Proton-coupled folate transporter explained

The proton-coupled folate transporter is a protein that in humans is encoded by the SLC46A1 gene.[1] [2] [3] The major physiological roles of PCFTs are in mediating the intestinal absorption of folate (Vitamin B9), and its delivery to the central nervous system.

Structure

PCFT is located on chromosome 17q11.2 and consists of five exons encoding a protein with 459 amino acids and a MW of ~50kDa. PCFT is highly conserved, sharing 87% identity to the mouse and rat PCFT and retaining more than 50% amino acid identity to the frog (XP415815) and zebrafish (AAH77859) proteins.[4] Structurally, there are twelve transmembrane helices with the N- and C- termini directed to the cytoplasm and a large internal loop that divides the molecule in half.[5] [6] There are two glycosylation sites (N58, N68) and a disulfide bond connecting residues C66, in the 1st and C298 in the 4th, external loop. Neither glycosylation nor the disulfide bond are essential for function.[5] [7] Residues have been identified that play a role in proton-coupling, proton binding, folate binding and oscillation of the carrier between its conformational states.[8] PCFT forms oligomers and some of the linking residues have been identified.[9] [10]

Regulation

PCFT-mediated transport into cells is optimal at pH 5.5. The low-pH activity and the structural specificity of PCFT (high affinity for folic acid, and low affinity for PT523 - a non-polyglutamable analog of aminopterin) distinguishes this transporter functionally from the other major folate transporter, the reduced folate carrier[11] (optimal activity at pH 7.4, very low affinity for folic acid and very high affinity for PT523), another member (SLC19A1) of the superfamily of solute transporters.[4] [11] [12] Influx mediated by PCFT is electrogenic and can be assessed by current, cellular acidification, and radiotracer uptake.[4] [12] [13] [14] Influx has a Km range of 0.5 to 3 μM for most folates and antifolates at pH 5.5. The influx Km rises and the influx Vmax falls as the pH is increased, least so for the antifolate, pemetrexed.[15] The transporter is specific for the monoglutamyl forms of folates.[12] A variety of organic anions inhibit PCFT-mediated transport at extremely high ratio of inhibitor to folate, the most potent are sulfobromophthalein, p-aminobenzylglutamate, and sulfathalazine.[14] [16] This may have pharmacological relevance in terms of the inhibitory effect of these agents on the intestinal absorption of folates. The PCFT minimal promoter has been defined[17] [18] and contains an NRF1 response element.[19] There is also evidence for a role of vitamin D in the regulation of PCFT with a VDR response element upstream of the minimal promoter.[20] PCFT mRNA was reported to be increased in folate-deficient mice.[12]

Tissue distribution

PCFT is expressed in the proximal jejunum with a lower level of expression elsewhere in the intestine.[4] [12] [21] Expression is localized to the apical membrane of intestinal [12] [14] [21] and polarized MDCK dog kidney cells.[22] PCFT is also expressed at the basolateral membrane of the choroid plexus. In view of the low levels of folate in the cerebrospinal fluid (CSF) in PCFT-null humans,[23] PCFT must play a role in transport of folates across the choroid plexus into the CSF; however, the underlying mechanism for this has not been established.[24] PCFT is expressed at the sinusoidal (basolateral) membrane of the hepatocyte, the apical brush-border membrane of the proximal tubule of the kidney, the basolateral membrane of the retinal pigment epithelium and the placenta.[5] [25] [26] There is a prominent low-pH folate transport activity in the cells and/or membrane vesicles derived from these tissues which, in some cases, has been shown to be indicative of a proton-coupled folate transport process.[27] [28] [29] [30] [31] However, it is unclear as to the extent that PCFT contributes to folate transport across these epithelia.

Loss-of-function

The physiological role of PCFT is known based upon the phenotype of subjects with loss-of-function mutations of this gene – the rare autosomal hereditary disorder, hereditary folate malabsorption (HFM).[4] [23] [32] These subjects have two major abnormalities: (i) severe systemic folate deficiency and (ii) a defect in the transport of folates from blood across the choroid plexus into the CSF with very low CSF folate levels even when the blood folate level is corrected or supranormal.[33] Severe anemia, usually macrocytic, always accompanies the folate deficiency. Sometimes there is pancytopenia and/or hypogammaglobulinemia and/or T-cell dysfunction which can result in infections such as Pneumocystis jirovecii pneumonia. There can be GI signs including diarrhea and mucositis. The CNS folate deficiency is associated with a variety of neurological findings including developmental delays and seizures. The phenotype of the PCFT-null mouse has been reported and mirrors many of the findings in humans.[34] PCFT was initially reported to be a low-affinity heme transporter.[21] However, a role for PCFT in heme and iron homeostasis is excluded by the observation that humans or mice with loss-of-function PCFT mutations are not iron or heme deficient and the anemia, and all other systemic consequences of the loss of this transporter, are completely corrected with high-dose oral, or low-dose, parenteral folate.[23] [32]

