Human intestinal folate transport: cloning, expression, and distribution of complementary RNA.
OBJECTIVE
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.
Nguyen
T T
TT
Department of Medicine, California College of Medicine, University of California, Irvine, USA.
Dyer
D L
DL
Dunning
D D
DD
Rubin
S A
SA
Grant
K E
KE
Said
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000378:metabolism*; D008969:Molecular Sequence Data; D018075:RNA, Complementary / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D011956:Receptors, Cell Surface; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D058973:Folate Receptors, GPI-Anchored; D018075:RNA, Complementary; D012333:RNA, Messenger; D011956:Receptors, Cell Surface