The micronutrient cobalamin ( Cbl, B12 ), which is essential for hematopoiesis and neurological function, is converted to active coenzyme forms by largely unknown and uncharacterized metabolic reactions. Impairment of the conversion process by mutation or chemical interdiction results in severe metabolic dysfunction and pathology. The predominant clinical manifestations are megaloblastic anemia, atherosclerosis and neurological disease which appear individually or combined. At least two biochemically distinct mutations have been described which affect the biosynthesis of the Cbl coenzymes, methylcobalamin (Me-Cbl) and adenosylcobalamin (Ado-Cbl). Several lines of evidence suggest that the genetic defects involve a common Cbl-reducing system that is required for the synthesis of both Me-Cbl and Ado-Cbl. The objective of this research proposal is to identify and characterize the Cbl-reducing system in normal and Cbl metabolic mutant cells. This proposal capitalizes on the observations that CN-Cbl decyanation and Cbl reduction are catalyzed by two enzymes with distinct subcellular localizations. CN-Cbl decyanase is a cytosolic enzyme utilizing FAD, NADPH and reduced glutathione as cofactors. The product of this reaction is likely to be glutathionylcobalamin (GS-Cbl) which recently has been identified as an intracellular form of Cbl. Cbl reductase is a microsomal enzyme and requires NADH as cofactor. Cbl C mutant fibroblasts have very low activities of both CN-Cbl decyanase and Cbl reductase. The Cbl D mutation also affects the same enzymes but less severely.
The specific aims to be accomplished are: (1) Identification, purification and characterization of the Cbl reductase required for Me-Cbl and Ado-Cbl formation in rat liver and/or human placenta; (2) Confirmation that the isolated Cbl reductase plays an in vivo role in Cbl coenzyme biosynthesis; this will be evaluated by inhibition studies using Cbl analogs introduced via transcobalamin H receptor-mediated transport; (3) Characterization of Cbl reductase in normal and mutant skin fibroblasts by immunoprecipitation techniques; and, (4) Identification of the physiological substrate for CN-Cbl decyanase. Cbl reductase will be purified by a combination of conventional and affinity chromatographic methods (use of immobilized Cbl and/or flavin). Antibodies to the purified reductase will be produced by standard immunological techniques, partially purified and used as immunoaffinity reagents (e.g., Western blots). Mutant forms of the reductase will be partially purified from extracts of cultured skin fibroblasts using immunoaffinity chromatography. Analysis of intracellular Cbls will be carried out by reverse-phase high-performance liquid chromatography with UV and radiochemical detection. Early diagnosis of Cbl reductase defects will advance prenatal and neonatal diagnosis of inborn errors of Cbl metabolism.
