Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

Abstract

Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodiumdependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 M. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.Nucleosides are essential components of DNA and RNA, the genomic memory devices, and also play pivotal roles as energy sources and in signal transductions (1, 2). In mammals, nucleosides can be synthesized via the de novo pathway from small precursor molecules, such as aspartate, glutamine, glycine, and 10-formyltetrahydrofolate, and via the salvage pathway from nucleobases. In the salvage pathway, the supply of nucleobases mainly occurs from extracellular sources, typically those produced by digestion of dietary nucleic acids in the intestinal lumen. There also occurs redistribution of nucleosides/nucleobases via bloodstream from the tissues that are highly active in de novo synthesizing and/or degrading nucleosides. Therefore, carrier-mediated transport systems are needed for the efficient trafficking across cellular membranes of nucleosides and nucleobases, which are hydrophilic and hence can little permeate otherwise (3, 4).The transporters involved in the cellular uptake of nucleosides have been well characterized in mammals, including humans (5-7). There are two families of transporters, which are sodium-coupled concentrative nucleoside transporters (CNT1/SLC28A1, CNT2/SLC28A2, and CNT3/SLC28A3) and equilibrative nucleoside transporters (ENT1/SLC29A1 and ENT2/SLC29A2...

Purine Substrate Recognition by the Nucleobase-Ascorbate Transporter Signature Motif in the YgfO Xanthine Permease

“…In any event, our ongoing Cys-scanning and substituted-Cys accessibility analysis of TM8, TM9a, and TM9b is expected to allow further refinement of the topology model in this permease region. 6 Given the key functional role of the NAT motif for purine substrate recognition in both a bacterial (this study) and a fungal (6) transporter and its sequence conservation across distantly related species, it would be interesting to examine whether such a role is also retained in the uracil-transporting NATs, including the quite recently characterized rSNBT1 from the rat small intestine (27), or in the mammalian homologs SVCT1 and SVCT2, which transport L-ascorbate and do not recognize nucleobases (1,26). In this respect, it is important that residues found to be irreplaceable (7)(8)(9)(10) or critical for function in the bacterial study model cluster at highly conserved positions of the motif (Ala-323, Gln-324, Asn-325, Gly-333) and neighboring sequence regions (Asp-304, Glu-272) (Fig.…”

“…Strikingly, as well, substrate selectivity differences of NAT transporters appear to be correlated with different side-chain occupation at specific conserved positions of the NAT motif. For example, the known purine-transporting NATs have a Gln at position of Gln-324 (10), the uraciltransporting NATs have a Glu, and the ascorbate-transporting mammalian NATs have a Pro at this position; the pattern is even retained for closely related pairs of paralogs, such as in the case of the recently recognized rSNBT1 (uracil transporter; Glu at position-324), which shares 50% sequence identity (67% similarity) with SVCT1 or SVCT2 (ascorbate transporter; Pro at position-324) (27). Rationalized site-directed mutagenesis approaches might help elucidate whether such side-chain differences are functionally relevant to specific shifts in substrate selectivity in the evolution of NAT transporters.…”

Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

“…The nucleobase-ascorbate transporter (NAT) 2 or nucleobase-cation symporter-2 (NCS2) family is an evolutionarily ubiquitous family of purine, pyrimidine, and L-ascorbate transporters, with members specific for cellular uptake of uracil, xanthine, or uric acid (microbial, plant and non-primate mammalian genomes) or vitamin C (mammalian genomes) (1)(2)(3). Despite their importance for the recognition and uptake of several frontline purine-related drugs, NAT/NCS2 members have not been studied systematically at the molecular level and highresolution structures or mechanistic models are missing.…”

The Role of Transmembrane Segment TM3 in the Xanthine Permease XanQ of Escherichia coli

“…The Nucleobase-Ascorbate Transporter (NAT) 2 or Nucleobase-Cation Symporter-2 (NCS2) family is evolutionarily ubiquitous and encompasses more than 2,000 putative members in all major taxa of organisms. Despite their relevance to the recognition and uptake of several frontline purine-related drugs, only 16 members have been characterized experimentally; these are specific for the cellular uptake of uracil, xanthine, or uric acid (microbial, plant and non-primate mammalian genomes) or vitamin C (mammalian genomes) (1-3).…”