Biology:Proton-coupled folate transporter

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Short description: Mammalian protein found in Homo sapiens


A representation of the 3D structure of the protein myoglobin showing turquoise α-helices.
Generic protein structure example

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 3µM 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]

References

  1. ↑ "Entrez Gene: PCFT proton-coupled folate transporter". https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=113235. 
  2. ↑ "Identification of an intestinal heme transporter". Cell 122 (5): 789–801. September 2005. doi:10.1016/j.cell.2005.06.025. PMID 16143108. 
  3. ↑ "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. doi:10.1016/j.yexcr.2007.01.019. PMID 17335806. 
  4. ↑ 4.0 4.1 4.2 4.3 4.4 "Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption". Cell 127 (5): 917–928. December 2006. doi:10.1016/j.cell.2006.09.041. PMID 17129779. 
  5. ↑ 5.0 5.1 5.2 "Membrane topological analysis of the proton-coupled folate transporter (PCFT-SLC46A1) by the substituted cysteine accessibility method". Biochemistry 49 (13): 2925–2931. April 2010. doi:10.1021/bi9021439. PMID 20225891. 
  6. ↑ "Delineating the extracellular water-accessible surface of the proton-coupled folate transporter". PLOS ONE 8 (10): e78301. 2013. doi:10.1371/journal.pone.0078301. PMID 24205192. Bibcode: 2013PLoSO...878301D. 
  7. ↑ "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. doi:10.1016/j.bbamem.2008.03.009. PMID 18405659. 
  8. ↑ "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. doi:10.1016/j.mam.2012.07.006. PMID 23506878. 
  9. ↑ "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. doi:10.1074/jbc.m111.306860. PMID 22179615. 
  10. ↑ "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. doi:10.1042/bj20150169. PMID 25877470. 
  11. ↑ 11.0 11.1 "Biology of the major facilitative folate transporters SLC19A1 and SLC46A1". Current Topics in Membranes 73: 175–204. 2014. doi:10.1016/B978-0-12-800223-0.00004-9. ISBN 9780128002230. PMID 24745983. 
  12. ↑ 12.0 12.1 12.2 12.3 12.4 12.5 "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. doi:10.1152/ajpcell.00202.2007. PMID 17898134. 
  13. ↑ "Cloning and functional characterization of the proton-coupled electrogenic folate transporter and analysis of its expression in retinal cell types". Investigative Ophthalmology & Visual Science 48 (11): 5299–5305. November 2007. doi:10.1167/iovs.07-0288. PMID 17962486. 
  14. ↑ 14.0 14.1 14.2 "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. doi:10.1124/jpet.107.122606. PMID 17475902. 
  15. ↑ 15.0 15.1 "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. doi:10.1124/mol.108.045443. PMID 18524888. 
  16. ↑ "The human proton-coupled folate transporter (hPCFT): modulation of intestinal expression and function by drugs". American Journal of Physiology. Gastrointestinal and Liver Physiology 298 (2): G248–G254. February 2010. doi:10.1152/ajpgi.00224.2009. PMID 19762432. 
  17. ↑ "Functional elements in the minimal promoter of the human proton-coupled folate transporter". Biochemical and Biophysical Research Communications 388 (1): 79–85. October 2009. doi:10.1016/j.bbrc.2009.07.116. PMID 19643086. 
  18. ↑ "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. doi:10.1158/1535-7163.mct-08-0938. PMID 19671745. 
  19. ↑ "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. doi:10.1074/jbc.m110.135640. PMID 20724482. 
  20. ↑ "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. doi:10.1124/mol.109.055392. PMID 19666701. 
  21. ↑ 21.0 21.1 21.2 "Identification of an intestinal heme transporter". Cell 122 (5): 789–801. September 2005. doi:10.1016/j.cell.2005.06.025. PMID 16143108. 
  22. ↑ "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. doi:10.1152/ajpcell.00468.2007. PMID 18003745. 
  23. ↑ 23.0 23.1 23.2 "Hereditary folate malabsorption: family report and review of the literature". Medicine 81 (1): 51–68. January 2002. doi:10.1097/00005792-200201000-00004. PMID 11807405. 
  24. ↑ "Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma". Nature Communications 4: 2123. 2013. doi:10.1038/ncomms3123. PMID 23828504. Bibcode: 2013NatCo...4.2123G. 
  25. ↑ "Molecular and biochemical characterization of folate transport proteins in retinal Müller cells". Investigative Ophthalmology & Visual Science 51 (6): 3226–3235. June 2010. doi:10.1167/iovs.09-4833. PMID 20053979. 
  26. ↑ "Folate transporter expression decreases in the human placenta throughout pregnancy and in pre-eclampsia". Pregnancy Hypertension 2 (2): 123–131. April 2012. doi:10.1016/j.preghy.2011.12.001. PMID 26105097. 
  27. ↑ "5-Methyltetrahydrofolate transport in basolateral membrane vesicles from human liver". The American Journal of Clinical Nutrition 58 (1): 80–84. July 1993. doi:10.1093/ajcn/58.1.80. PMID 8317394. 
  28. ↑ "Expression and differential polarization of the reduced-folate transporter-1 and the folate receptor alpha in mammalian retinal pigment epithelium". The Journal of Biological Chemistry 275 (27): 20676–20684. July 2000. doi:10.1074/jbc.m002328200. PMID 10787414. 
  29. ↑ "Mechanisms of membrane transport of folates into cells and across epithelia". Annual Review of Nutrition 31: 177–201. August 2011. doi:10.1146/annurev-nutr-072610-145133. PMID 21568705. 
  30. ↑ "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. doi:10.1139/y05-129. PMID 16900951. 
  31. ↑ "Folate binding and transport by rat kidney brush-border membrane vesicles". Biochimica et Biophysica Acta (BBA) - Biomembranes 937 (2): 211–218. January 1988. doi:10.1016/0005-2736(88)90243-x. PMID 2892531. 
  32. ↑ 32.0 32.1 "Hereditary Folate Malabsorption". GeneReviews (Seattle (WA): University of Washington, Seattle). 1993. PMID 20301716. 
  33. ↑ "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. doi:10.1007/8904_2015_445. ISBN 978-3-662-48226-1. PMID 26006721. 
  34. ↑ "A mouse model of hereditary folate malabsorption: deletion of the PCFT gene leads to systemic folate deficiency". Blood 117 (18): 4895–4904. May 2011. doi:10.1182/blood-2010-04-279653. PMID 21346251. 
  35. ↑ "Therapeutic targeting of a novel 6-substituted pyrrolo [2,3-dpyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter"]. Molecular Pharmacology 80 (6): 1096–1107. December 2011. doi:10.1124/mol.111.073833. PMID 21940787. 
  36. ↑ "A prominent low-pH methotrexate transport activity in human solid tumors: contribution to the preservation of methotrexate pharmacologic activity in HeLa cells lacking the reduced folate carrier". Clinical Cancer Research 10 (2): 718–727. January 2004. doi:10.1158/1078-0432.ccr-1066-03. PMID 14760095. 
  37. ↑ 37.0 37.1 "The human proton-coupled folate transporter: Biology and therapeutic applications to cancer". Cancer Biology & Therapy 13 (14): 1355–1373. December 2012. doi:10.4161/cbt.22020. PMID 22954694. 



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