As a drug target

Because of the Warburg effect, and a compromised blood supply, human epithelial cancers grow within an acidic milieu, as lactate is produced during anaerobic glycolysis. Because PCFT activity is optimal at low pH, and its expression and a prominent low-pH transport activity are present in human cancers,[35] [36] there is interest in exploiting these properties by the development of antifolates that have a high affinity for this transporter and a very low affinity for the reduced folate carrier which delivers antifolates to normal tissues and thereby mediates the toxicity of these agents.[37] A novel class of inhibitors of one carbon incorporation into purines is being developed with these properties.[37] Pemetrexed, an antifolate inhibitor primarily of thymidylate synthase, is a good substrate for PCFT even at neutral pH as compared to other antifolates and folates.[15]

Notes and References

  1. Web site: Entrez Gene: PCFT proton-coupled folate transporter.
  2. Shayeghi M, Latunde-Dada GO, Oakhill JS, Laftah AH, Takeuchi K, Halliday N, Khan Y, Warley A, McCann FE, Hider RC, Frazer DM, Anderson GJ, Vulpe CD, Simpson RJ, McKie AT . 6 . Identification of an intestinal heme transporter . Cell . 122 . 5 . 789–801 . September 2005 . 16143108 . 10.1016/j.cell.2005.06.025 . 9130882 . free .
  3. Sharma S, Dimasi D, Bröer S, Kumar R, Della NG . Heme carrier protein 1 (HCP1) expression and functional analysis in the retina and retinal pigment epithelium . Experimental Cell Research . 313 . 6 . 1251–1259 . April 2007 . 17335806 . 10.1016/j.yexcr.2007.01.019 .
  4. Qiu A, Jansen M, Sakaris A, Min SH, Chattopadhyay S, Tsai E, Sandoval C, Zhao R, Akabas MH, Goldman ID . 6 . Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption . Cell . 127 . 5 . 917–928 . December 2006 . 17129779 . 10.1016/j.cell.2006.09.041 . 1918658 . free .
  5. Zhao R, Unal ES, Shin DS, Goldman ID . Membrane topological analysis of the proton-coupled folate transporter (PCFT-SLC46A1) by the substituted cysteine accessibility method . Biochemistry . 49 . 13 . 2925–2931 . April 2010 . 20225891 . 2866095 . 10.1021/bi9021439 .
  6. Duddempudi PK, Goyal R, Date SS, Jansen M . Delineating the extracellular water-accessible surface of the proton-coupled folate transporter . PLOS ONE . 8 . 10 . e78301 . 2013 . 24205192 . 3799626 . 10.1371/journal.pone.0078301 . free . 2013PLoSO...878301D .
  7. Unal ES, Zhao R, Qiu A, Goldman ID . N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT) . Biochimica et Biophysica Acta (BBA) - Biomembranes . 1778 . 6 . 1407–1414 . June 2008 . 18405659 . 2762823 . 10.1016/j.bbamem.2008.03.009 .
  8. Zhao R, Goldman ID . Folate and thiamine transporters mediated by facilitative carriers (SLC19A1-3 and SLC46A1) and folate receptors . Molecular Aspects of Medicine . 34 . 2–3 . 373–385 . 2013 . 23506878 . 3831518 . 10.1016/j.mam.2012.07.006 .
  9. Hou Z, Kugel Desmoulin S, Etnyre E, Olive M, Hsiung B, Cherian C, Wloszczynski PA, Moin K, Matherly LH . 6 . Identification and functional impact of homo-oligomers of the human proton-coupled folate transporter . The Journal of Biological Chemistry . 287 . 7 . 4982–4995 . February 2012 . 22179615 . 3281668 . 10.1074/jbc.m111.306860 . free .
  10. Wilson MR, Kugel S, Huang J, Wilson LJ, Wloszczynski PA, Ye J, Matherly LH, Hou Z . 6 . Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6 . The Biochemical Journal . 469 . 1 . 33–44 . July 2015 . 25877470 . 4713120 . 10.1042/bj20150169 .
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  12. Qiu A, Min SH, Jansen M, Malhotra U, Tsai E, Cabelof DC, Matherly LH, Zhao R, Akabas MH, Goldman ID . 6 . Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction . American Journal of Physiology. Cell Physiology . 293 . 5 . C1669–C1678 . November 2007 . 17898134 . 10.1152/ajpcell.00202.2007 . 7250544 .
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  14. Nakai Y, Inoue K, Abe N, Hatakeyama M, Ohta KY, Otagiri M, Hayashi Y, Yuasa H . 6 . Functional characterization of human proton-coupled folate transporter/heme carrier protein 1 heterologously expressed in mammalian cells as a folate transporter . The Journal of Pharmacology and Experimental Therapeutics . 322 . 2 . 469–476 . August 2007 . 17475902 . 10.1124/jpet.107.122606 . 23277839 .
