MOLECULAR ANALYSIS OF CYSTATHIONE BETA SYNTHASE DISORDERS IN HUMAN DISEASE

这项提案的长期目标是确定 胱硫醚β-合酶（CBS）基因的表达在 人类疾病，特别是高胱氨酸尿症和血管闭塞性疾病 因为高同型半胱氨酸血症 CBS是一种位于细胞分支点的中心酶， 硫代谢，是63 kDa亚基的四聚体;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，PLMet， 血红素 它在人类21号染色体上编码;它的遗传缺陷 导致最常见的同型胱氨酸尿症 此外，约三分之一的 据报道， CBS缺陷杂合子。 我们分离了大鼠、小鼠和人类CBS cDNA克隆及其相应的基因。 我们还发现，哥伦比亚广播公司前- mRNA经历选择性剪接，产生不同的酶同工型。 我们将正常和突变CBS cDNA克隆到融合表达载体中， 开发了将大量的酶纯化至均一的方法。 我们 证明CBS是一种含血红素的酶，卟啉 是PLP结合和活性所必需的;至少一个突变 破坏血红素结合，导致B6无反应性同型胱氨酸尿症。 我们 开发了一种快速筛查系统，并在 高胱氨酸尿症 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; 3）利用E.大肠杆菌来制备大量的 可随后切割的CBS/β-半乳糖苷酶融合蛋白 产生足够的CBS用于生物化学、生物物理和X射线研究; 4） 测定辅酶吡哆醛、5 '磷酸和S-腺苷蛋氨酸 区域;并定义酶的催化中心; 5）以 研究PLP、蛋氨酸和血红素的氨基酸序列要求 通过定点诱变结合;和6）继续定位， 表征突变并开发针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 这些信息对于描述 疾病的表现和发展有效的治疗模式，无论是通过 酶和最终的基因治疗。

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.