A human sodium-dependent vitamin C transporter 2 isoform acts as a dominant-negative inhibitor of ascorbic acid transport

Abstract

Vitamin C is transported as ascorbic acid (AA) through the sodium-ascorbate cotransporters (SVCT1 and -2) and as dehydroascorbic acid (DHA) through the facilitative glucose transporters. All cells have glucose transporters and take up DHA that is trapped intracellularly by reduction and accumulated as AA. SVCT2 is widely expressed in cells and tissues at the mRNA level; however, only specialized cells directly transport AA. We undertook a molecular analysis of SVCT2 expression and discovered a transcript encoding a short form of human SVCT2 (hSVCT2-short) in which 345 bp is deleted without a frame shift. The deletion involves domains 5 and 6 and part of domain 4. cDNA encoding this isoform was isolated and expressed in 293T cells, where the protein was detected on the plasma membrane. Transport studies, however, revealed that hSVCT2-short gave rise to a nonfunctional transporter protein. hSVCT2-short arises by alternative splicing and encodes a protein that strongly inhibited the function of SVCT2 and, to a lesser extent, SVCT1 in a dominant-negative manner, probably by protein-protein interaction. The expression of hSVCT2-short varies among cells. PCR analysis of cDNA isolated from melanocytes capable of transporting AA revealed a predominance of the full-length isoform, while HL-60 cells, which express SVCT2 at the mRNA level and were incapable of transporting AA, showed a predominance of the short isoform. These findings suggest a mechanism of AA uptake regulation whereby an alternative SVCT2 gene product inhibits transport through the two known AA transporters.

FIG. 1.

Cloning and distribution of full-length hSVCT2 and its short isoform. (A) Identification of full-length and short isoforms of hSVCT2. cDNA from fetal brain tissue was amplified using hSVCT2-3 and hSVCT2-5 primers (see Materials and Methods). The PCR products were separated in a 1% agarose gel, and bands at 2 kb were excised and cloned into the T/A vector. DNA isolated from the clones was digested with EcoRI. Lane 1, molecular weight marker; lane 2, empty T/A; lanes 3 and 4, T/A vector with full-length hSVCT2; lane 5, T/A vector with hSVCT2-short. Arrows, short and long isoforms. (B) Sequencing analysis of full-length hSVCT2 and its short isoform. (C) Northern blot analysis of hSVCT2 distribution in cell lines. A poly(A⁺) RNA master blot prepared from different cancer cell lines was probed with a DNA fragment obtained by BamHI digestion of full-length hSVCT2. Arrow, hSVCT2 RNA product.

FIG. 2.

Expression and functional characterization of hSVCT2 and its short isoform. (A) Western blot analysis of the expression of full-length hSVCT2 and its short isoform. 293T cells were transfected with 4 μg of a plasmid carrying full-length hSVCT2 or its short isoform. The membrane was probed with commercially available antibodies against hSVCT2. Molecular masses are in kilodaltons. (B) Uptake studies in 293T cells. Cells were untransfected or transfected with full-length hSVCT2 (SVC2) or its short isoform (SVCT2-s). Uptake of radiolableled AA was performed at different time intervals. (Inset) Accumulation of ascorbic acid during longer incubations. (C) Sodium dependency of AA uptake mediated by hSVCT2. Cells were transfected with full-length hSVCT2, and uptakes were done with (+) and without (−) sodium in the buffer at 0 and 10 min. The ratios between accumulated AA at 10 min and that at 0 min are shown. The error bars indicate standard deviations.

FIG. 3.

Dominant-negative effect of hSVCT2-short isoform on AA uptake in 293T cells expressing full-length hSVCT2. (A) Cells were transfected with full-length hSVCT2 (SVCT2) or cotransfected with full-length hSVCT2 and increasing amounts of its short isoform (SVCT2-s). AA uptake was done at 0 and 10 min. The error bar indicates the standard deviation. (B) Stable 293T cells expressing full-length hSVCT2 were transfected with increasing amounts of the hSVCT2-short isoform. AA uptake was measures at 0, 5, and 10 min. (C) Cells were cotransfected with full-length hSVCT2 and its short isoform carrying FLAG and His tags, respectively. Immunostaining was performed the next day using either anti-His or anti-FLAG antibody. (Top) Cells cotransfected with full-length hSVCT2 and hSVCT2-short. (Bottom) Negative control experiment with nontransfected cells probed with either anti-His or anti-FLAG antibody.

FIG. 4.

Dominant-negative effect of hSVCT2-short isoform on AA uptake in 293T cells expressing full-length hSVCT1. (A) Cells were transfected with full-length hSVCT1 (SVCT1) or cotransfected with hSVCT1 and increasing amounts of the hSVCT2-short isoform (SVCT2-s). AA uptakes were measured at 0 and 10 min. (B) Cells were cotransfected with hSVCT1 and increasing amounts of hSVCT2-short isoform. DHA uptakes were measures at 0 and 30 min. Data are shown as percentages of control DHA uptake in cells transfected with hSVCT1. (C) Sodium dependency of AA uptake mediated by hSVCT1. Cells were transfected with full-length hSVCT1, and uptakes were done in the presence (+) or absence (−) of sodium in the buffer. Ratios between accumulated AA at 10 min and that at 0 min are shown. The error bar indicates the standard deviation.

FIG. 5.

Correlation between levels of short isoform of hSVCT2 and ability to transport ascorbate. (A) Melanocytes were incubated with 100 μM [¹⁴C]AA with and without sodium in the buffer. The cells were washed in cold PBS buffer, and the remaining radioactivity was counted and expressed as millimolar AA per million cells. (B) HL-60 cells were incubated with 100 μM [¹⁴C]AA with and without sodium in the buffer. The cells were washed in cold PBS buffer, and the remaining radioactivity was counted and expressed as millimolar AA per million cells. (C) PCR was performed on cDNAs isolated from melanocytes and HL-60 cells using AAT2-300 and AAT2-R900 primers. The products were separated on a 1% agarose gel and visualized by ethidium bromide staining.