BACKGROUND & AIMS: Despite intensive investigations, very little is known about the molecular identity(ies) of the intestinal folate transport system(s), especially in humans. The aim of this study was to isolate a functional human intestinal folate carrier complementary DNA (cDNA) clone and determine the distribution of complementary RNA at the tissue and cellular levels. METHODS: Hybridization screening, modified Marathon cDNA amplification, expression in Xenopus oocytes, Northern analysis, and in situ hybridization were used. RESULTS: The hIFC-1 cDNA contains an open reading frame for 591 amino acids (relative molecular mass = 64,826, pI = 9.4, 12 transmembrane domains, three protein kinase C phosphorylation sites, and one N-glycosylation site) with 74% DNA and 66% amino acid sequence homologies with the mouse cDNA counterpart. Xenopus oocytes injected with hIFC-1 cRNA show induced folate uptake that was (1) saturable with substrate concentration (apparent Michaelis constant = 0.71 +/- 0.06 micromol/L; maximum velocity = 128 +/- 3 fmol x h(-1) x oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (Ki = 0.84 micromol/L, 0.71 micromol/L, and 10 micromol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki = 0.29 mmol/L). Northern analysis showed wide distribution of hIFC1-complementary messenger RNA species in various human tissues. In situ hybridization on sections of human jejunum showed preferential hIFC-1 expression in epithelial cells, especially in the upper half of the villi. CONCLUSIONS: These results represent the first molecular characterization of a human small intestinal folate carrier.
BACKGROUND & AIMS: Despite intensive investigations, very little is known about the molecular identity(ies) of the intestinal folate transport system(s), especially in humans. The aim of this study was to isolate a functional human intestinal folate carrier complementary DNA (cDNA) clone and determine the distribution of complementary RNA at the tissue and cellular levels. METHODS: Hybridization screening, modified Marathon cDNA amplification, expression in Xenopus oocytes, Northern analysis, and in situ hybridization were used. RESULTS: The hIFC-1 cDNA contains an open reading frame for 591 amino acids (relative molecular mass = 64,826, pI = 9.4, 12 transmembrane domains, three protein kinase C phosphorylation sites, and one N-glycosylation site) with 74% DNA and 66% amino acid sequence homologies with the mouse cDNA counterpart. Xenopus oocytes injected with hIFC-1 cRNA show induced folate uptake that was (1) saturable with substrate concentration (apparent Michaelis constant = 0.71 +/- 0.06 micromol/L; maximum velocity = 128 +/- 3 fmol x h(-1) x oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (Ki = 0.84 micromol/L, 0.71 micromol/L, and 10 micromol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki = 0.29 mmol/L). Northern analysis showed wide distribution of hIFC1-complementary messenger RNA species in various human tissues. In situ hybridization on sections of human jejunum showed preferential hIFC-1 expression in epithelial cells, especially in the upper half of the villi. CONCLUSIONS: These results represent the first molecular characterization of a human small intestinal folate carrier.
Authors: Simone J P M Eussen; Stein Emil Vollset; Jannicke Igland; Klaus Meyer; Ase Fredriksen; Per Magne Ueland; Mazda Jenab; Nadia Slimani; Paolo Boffetta; Kim Overvad; Anne Tjønneland; Anja Olsen; Françoise Clavel-Chapelon; Marie-Christine Boutron-Ruault; Sophie Morois; Cornelia Weikert; Tobias Pischon; Jakob Linseisen; Rudolf Kaaks; Antonia Trichopoulou; Demosthenes Zilis; Michael Katsoulis; Domenico Palli; Franco Berrino; Paolo Vineis; Rosario Tumino; Salvatore Panico; Petra H M Peeters; H Bas Bueno-de-Mesquita; Fränzel J B van Duijnhoven; Inger Torhild Gram; Guri Skeie; Eiliv Lund; Carlos A González; Carmen Martínez; Miren Dorronsoro; Eva Ardanaz; Carmen Navarro; Laudina Rodríguez; Bethany Van Guelpen; Richard Palmqvist; Jonas Manjer; Ulrika Ericson; Sheila Bingham; Kay-Tee Khaw; Teresa Norat; Elio Riboli Journal: Cancer Epidemiol Biomarkers Prev Date: 2010-05 Impact factor: 4.254
Authors: Sook Wah Yee; Li Gong; Ilaria Badagnani; Kathleen M Giacomini; Teri E Klein; Russ B Altman Journal: Pharmacogenet Genomics Date: 2010-11 Impact factor: 2.089