2.A.41 The Concentrative Nucleoside Transporter (CNT) Family

Sequenced members of the CNT family are derived from Gram-negative and Gram-positive bacteria as well as yeast and animals. They are of about 400 residues (bacterial) or about 600-700 residues (eukaryotes) with 10-14 transmembrane α-helical spanners (TMSs). The rat CNT1 has been shown to have 13 TMSs with the hydrophilic N-terminus in the cytoplasm and the C-terminus on the extracellular side of the intestinal or renal brush border membranes of these polarized epithelial cells (Hamilton et al., 2001). The C. albicans homologue is probably of the same topology. The first 3 TMSs of the mammalian CNT1 are non-essential and are, in fact, absent from the bacterial systems (Hamilton et al., 2001). Nucleoside drug analogues and inhibitors used in cancer chemotherapy include docetaxel, uridine-furane and S-(4-nitrobenzyl)-6-thioinosine (Drápela et al. 2018).

In bacteria and yeast, CNT family members are energized by H+ symport, but in mammals they are energized by Na+ symport. The different transporters exhibit differing specificities for nucleosides. Thus, the E. coli NupC permease transports all nucleosides (both ribo- and deoxyribonucleosides) except hypoxanthine and guanine nucleosides. Another system in E. coli, NupG, a member of the MFS (TC #2.A.1.10.1), transports all ribo- and deoxyribonucleosides, while a NupG homologue, XapB (TC #2.A.1.10.2), apparently transports only xanthine. Similarly, in B. subtilis, there is evidence for three distinct nucleoside permeases, one specific for hypoxanthine and guanine nucleosides, a second specific for adenine nucleosides, and a third (B. subtilis NupC) specific for pyrimidine nucleosides (cytidine and uridine and the corresponding deoxyribonucleosides).

The mammalian permease members of the CNT family also exhibit differing specificities. Thus, rats possess at least two NupC homologues, one specific for both purine and pyrimidine nucleosides (gbU10279) and one specific for purine nucleosides (gbU25055). At least three paralogues have been characterized from humans. One human homologue (CNT1) transports pyrimidine nucleosides and adenosine, but deoxyadenosine and guanosine are poor substrates of this permease. Another (CNT2) is selective for purine nucleosides. Alteration of just a few amino acyl residues in TMSs 7 and 8 interconverts their specificities. A third homologue (CNT3) transports both purine and pyrimidine nucleosides with broad specificity (Ritzel et al., 2001). All of these transporters also accumulate various nucleoside analogue drugs such as cladribrine, 2CdA. The rat CNT2 transports this drug much better than the human orthologue due to residue substitutions in the C-terminal half of the proteins (Owen et al., 2006).

The phylogenetic tree for the CNT family shows three clusters. One includes the NupC proteins of E. coli and B. subtilis, the second includes all mammalian symporters, and the third includes functionally uncharacterized bacterial homologues (Saier et al., 1999).

The 7 known human nucleosides transporters (hNTs) exhibit varying permeant selectivities and are found into 2 protein families: the solute carrier (SLC) 29 (SLC29A1, SLC29A2, SLC29A3, SLC29A4) and SLC28 (SLC28A1, SLC28A2, SLC28A3) proteins, otherwise known, respectively, as the human equilibrative NTs (hENTs, hENT1, hENT2, hENT3, hENT4) and human concentrative NTs (hCNTs, hCNT1, hCNT2, hCNT3) (Elwi et al., 2006). The well characterized hENTs (hENT1 and hENT2) are bidirectional facilitative diffusion transporters in plasma membranes; hENT3 and hENT4 are much less well known, although hENT3, found in lysosomal membranes, transports nucleosides and is pH dependent.  hENT4-PMAT is a H+/adenosine cotransporter as well as a monoamine-organic cation transporter. The 3 hCNTs are unidirectional secondary active Na+/nucleoside cotransporters. In renal epithelial cells, hCNT1, hCNT2, and hCNT3, at apical membranes, and hENT1 and hENT2 at basolateral membranes, apparently work in concert to mediate reabsorption of nucleosides from lumen to blood, driven by Na+ gradients. Secretion of some physiological nucleosides, therapeutic nucleoside analog drugs, and nucleotide metabolites of therapeutic nucleoside and nucleobase drugs likely occurs through various xenobiotic transporters in renal epithelia, including organic cation transporters, organic anion transporters, multidrug resistance related proteins, and multidrug resistance proteins. Mounting evidence suggests that hENT1 may have a presence at both apical and basolateral membranes of renal epithelia, and thus may participate in both selective secretory and reabsorptive fluxes of nucleosides (Elwi et al., 2006).