  15. Zhao R, Qiu A, Tsai E, Jansen M, Akabas MH, Goldman ID . The proton-coupled folate transporter: impact on pemetrexed transport and on antifolates activities compared with the reduced folate carrier . Molecular Pharmacology . 74 . 3 . 854–862 . September 2008 . 18524888 . 2716086 . 10.1124/mol.108.045443 .
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  17. Stark M, Gonen N, Assaraf YG . Functional elements in the minimal promoter of the human proton-coupled folate transporter . Biochemical and Biophysical Research Communications . 388 . 1 . 79–85 . October 2009 . 19643086 . 10.1016/j.bbrc.2009.07.116 .
  18. Diop-Bove NK, Wu J, Zhao R, Locker J, Goldman ID . Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal transcriptional regulatory region in an antifolate-resistant HeLa cell line . Molecular Cancer Therapeutics . 8 . 8 . 2424–2431 . August 2009 . 19671745 . 2735101 . 10.1158/1535-7163.mct-08-0938 .
  19. Gonen N, Assaraf YG . The obligatory intestinal folate transporter PCFT (SLC46A1) is regulated by nuclear respiratory factor 1 . The Journal of Biological Chemistry . 285 . 44 . 33602–33613 . October 2010 . 20724482 . 2962458 . 10.1074/jbc.m110.135640 . free .
  20. Eloranta JJ, Zaïr ZM, Hiller C, Häusler S, Stieger B, Kullak-Ublick GA . Vitamin D3 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter . Molecular Pharmacology . 76 . 5 . 1062–1071 . November 2009 . 19666701 . 10.1124/mol.109.055392 . 11155598 .
  21. Shayeghi M, Latunde-Dada GO, Oakhill JS, Laftah AH, Takeuchi K, Halliday N, Khan Y, Warley A, McCann FE, Hider RC, Frazer DM, Anderson GJ, Vulpe CD, Simpson RJ, McKie AT . 6 . Identification of an intestinal heme transporter . Cell . 122 . 5 . 789–801 . September 2005 . 16143108 . 10.1016/j.cell.2005.06.025 . 9130882 . free .
  22. Subramanian VS, Marchant JS, Said HM . Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia . American Journal of Physiology. Cell Physiology . 294 . 1 . C233–C240 . January 2008 . 18003745 . 10.1152/ajpcell.00468.2007 . 7730829 .
  23. Geller J, Kronn D, Jayabose S, Sandoval C . Hereditary folate malabsorption: family report and review of the literature . Medicine . 81 . 1 . 51–68 . January 2002 . 11807405 . 10.1097/00005792-200201000-00004 . 27156694 . free .
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  26. Williams PJ, Mistry HD, Morgan L . Folate transporter expression decreases in the human placenta throughout pregnancy and in pre-eclampsia . Pregnancy Hypertension . 2 . 2 . 123–131 . April 2012 . 26105097 . 10.1016/j.preghy.2011.12.001 .
  27. Horne DW, Reed KA, Hoefs J, Said HM . 5-Methyltetrahydrofolate transport in basolateral membrane vesicles from human liver . The American Journal of Clinical Nutrition . 58 . 1 . 80–84 . July 1993 . 8317394 . 10.1093/ajcn/58.1.80 . free .
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  30. Keating E, Lemos C, Azevedo I, Martel F . Comparison of folic acid uptake characteristics by human placental choriocarcinoma cells at acidic and physiological pH . Canadian Journal of Physiology and Pharmacology . 84 . 2 . 247–255 . February 2006 . 16900951 . 10.1139/y05-129 .
  31. Bhandari SD, Joshi SK, McMartin KE . Folate binding and transport by rat kidney brush-border membrane vesicles . Biochimica et Biophysica Acta (BBA) - Biomembranes . 937 . 2 . 211–218 . January 1988 . 2892531 . 10.1016/0005-2736(88)90243-x .
  32. Goldman ID . Hereditary Folate Malabsorption . Adam MP, Mirzaa GM, Pagon RA, Wallace SE, Bean LJ, Gripp KW, Amemiya A . Seattle (WA) . University of Washington, Seattle . GeneReviews . 1993 . 20301716 .
  33. Torres A, Newton SA, Crompton B, Borzutzky A, Neufeld EJ, Notarangelo L, Berry GT . CSF 5-Methyltetrahydrofolate Serial Monitoring to Guide Treatment of Congenital Folate Malabsorption Due to Proton-Coupled Folate Transporter (PCFT) Deficiency . JIMD Reports . 24 . 91–96 . 26 May 2015 . 26006721 . 4582027 . 10.1007/8904_2015_445 . 978-3-662-48226-1 .
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  35. Kugel Desmoulin S, Wang L, Hales E, Polin L, White K, Kushner J, Stout M, Hou Z, Cherian C, Gangjee A, Matherly LH . 6 . Therapeutic targeting of a novel 6-substituted pyrrolo [2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter . Molecular Pharmacology . 80 . 6 . 1096–1107 . December 2011 . 21940787 . 3228537 . 10.1124/mol.111.073833 .
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