Expression,Purification and Low-Resolution Structure of Human Vitamin C Transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.     

A C-terminal region dictates the apical plasma membrane targeting of the human sodium-dependent vitamin C transporter-1 in polarized epithelia

The human sodium-dependent vitamin C transporter (hSVCT1) mediates sodium-dependent cellular uptake of the essential micronutrient l-ascorbic acid (vitamin C). However, the molecular determinants that control the cell surface expression, subcellular distribution, and dynamics of hSVCT1 remain undefined. To identify molecular determinants involved in hSVCT1 targeting in polarized epithelia, we used live cell imaging approaches to resolve the targeting and trafficking dynamics of hSVCT1 truncation mutants in renal and intestinal cells. Confocal imaging demonstrated that hSVCT1 was expressed at the apical cell surface and video rate measurements revealed hSVCT1 also resided in a heterogeneous population of intracellular organelles with discrete dynamic properties. By progressive truncation of the cytoplasmic C-terminal tail of hSVCT1, we delimited an essential role for an embedded ten amino acid sequence PICPVFKGFS (amino acids 563-572) in defining the physiological targeting of hSVCT1. Intriguingly, this sequence bears significant homology to recently identified apical targeting motifs in two other sodium-dependent transporters, and we suggest this conservation is reflected topologically through the adoption of a beta-turn confirmation in the cytoplasmic C-tail of each transporter. Our results provide the first direct resolution of functional hSVCT1 expression at the apical cell surface of polarized epithelia and define an apical targeting signal of relevance to transporters of diverse substrate specificity.     

Sodium-dependent vitamin C transporter isoforms in skin: Distribution, kinetics, and effect of UVB-induced oxidative stress

Two sodium-dependent vitamin C transporter isoforms (SVCT1 and SVCT2) were identified as ascorbic acid transporters, but their roles in skin have, as yet, not been elucidated. Here we analyze the expression and function of SVCTs in healthy human skin cells and skin tissues, and in UVB-induced cutaneous tissue injury. SVCT1 was primarily found in the epidermis expressed by keratinocytes, whereas SVCT2 expression was in the epidermis and dermis in keratinocytes, fibroblasts, and endothelial cells. Uptake experiments revealed that ascorbic acid affinity of SVCT1 was lower than SVCT2 (K(m)=75 muM and K(m)=44 muM, respectively), but maximal velocity was 9-times higher (36 nmol/min/well). In keratinocytes, SVCT1 was found to be responsible for vitamin C transport, although SVCT2 gene expression was higher. On UVB irradiation, SVCT1 mRNA expression in murine skin declined significantly in a time- and dose-dependent manner, whereas SVCT2 mRNA levels were unchanged. Furthermore, UVB irradiation of keratinocytes in vitro was accompanied by reduced ascorbic acid transport. In summary, these data indicate that the two vitamin C transporter isoforms fulfill specific functions in skin: SVCT1 is responsible for epidermal ascorbic acid supply, whereas SVCT2 mainly facilitates ascorbic acid transport in the dermal compartment. UVB-induced oxidative stress in mice resulted in depletion of SVCT1 mRNA levels and led to significantly decreased ascorbic acid uptake in keratinocytes, providing evidence on why ascorbic acid levels are decreased on UVB irradiation in vivo.     

N-Glycosylation is required for Na+-dependent vitamin C transporter functionality

The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased ¹⁴C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.     

Acetylation regulates the stability of glutamate carboxypeptidase II protein in human astrocytes