The generalized transport reaction for permeases of the CNT family is:

Nucleoside (out) + n[H+ or Na+] (out) → Nucleoside (in) + n[H+ or Na+] (in)



This family belongs to the .

 

References:

Beaman, T.C., A.D. Hitchins, K. Ochi, N. Vasantha, T. Endo, and E. Freese. (1983). Specificity and control of uptake of purines and other compounds in Bacillus subtilis. J. Bacteriol. 156: 1107-1117.

Che, M., D.F. Ortiz, and I.M. Arias. (1995). Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na+-nucleoside cotransporter. J. Biol. Chem. 270: 13596-13599.

Craig, J.E., Y. Zhang, and M.P. Gallagher. (1994). Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186. Mol. Microbiol. 11: 1159-1168.

Drápela, S., R. Fedr, P. Khirsariya, K. Paruch, M. Svoboda, and K. Souček. (2018). Flow Cytometric Analysis of Nucleoside Transporters Activity in Chemoresistant Prostate Cancer Model. Klin Onkol 31: 140-144.

Elwi, A.N., V.L. Damaraju, S.A. Baldwin, J.D. Young, M.B. Sawyer, and C.E. Cass. (2006). Renal nucleoside transporters: physiological and clinical implications. Biochem Cell Biol 84: 844-58.

Hamilton, S.R., S.Y.M. Yao, J.C. Ingram, D.A. Hadden, M.W.L. Ritzel, M.P. Gallagher, P.J.F. Henderson, C.E. Cass, J.D. Young, and S.A. Baldwin. (2001). Subcellular distribution and membrane topology of the mammalian concentrative Na+-nucleoside cotransporter rCNT1. J. Biol. Chem. 276: 27981-27988.

Hesler, R.A., J.J. Huang, M.D. Starr, V.M. Treboschi, A.G. Bernanke, A.B. Nixon, S.J. McCall, R.R. White, and G.C. Blobe. (2016). TGF-β-Induced Stromal CYR61 Promotes Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma Through Down-Regulation of the Nucleoside Transporters hENT1 and hCNT3. Carcinogenesis. [Epub: Ahead of Print]

Huang, Q.-Q., S.Y.M. Yao, M.W.L. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. (1994). Cloning and functional expression of a complementary DNA encoding a mammalian nucleoside transport protein. J. Biol. Chem. 269: 17757-17760.

Johansen, L.E., P. Nygaard, C. Lassen, Y. Agerso, and H.H. Saxild. (2003). Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL). J. Bacteriol. 185: 5200-5209.

Larráyoz, I.M., F.J. Casado, M. Pastor-Anglada, and M.P. Lostao. (2004). Electrophysiological characterization of the human Na+/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function. J. Biol. Chem. 279: 8999-9007.

Latek, D. (2017). Rosetta Broker for membrane protein structure prediction: concentrative nucleoside transporter 3 and corticotropin-releasing factor receptor 1 test cases. BMC Struct Biol 17: 8.

Loewen, S., A. Ng, S. Yao, C. Cass, S. Baldwin, and J. Young. (1999). Identification of amino acid residues responsible for the pyrimidine and purine nucleoside specificities of human concentrative Na+ nucleoside cotransporters hCNT1 and hCNT2. J. Biol. Chem. 274: 24475-24484.

