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.
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