MOLECULAR ANALYSIS OF CYSTATHIONE BETA SYNTHASE DISORDERS IN HUMAN DISEASE

这项提案的长期目标是定义 胱硫醚β-合成酶(CBS)基因的表达在血管紧张素转换酶中的作用 人类疾病，特别是同型半胱氨酸尿症和血管闭塞性疾病 由于高同型半胱氨酸血症。CBS，一种位于分支点的中心酶 硫代谢，是一个由63 kDa亚基组成的四聚体；该酶结合两个 底物，同型半胱氨酸和丝氨酸，以及三种配体，PLP，ADOMet和 亚铁血红素。它在人类身上编码在21号染色体上；它的遗传缺陷 导致最常见的同型半胱氨酸尿症。此外，约三分之一的 据报道，未被选中的外周动脉疾病患者有 CBS缺乏症的杂合子。我们分离了大鼠、小鼠和人类CBS CDNA克隆及其相应基因。我们还展示了CBS的预览版 信使核糖核酸经历选择性剪接，产生不同的同工酶。 我们将正常和突变的CBS基因克隆到融合表达载体中 开发了将大量酶提纯为均一的方法。我们 证明了CBS是一种含血红素的酶，而卟啉 是PLP结合和活性所必需的；至少有一个突变 破坏血红素结合，导致B6无反应性同型半胱氨酸尿症。我们 开发了一种快速筛查系统，并已确定了26个突变 目前为止是同型半胱氨酸尿症。我们的具体目标是：1)提供详细的 人CBS基因的分析；2)CBS的分离和鉴定 启动子区域；3)利用大肠杆菌大量制备 CBS/β-半乳糖苷酶融合蛋白，可随后切割 生产足够的CBS，用于生化、生物物理和X射线研究；4) 辅酶吡哆醛-5‘-磷酸和S-腺苷蛋氨酸的测定 区域；以及确定酶的催化中心；5) 研究PLP、ADOMet和HEME的氨基酸序列需求 通过定点突变结合；以及6)继续定位和 确定突变的特征，并开发对频繁突变的直接测试。 这个项目的作用是将我们对小学的知识扩展到 三级酶结构。这一信息对于描述 疾病表现和开发有效的治疗范例，既通过 酶，最终是基因疗法。

The long-term objectives of this proposal are to define how abnormal expression of the cystathionine beta-synthase (CBS) gene plays a role in human disease, in particular, homocystinuria and vascular occlusive disease due to hyperhomocysteinemia. CBS, a central enzyme at a branch point in sulfur metabolism, is a tetramer of 63 kDa subunits; the enzyme binds two substrates, homocysteine and serine, and three ligands, PLP, AdoMet, and heme. It is encoded in humans on chromosome 21; its inherited deficiency causes the most common form of homocystinuria. Further, about one third of unselected patients with peripheral arterial disease reportedly are heterozygous for CBS deficiency. We isolated rat, mouse, and human CBS cDNA clones and their corresponding genes. We also showed that CBS pre- mRNA undergoes alternative splicing, yielding different enzyme isoforms. We cloned normal and mutant CBS cDNA into fusion expression vectors and developed methods to purify large amounts of the enzyme to homogeneity. We demonstrated that CBS is a heme-containing enzyme, and that the porphyrin is required for both PLP binding and activity; at least one mutations disrupts heme binding, resulting in B6-nonresponsive homocystinuria. We developed a rapid screening system and have identified 26 mutations in homocystinuria so far. Our specific aims are; 1) to provide detailed analysis of the human CBS gene; 2) to isolate and characterize the CBS promoter region; 3) to employ E. coli to prepare large amounts of CBS/beta-galactosidase fusion protein which can be subsequently cleaved yielding sufficient CBS for biochemical, biophysical and X-ray studies; 4) to determine the coenzyme pyridoxal; 5'phosphate and S-adenosylmethionine regions; and to define the catalytic center of the enzyme; 5) to investigate the amino acid sequence requirements for PLP, AdoMet, and heme binding through site-directed mutagenesis; and 6) to continue to locate and characterize mutations and to develop direct tests for frequent mutations. The role of this project is to extend our knowledge of the primary to the tertiary enzyme structure. This information is crucial to delineate disease manifestation and develop efficacious treatment paradigms, both by enzyme and, ultimately, gene therapy.