Glutamate carboxypeptidase II (GCPII) is known to be implicated in brain diseases such as schizophrenia and bipolar disorder, and dramatically increases in prostate cancer. Here, we investigated the regulation of GCPII expression in astrocytes and examined whether GCPII is epigenetically regulated through histone modification. In this study, valproic acid (VPA), a drug used for bipolar disorder and epilepsy and a known histone deacetylase (HDAC) inhibitor was used. We found that acute exposure of VPA for 4–6 h increased the GCPII protein level in human astrocyte U87MG cells but did not have a similar effect after 12–24 h exposure. Real-time polymerase chain reaction analysis revealed that VPA did not affect the GCPII mRNA expression. In contrast, decrease in GCPII protein level by cycloheximide treatment was blocked by VPA, indicating that VPA increases GCPII protein stability. Treatment with MG132, a proteasome inhibitor, suggested that the VPA-induced increase of GCPII protein level is dependent on the ubiquitin/proteasome pathway. In addition, immunoprecipitation analysis revealed that VPA increased the acetylation of GCPII protein at the lysine residues and facilitated a decrease of the poly-ubiquitinated GCPII level. Similarly, M344, a specific HDAC 1/6 inhibitor, also increased the GCPII protein level. In contrast, treatment with C646, a histone acetyltransferase inhibitor of p300/CBP, significantly reduced the level of GCPII protein. Taken together, this study demonstrated that the increase in GCPII induced by VPA is not due to the classical epigenetic mechanism, but via enhanced acetylation of lysine residues in GCPII.     

Polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells

Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.     

Human vitamin C (L-ascorbic acid) transporter SVCT1

In human, vitamin C (l-ascorbic acid) is an essential micronutrient required for an array of biological functions including enzymatic reactions and antioxidation. We describe here the molecular cloning of a novel human cDNA encoding a vitamin C transporter SVCT1. SVCT1 is largely confined to bulk-transporting epithelia (e.g., kidney and small intestine) with a putative alternative-splice product present in thymus. Applying radiotracer and voltage-clamp approaches in cRNA-injected Xenopus oocytes, we found that SVCT1 mediates saturable, concentrative, high-affinity l-ascorbic acid transport (K(0.5) = 50-100 microM) that is electrogenic and can be inhibited by phloretin. SVCT1 displays exquisite substrate selectivity, greatly favoring l-ascorbic acid over its isomers d-isoascorbic acid and dehydroascorbic acid and 2- or 6-substituted analogues, whereas glucose and nucleobases are excluded. We have mapped the SLC23A2 gene (coding for SVCT1) to human chromosome 5 in band 5q31.2-31.3, within a region commonly deleted in malignant myeloid (leukemia) diseases. In addition, we have demonstrated that the human SLC23A1 gene product is a related high-affinity l-ascorbic acid transporter (SVCT2) that is widely distributed in brain, retina, and a host of endocrine and neuroendocrine tissues. The molecular identification of the human l-ascorbic acid transporters now provides the tools with which to investigate their roles in vitamin C metabolism in health and disease.     

HDAC1 Regulates the Proliferation of Radial Glial Cells in the Developing Xenopus Tectum

In the developing central nervous system (CNS), progenitor cells differentiate into progeny to form functional neural circuits. Radial glial cells (RGs) are a transient progenitor cell type that is present during neurogenesis. It is thought that a combination of neural trophic factors, neurotransmitters and electrical activity regulates the proliferation and differentiation of RGs. However, it is less clear how epigenetic modulation changes RG proliferation. We sought to explore the effect of histone deacetylase (HDAC) activity on the proliferation of RGs in the visual optic tectum of Xenopus laevis. We found that the number of BrdU-labeled precursor cells along the ventricular layer of the tectum decrease developmentally from stage 46 to stage 49. The co-labeling of BrdU-positive cells with brain lipid-binding protein (BLBP), a radial glia marker, showed that the majority of BrdU-labeled cells along the tectal midline are RGs. BLBP-positive cells are also developmentally decreased with the maturation of the brain. Furthermore, HDAC1 expression is developmentally down-regulated in tectal cells, especially in the ventricular layer of the tectum. Pharmacological blockade of HDACs using Trichostatin A (TSA) or Valproic acid (VPA) decreased the number of BrdU-positive, BLBP-positive and co-labeling cells. Specific knockdown of HDAC1 by a morpholino (HDAC1-MO) decreased the number of BrdU- and BLBP-labeled cells and increased the acetylation level of histone H4 at lysine 12 (H4K12). The visual deprivation-induced increase in BrdU- and BLBP-positive cells was blocked by HDAC1 knockdown at stage 49 tadpoles. These data demonstrate that HDAC1 regulates radial glia cell proliferation in the developing optical tectum of Xenopus laevis.     