Loewen, S.K., A.M. Ng, N.N. Mohabir, S.A. Baldwin, C.E. Cass, and J.D. Young. (2003). Functional characterization of a H+/nucleoside co-transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins. Yeast 20: 661-675.

Owen, R.P., I. Badagnani, and K.M. Giacomini. (2006). Molecular determinants of specificity for synthetic nucleoside analogs in the concentrative nucleoside transporter, CNT2. J. Biol. Chem. 281: 26675-26682.

Patching, S.G., S.A. Baldwin, A.D. Baldwin, J.D. Young, M.P. Gallagher, P.J. Henderson, and R.B. Herbert. (2005). The nucleoside transport proteins, NupC and NupG, from Escherichia coli: specific structural motifs necessary for the binding of ligands. Org Biomol Chem 3: 462-470.

Ritzel, M.W., S.Y.M. Yao, A.M.L. Ng, J.R. Mackey, C.E. Cass, and J.D. Young. (1998). Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol. Membrane Biol. 15: 203-211.

Ritzel, M.W.L., A.M.L. Ng, S.Y.M. Yao, K. Graham, S.K. Loewen, K.M. Smith, R.G. Ritzel, D.A. Mowles, P. Carpenter, X.Z. Chen, E. Karpinski, R.J. Hyde, S.A. Baldwin, C.E. Cass, and J.D. Young. (2001). Molecular identification and characterization of novel human and mouse concentrative Na+ nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J. Biol. Chem. 276: 2914-2927.

Ritzel, M.W.L., S.Y.M. Yao, M.-Y. Huang, J.F. Elliott, C.E. Cass, and J.D. Young. (1997). Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). Am. J. Physiol. 272: C707-C714.

Saier, M.H., Jr., B.H. Eng, S. Fard, J. Garg, D.A. Haggerty, W.J. Hutchinson, D.L. Jack, E.C. Lai, H.J. Liu, D.P. Nusinew, A.M. Omar, S.S. Pao, I.T. Paulsen, J.A. Quan, M. Sliwinski, T.-T. Tseng, S. Wachi, and G.B. Young. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422: 1-56.

Smith, K.M., M.D. Slugoski, S.K. Loewen, A.M. Ng, S.Y. Yao, X.Z. Chen, E. Karpinski, C.E. Cass, S.A. Baldwin, and J.D. Young. (2005). The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics. J. Biol. Chem. 280: 25436-25449.

Sun, L., H. Xie, J.C. Ingram, R.J. Hope, S.A. Baldwin, and S.G. Patching. (2017). Characterization of the Escherichia coli Concentrative Nucleoside Transporter NupC Using Computational, Biochemical, and Biophysical Methods. Biochemistry. [Epub: Ahead of Print]

Vergara-Jaque, A., C. Fenollar-Ferrer, D. Kaufmann, and L.R. Forrest. (2015). Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms. Front Pharmacol 6: 183.

Yao, S.Y., A.M. Ng, S.K. Loewen, C.E. Cass, S.A. Baldwin, and J.D. Young. (2002). An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti). Am. J. Physiol. Cell Physiol. 283: C155-168.

Examples:

TC#NameOrganismal TypeExample
2.A.41.1.1

Pyrimidine nucleoside:H+ symporter, NupC (Craig et al. 1994; Patching et al. 2005). Wild-type NupC had an apparent affinity for uridine of 22.2 +/- 3.7 muM and an apparent binding affinity of 1.8-2.6 mM, and various mutants with alterred properties were isolated and characterized (Sun et al. 2017).

