Studies of Human Folate Hydrolase

膳食聚谷氨酰叶酸在叶酸衍生物吸收之前在肠膜的刷状缘处经历消化。 我们的特点是人肠道叶酸水解酶，这是谷氨酸羧肽酶II（GCPII）的表达的分子特性。 我们确定了GCPII催化位点的H475 Y多态性，该多态性降低了细胞转染子中的酶活性，并与老年高加索人群中相对较低的叶酸和升高的同型半胱氨酸水平相关。 我们还发现了一个外显子18缺失剪接变异体（GCP-18），它与野生型GCPII一起在正常人空肠粘膜中表达，并普遍存在于各种胃肠道疾病患者的十二指肠活检组织中。 我们认为H475 Y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H475 Y和GCP-18变体均调节GCPII在肠道中的表达。拟议研究的总体目标是确定H475 Y等位基因在与高同型半胱氨酸血症相关的临床病症中的潜在意义，并研究哺乳动物细胞模型中GCPII转录和表达的调节。 具体目标我将解决的发病率和H475 Y等位基因的神经管缺陷的父母和受影响的儿童，在老年人与认知改变，包括阿尔茨海默病，并在已知的冠状动脉疾病的患者的临床意义。将通过特异性PCR-限制性内切酶方法筛选这些患者组和对照组的DNA样本中的H475 Y和潜在纯合子，并将发病率与疾病诊断、叶酸和同型半胱氨酸水平相关。 将在这些组内的非高加索受试者中寻找其他多态性。 Specific Aim II将利用已知表达GCPII的人肠Caco-2细胞系，以研究使用不同长度的启动子区和荧光素酶报告基因的转录调控。 转录后调控的其他研究将利用已知不表达GCPII的单独Cos-7哺乳动物细胞系和肠Caco-2细胞，以及细胞转染和与GCPII、H475 Y或GCP-18变体共转染。 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，并使用针对每种标签的单独抗体通过免疫沉淀研究蛋白质相互作用。 总的来说，拟议的研究将加强对GCPII在调节叶酸利用率方面的核心作用的理解，以及与高同型半胱氨酸血症相关的GCPII新多态性的临床意义。

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.