f = formycin B or purine selective) nucleoside transport, not previously described in human cells. © 1996 Wiley-Liss, Inc.     

Fatty acids alter monolayer integrity, paracellular transport, and iron uptake and transport in Caco-2 cells

The Caco-2 cell line was used as a model to determine if the type of fatty acid, unsaturated versus saturated, differentially altered the uptake and transport of iron in the human intestine and if the changes were the result of alterations in monolayer integrity and paracellular transport. Cells were cultured in either a lower-iron or normal-iron medium and incubated with a bovine serum albumin control, linoleate, oleate, palmatate, or stearate. Oleate, palmatate, and stearate enhanced (p<0.05) iron uptake in cells grown in lower iron. The fatty acid effect on iron uptake by cells grown in normal iron was not as pronounced. Iron transport was not affected (p>0.05) by an interaction between the type of medium (iron concentration) and the type of fatty acid. Iron transport was enhanced (p<0.05) with palmatate and stearate. Various indicators of monolayer integrity and paracellular transport were also affected by the fatty acids, thus impacting iron uptake and transport. These results indicate that oleate, palmatate, and stearic can enhance iron uptake and transport; however, this enhancement may be the result of alterations in the integrity of the intestine. A portion of the data was presented at Experimental Biology 96 as a poster session. E. A. Droke, L. K. Johnson, and H. C. Lukaski. Fatty acids affect iron uptake and transport in Caco-2 cells. FASEB J. 10, 1431 (1996).     

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.     

Cell-cycle-dependent regulation of CNT1, a concentrative nucleoside transporter involved in the uptake of cell-cycle-dependent nucleoside-derived anticancer drugs

Most nucleoside-derived anticancer drugs are taken up by the high-affinity Na-dependent nucleoside transporter CNT1. Since such drugs are to some extent cell-cycle-dependent in their cytotoxic action, we examined the relationship between CNT1 expression and cell-cycle progression in the rat hepatoma cell line FAO. Cell cultures were synchronized either at late G1 or early S stages by combining mimosin treatment with either previous synchronization or not by serum starvation. Cell-cycle progression was then assessed by measuring [methyl-3H]thymidine incorporation into DNA and monitoring cyclin E and A protein levels. In these conditions, CNT1 protein amounts increase at the G1-S transition. When cells were synchronized using hydroxyurea (HU), which directly interacts with nucleotide metabolism by inhibiting ribonucleotide reductase, CNT1 protein amounts increased in synchronized cells and remained high during cell-cycle progression. These data indicate that CNT1 adapts to cell-cycle progression and responds to nucleos(t)ide metabolism status, a feature that might contribute to the cytotoxic action of cell-cycle-dependent anticancer drugs.     

Inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hCNT3) predicts an elevator-type transport mechanism

The human SLC28 family of concentrative (Na⁺-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNT_NW to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter’s outward-facing conformation. In addition, both the Na⁺ and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.     

Immunohistochemical localization of alpha1-acid-glycoprotein in human liver parenchymal cells.

alpha1-Acid glycoprotein, a major human serum glycoprotein was detected and localized in human liver parenchymal cells of a biopsy specimen. A heavy metal salt containing fixative was required to retain sufficient antigen determinants of alpha1-acid glycoprotein in order to visualize this protein by the peroxidase-anti-peroxidase unlabeled antibody enzyme method.     

Cellular localization of sulfobromophthalein transport activity in rat liver

The movement of sulfobromophthalein is measured in rat liver plasma-membrane vesicles by direct dual-wavelength spectrophotometry. The technique is based on the principle that the dye, when entering a more acidic compartment, changes its absorption in the visible region. From this study it may be concluded that, among the different cellular subfractions, only liver plasma-membrane vesicles can catalyze electrogenic transport of sulfobromophthalein. Plasma membranes from erythrocytes are unable to perform such a function. The movement follows the distribution pattern of (Na⁺+K⁺-ATPase and it is therefore concluded that this process occurs exclusively at the sinusoidal membrane level. Inhibition studies confirm that the process is catalyzed by bilitranslocase.     

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods

The Na⁺-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K_0.5) was in the low micromolar range (12–34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I_max) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I_max. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I_max. Fludarabine and formycin B were also transported with higher I_max than uridine and moderate affinity (102 ± 10 and 66 ± 6 µM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about –39 mV and a valence of about –0.8. K_0.5 for Na⁺ was 2.3 mM at –50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na⁺ per nucleoside. This discrepancy in the number of Na⁺ molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na⁺ binding sites. two-electrode voltage clamp; concentrative nucleoside transport; presteady-state currents     

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.     

GII型诺如病毒VP2蛋白在昆虫细胞中的表达与定位研究

【目的】利用杆状病毒表达系统表达诺如病毒(GenegroupⅡ)VP2蛋白,分析其亚细胞定位,为深入研究VP2蛋白的功能奠定基础。【方法】设计可扩增完整ORF3基因片段的引物P1和P2,在下游引物中引入6×His标签的编码序列,从质粒pMD-ORF3中克隆了含有6×His编码序列的ORF3基因,与pFastBac1载体连接,构建重组质粒pFB-ORF3,转化DH10Bac感受态细胞获得重组杆状病毒基因组Bac-ORF3,脂质体介导转染sf9昆虫细胞获得表达VP2蛋白的重组杆状病毒Ac-VP2,感染sf9细胞后,收集病变细胞,采用抗6×His标签的单克隆抗体作为一抗进行Western blot与间接免疫荧光实验鉴定。【结果】Western blot实验证实Ac-VP2感染的sf9细胞在约29 kD处出现特异性条带;间接免疫荧光实验证实Ac-VP2感染的sf9细胞出现特异性绿色荧光,并且VP2主要定位于sf9的细胞核与细胞膜。【结论】诺如病毒VP2蛋白在Ac-VP2感染的sf9细胞中获得成功表达,并且主要定位于sf9细胞的细胞核与细胞膜。     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

Summary The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.