Bacteria

NupC of E. coli (P0AFF2)

 
2.A.41.1.2Pyrimidine-specific nucleoside:H+ symporter, NupC Bacteria NupC of Bacillus subtilis
 
2.A.41.1.3The purine nucleoside uptake transporter NupG (YxjA) (Johansen et al., 2003)BacteriaNupG of Bacillus subtilis (P42312)
 
Examples:

TC#NameOrganismal TypeExample
2.A.41.2.1Purine-specific nucleoside:Na+ symporter Mammals The bile canalicular purine-specific nucleoside:Na+ symporter of Rattus norvegicus (Q62773)
 
2.A.41.2.10

Nucleoside permease NupX

Bacteria

NupX of Escherichia coli

 
2.A.41.2.11

Concentrative nucleoside transporter, CNT, of 418 aas and 12 TMSs. A repeat-swapped model of VcCNT predicts that nucleoside transport occurs via a mechanism involving an elevator-like substrate binding domain movement across the membrane (Vergara-Jaque et al. 2015).

CNT of Vibrio cholerae

 
2.A.41.2.2Nonspecific nucleoside:Na+ symporter Mammals The nucleoside:Na+ symporter of Rattus norvegicus (Q62674)
 
2.A.41.2.3

Pyrimidine-preferring nucleoside:Na+ symporter, CNT1 (Na+/nucleoside = 2)(transports uridine, gemcitabine and 5'-deoxy-5'-fluorouridine) (Larráyoz et al., 2004)

AnimalsSLC28A1 of Homo sapiens
 
2.A.41.2.4Purine nucleoside, uridine, and 2'3'dideoxyinosine cladribrine:Na+ symporter, CNT2 (Na+/nucleoside = 1) (Owen et al., 2006)AnimalsSLC28A2 of Homo sapiens
 
2.A.41.2.5Broadly selective nucleoside:Na+ cotransporter, hfCNT (transports uridine, thymidine, inosine, 3'-azido-3'deoxythymidine, 2'3'dideoxycytidine, and 2'3'dideoxyinosine) (Na+/uridine = 2)Animals hfCNT of Eptatretus stouti (Q9UA35)
 
2.A.41.2.6

Broad-specificity nucleoside:Na+, H+ and Li+ symporter, hCNT3 (Slc28a3) transports a broad range of both purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, but guanosine, 3'azido-3-deoxythymidine and 2',3'-dideoxycytidine, which are substrates with Na+, are not substrates with H+. Both of the two cation-binding sites can apparently bind Na+, but only one can bind H+, and the Na+ and H+ forms transport different ranges of substrates. (Note: Cnt1 and Cnt2 are Na+-specific.) (Smith et al., 2005).  (Na+/nucleoside = 2; Na+ + H+/nucleoside = 2; H+/nucleoside = 1).  The matricellular protein, cysteine-rich angiogenic inducer 61 (CYR61), negatively regulates synthesis of the nucleoside transporters hENT1 and hCNT3, both of which transport the anti-cancer agent, gemcitabine (Hesler et al. 2016). Also probably transports gemcitabine, 3'-azido-3'-deoxythymidine (AZT), ribavirin and 3-deazauridine. Modeling revealed mobility of selected binding site and homotrimer interface residues (Latek 2017).

Mammals

CNT3 of Homo sapiens (Q9ERH8)

 
2.A.41.2.7

Broad specificity nucleoside:H+ symporter (1:1 stoichiometry). Adenosine, uridine, inosine, and guanosine are transported but not cytidine, thymidine or the nucleobase hypoxanthine (Km range: 15-65 μM). Purine and uridine nucleoside drug analogues including cordycepin (3'-deoxyadenosine) are substrates.

Yeast

CNT of Candida albicans, (Q874I3)

 
2.A.41.2.8

Solute carrier family 28 member 3 (Concentrative Na+-nucleoside cotransporter 3) (CNT 3) (hCNT3).  This protein is distinct from TC# 2.A.41.2.6 (78% identity) although these two proteins are called Slc28a3 and CNT3 and have the same description in UniProt (see 2.A.41.2.6 for a more complete description). 

Animals

SLC28A3 of Homo sapiens

 
2.A.41.2.9

Putative pseudouridine transporter, PsuT

Bacteria

PsuT of Escherichia coli

 
Examples:

TC#NameOrganismal TypeExample