Human intestinal folate transport: Cloning, expression, and distribution of complementary RNA
Human intestinal folate transport: Cloning, expression, and distribution of complementary RNA
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DOI：
10.1053/gast.1997.v112.pm9041240
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发表时间：
1997-03-01
期刊：
影响因子：
29.4
通讯作者：
Said, HM
中科院分区：
文献类型：
--
作者：
Nguyen, TT;Dyer, DL;Said, HM
背景和目标：尽管进行了深入的研究，但对肠道叶酸转运系统的分子特性知之甚少，特别是在人类中。本研究的目的是分离一个功能性的人肠道叶酸载体互补DNA（cDNA）克隆，并确定互补RNA在组织和细胞水平上的分布。研究方法：杂交筛选，改良的马拉松cDNA扩增，在非洲爪蟾卵母细胞中的表达，北方分析，和原位杂交。结果如下：hIFC-1 cDNA含有591个氨基酸的开放阅读框（相对分子质量= 64，826，pi = 9.4，12个跨膜结构域，3个蛋白激酶C磷酸化位点和1个N-糖基化位点），与小鼠cDNA对应物具有74%的DNA和66%的氨基酸序列同源性。用hIFC-1 cRNA注射的非洲爪蟾卵母细胞显示出诱导的叶酸摄取，其（1）可被底物浓度饱和（表观米氏常数= 0.71 +/- 0.06 μ mol/L;最大速度= 128 +/- 3 fmol）。h（-1）。卵母细胞（-1））;（2）被甲氨蝶呤、亚叶酸和叶酸抑制（Ki分别为0.84 μ mol/L、0.71 μ mol/L和10 μ mol/L）;（3）对4，4 ′-二异硫氰基二苯乙烯-2，2 ′-二磺酸敏感（Ki = 0.29 mmol/L）。北方分析显示hIFC 1互补信使RNA种类在各种人体组织中广泛分布。原位杂交显示hIFC-1优先表达于上皮细胞，尤其是在绒毛的上半部分。结论：这些结果代表了第一个人小肠叶酸载体的分子特征。
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 mu mol/L; maximum velocity = 128 +/- 3 fmol . h(-1). oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (K-i = 0.84 mu mol/L, 0.71 mu mol/L, and 10 mu mol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (K-i = 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.