A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function

Abstract

Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [ 3 H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24-to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12-to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport. IntroductionFolates are essential micronutrients that serve as one-carbon donors and acceptors in a multitude of biosynthetic reactions including de novo biosynthesis of purines, thymidylate, methionine, glycine and methylation reactions. 1,2 Hence, folate vitamins are essential for DNA replication and cellular proliferation. However, mammalian organisms are devoid of folate biosynthesis and thus dietary sources must meet their metabolic requirement for folate cofactors. As such, mammalians rely on an efficient intestinal transport of folates that proceeds via a carrier-mediated system displaying optimal transport activity at acidic pH occurring primarily in the acidic microenvironment of the upper intestinal mucosal epithelium including that of the duodenum and the upper jejunum. [3][4][5] Recently, the human proton-coupled folate transporter (PCFT) gene, PCFT/SLC46A1, was identified that encodes for an intestinal folate transporter displaying optimal transport activity at acidic pH. 6 PCFT exhibits high affinities (Kt ϭ 0.5-1 M) for oxidized folates (folic acid) and reduced folates [eg, (6S)5-methylTHF and 5-formylTHF], as opposed to the reduced folate carrier (RFC/ SLC19A1) that has high affinity for reduced folates (Kt ϭ 2-4 M) but low affinity for folic acid (Ki ϭ 200-400 M). [6][7][8] Recently, several loss-of-function mutations were identified in the PCFT gene from patients with hereditary folate malabsorption (HFM), a congenital disease (OMIM 229050). 6,9 HFM is a rare autosomal recessive disorder caused by impaired intestinal folate absorption. 6,7,[9][10][11] Patients with HFM presen...

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…1). In addition, residues mapping to a well conserved ␤-turn in a region between TMDs 2 and 3 (DXXGRR; positions 109 -114) were implicated as functionally important (6,8,13,32,33) (Fig. 1).…”

“…The role of hPCFT in intestinal folate absorption was established by demonstrating loss-of-function mutations in hPCFT in patients with the rare autosomal inherited disorder, hereditary folate malabsorption (HFM) (5). To date, 17 unique hPCFT mutations have been reported in ethnically varied kindreds (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Although proton-coupled, this transporter is also func-tional at more physiologic pH, at which it retains appreciable affinity for pemetrexed (16), a newer antifolate currently approved for treating mesothelioma and non-squamous, nonsmall cell lung cancer (17)(18)(19).…”

CSF 5-Methyltetrahydrofolate Serial Monitoring to Guide Treatment of Congenital Folate Malabsorption Due to Proton-Coupled Folate Transporter (PCFT) Deficiency

“…It presents in early infancy with megaloblastic anemia, poor growth, diarrhea, infections, stomatitis, hypotonia, and progressive neurological deterioration including severe developmental delay and seizures that could be refractory to treatment (Diop-Bove et al 1993Fernandes et al 2012;Scriver et al 1995;Zhao et al 2012). To date 31 patients have been reported in the literature (Atabay et al 2010;Borzutzky et al 2009;Corbeel et al 1985;Diop-Bove et al 2013;Geller et al 2002;Hansen and Blau 2005;Jebnoun et al 2001;Kishimoto et al 2014;Lanzkowsky et al 1969;Lasry et al 2008;Malatack et al 1999;Ponz et al 1898;Rosenblatt and Fenton 2001;Santiago-Borrero et al 1973;Shin et al 2011;Sofer et al 2007;Steinschneider et al 1990;Su 1976;Urbach et al 1987;Wang et al 2014;Zhao et al 2007). It is imperative that the patients be treated very early and effectively with 5-formyltetrahydrofolate (folinic acid) in order to prevent irreversible neurological disease (Surtees 2001;Urbach et al 1987;Wang et al 2014;Whitehead 2006).…”

Random Mutagenesis of the Proton-coupled Folate Transporter (SLC46A1), Clustering of Mutations, and the Bases for Associated Losses of Function

“…Loss-of-function mutations in the pcft gene lead to the rare autosomal recessive disorder, hereditary folate malabsorption (HFM) characterized by markedly reduced folate levels in blood and cerebrospinal fluid (1)(2)(3)(4). A homozygous mutation in most cases or two compound heterozygous mutations in two cases have been identified in all subjects with the clinical diagnosis of HFM indicating that this disease is caused solely by alterations of the pcft gene (1,2,(5)(6)(7)(8)(9)(10)(11)(12).…”

“…Ten remaining mutations resulted in a single amino acid substitution in the PCFT protein (missense). Five mutations occurred at charged resides (p.R113C, p.R113S, p.R376W, p.R376Q, and p.D156Y) (2,6,7,11), whereas the other five, p.G147R, p.S318R, p.A335D, p.G338R, and p.P425R, involved substitutions of a non-charged, with a charged, residue (2,12). There appear to be hot spots for both nonsense and missense mutations.…”