Functional role of conserved transmembrane segment 1 residues in human sodium-dependent vitamin C transporters

Sodium-dependent vitamin C transporters, SVCT1 and SVCT2, are the only two known proteins for the uptake of ascorbate, the active form of vitamin C. Little structural information is available for SVCTs, although a transport activity increase from pH 5.5 to 7.5 suggests a functional role of one or more conserved histidines (p K a approximately 6.5). Confocal fluorescence microscopy and uptake kinetic analyses were used here to characterize cells transfected with mutants of EGFP-tagged hSVCTs. Mutating any of the four conserved histidine residues (His51, 147, 210, or 354) in hSVCT1 to alanine did not affect the apical membrane localization in polarized MDCK cells. His51Ala (in putative transmembrane segment 1, TM1) was the only mutation that resulted in a significant loss of ascorbate transport and an increase in apparent Km with no significant effect on Vmax. The corresponding mutation in hSVCT2, His109Ala, also led to a loss of transport activity. Among eight other mutations of His51 in hSVCT1, significant sodium-dependent ascorbate transport activity was only observed with asparagine or tyrosine replacement. Thus, our results suggest that uncharged His51, directly or indirectly, contributes to substrate binding through the hydrogen bond. His51 cannot account for the observed pH dependence as neutral amino acid substitutions failed to abolish the pH-dependent activity increase. The importance of TM1 is further strengthened by the comparable loss of sodium-dependent ascorbate transport activity upon the mutation of adjacent conserved Gln50 and the apparent change in substrate specificity in the hSVCT1-His51Gln mutation, which showed a specific increase in sodium-independent dehydroascorbate transport.     

Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and Glucose

Vitamin C and flavonoids, polyphenols with uncertain function, are abundant in fruits and vegetables. We postulated that flavonoids have a novel regulatory action of delaying or inhibiting absorption of vitamin C and glucose, which are structurally similar. From six structural classes of flavonoids, at least 12 compounds were chosen for studies. We investigated the effects of selected flavonoids on the intestinal vitamin C transporter SVCT1(h) by transfecting and overexpressing SVCT1(h) in Chinese hamster ovary cells. Flavonoids reversibly inhibited vitamin C transport in transfected cells with IC(50) values of 10-50 microm, concentrations expected to have physiologic consequences. The most potent inhibitor class was flavonols, of which quercetin is most abundant in foods. Because Chinese hamster ovary cells have endogenous vitamin C transport, we expressed SVCT1(h) in Xenopus laevis oocytes to study the mechanism of transport inhibition. Quercetin was a reversible and non-competitive inhibitor of ascorbate transport; K(i) 17.8 microm. Quercetin was a potent non-competitive inhibitor of GLUT2 expressed in Xenopus oocytes; K(i) 22.8 microm. When diabetic rats were administered glucose with quercetin, hyperglycemia was significantly decreased compared with administration of glucose alone. Quercetin also significantly decreased ascorbate absorption in normal rats given ascorbate plus quercetin compared with rats given ascorbate alone. Quercetin was a specific transport inhibitor, because it did not inhibit intestinal sugar transporters GLUT5 and SGLT1 that were injected and expressed in Xenopus oocytes. Quercetin inhibited but was not transported by SVCT1(h). Considered together, these data show that flavonoids modulate vitamin C and glucose transport by their respective intestinal transporters and suggest a new function for flavonoids.     

Poster Sessions CP10: Blood–Brain Barrier. Expression of vitamin C transporter SVCT2 in hypothalamic glial cells. An immunohistochemical and kinetic analysis

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium‐AA cotransporters. Recently, two different isoforms of sodium‐vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na⁺‐dependent l ‐ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μm and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area. Acknowledgements: Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006‐1.4 from Concepción University.