Studies of Human Folate Hydrolase

膳食中的聚谷氨酰叶酸在吸收叶酸衍生物之前，在肠膜的刷状边缘进行消化。我们对谷氨酸羧肽酶II(GCPII)表达的人肠道叶酸水解酶的分子性质进行了研究。我们发现了GCPII催化位点的H475Y多态，它降低了细胞转染体中的酶活性，并与老年高加索人群中相对较低的叶酸和较高的同型半胱氨酸水平有关。我们还发现了一个外显子18缺失的剪接变异体(GCP-18)，它与野生型GCPII一起在正常人的空肠粘膜和各种胃肠道疾病患者的十二指肠活检组织中普遍表达。我们认为，H475Y等位基因减少叶酸吸收，导致叶酸水平降低和高同型半胱氨酸血症，而H475Y和GCP-18变体都调节肠道中GCPII的表达。这项研究的总体目标是确定H475Y等位基因在与高同型半胱氨酸血症相关的临床条件下的潜在意义，并研究GCPII在哺乳动物细胞模型中的转录和表达调节。具体目的I将阐述H475Y等位基因在患有神经管缺陷的父母和受影响的儿童、认知改变的老年患者(包括阿尔茨海默病)和已知冠状动脉疾病患者中的发生率和临床意义。来自这些患者组和对照组的DNA样本将通过特定的PCR-限制性内切酶方法筛查H475Y和潜在的纯合子，发病率将与疾病诊断、叶酸和同型半胱氨酸水平相关。将在这些群体中的非高加索受试者中寻找额外的多态。特殊目的II将利用已知表达GCPII的人肠道Caco-2细胞系，以研究使用不同长度的启动子区域和荧光素酶报告基因的转录调控。对转录后调控的进一步研究将利用已知不表达GCPII和肠道Caco-2细胞的单独Cos-7哺乳动物细胞系，以及细胞转染和与GCPII、H475Y或GCP-18变体的共转染。在用绿色和红色荧光标签标记每种形式后，将研究细胞运输和膜插入，并将使用针对每种标签的单独抗体通过免疫沉淀来研究蛋白质相互作用。总体而言，建议的研究将加强对GCPII在调节叶酸供应方面的核心作用的理解，以及GCPII与高同型半胱氨酸血症相关的新的多态性的临床意义。

Dietary polyglutamyl folates undergo digestion at the brush border of the intestinal membrane prior to absorption of folic acid derivatives. We characterized the molecular properties of human intestinal folate hydrolase, which is an expression of Glutamate Carboxypeptidase II (GCPII). We identified a H475Y polymorphism in the catalytic site of GCPII, which reduces enzyme activity in cell transfectants and was associated with relatively low folate and elevated homocysteine levels in an aging Caucasian population. Also we found an exon 18-deletion splice variant (GCP-18) that is expressed together with wild type GCPII in normal human jejunal mucosa and universally in duodenal biopsies from patients with various gastrointestinal disorders. We propose that the H475Y allele reduces folate absorption resulting in lower folate levels and hyperhomocysteinemia, while both H475Y and GCP-18 variants modulate the expression of GCPII in the intestine. The overall goal of the proposed studies is to determine the potential significance of the H475Y allele in clinical conditions associated with hyperhomocysteinemia and to study the regulation of GCPII transcription and expression in mammalian cell models. Specific Aim I will address the incidence and clinical significance of the H475Y allele in parents and affected children with neural tube defects, in aging subjects with altered cognition including Alzheimer's disease, and in patients with known coronary artery disease. DNA samples from these patient groups and controls will be screened for H475Y and potential homozygotes by a specific PCR-restriction enzyme method and incidence will be correlated with disease diagnosis, folate and homocysteine levels. Additional polymorphisms will be sought in non- Caucasian subjects within these groups. Specific Aim II will utilize a human intestinal Caco-2 cell line known to express GCPII in order to study transcription regulation using promoter regions of different length and luciferase reporter. Additional studies of post-transcriptional regulation will utilize both a separate Cos-7 mammalian cell line known not to express GCPII and intestinal Caco-2 cells, and cell transfection and co-transfection with GCPII, H475Y, or GCP-18 variants. Cellular trafficking and membrane insertions will be studies after labeling each form with green and red fluorescent tags, and protein interactions will be studied by immunoprecipitation using separate antibodies to each tag. Overall, the proposed studies will enhance understanding of the central role of GCPII in regulating folate availability and the clinical significance of a novel polymorphism in GCPII associated with hyperhomocysteinemia.