In mammalian cells the sequence of events and the players involved in the biosynthesis of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) from vitamin B12 (cyanocobalamin, CNCbl) are only partially understood. A central objective of this work was to gain a mechanistic understanding of how mammalian cells process incoming cobalamins for coenzyme biosynthesis.
In Specific Aim 1 cobalamins with potential biological activity were synthesized and characterized. Nitrosylcobalamin, NOCbl, the elusive complex formed between nitric oxide and cobalamin, was synthesized via a novel reaction between aquacobalamin and the nitric oxide donor DEA-NONOate. NOCbl was obtained in high yield (85%) and purity (≥ 95% by 1H NMR spectroscopy) under alkaline and strictly anaerobic conditions. In addition to NOCbl, the synthesis and characterization of a number of other cobalamin forms were also carried out, some of which were utilized to assess the mechanisms of intracellular cobalamin processing in mammalian cells.
In Specific Aim 2, a method for the accurate assessment of intracellular cobalamins, hereafter referred to as “cold-trapping”, was developed. The procedure, which was tested in cultured cells, facilitated the identification and quantification of intracellular cobalamin forms that present exchangeable beta-axial ligands. A series of in vivo and in vitro experiments describing a new role for the MMACHC gene product (cblC protein) is also presented. Our in vivo studies strongly suggested that the cblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation). Our in vitro studies confirmed that the cblC protein catalyzed the dealkylation of Co-C bonded cobalamins by a reaction involving the nucleophilic attack of the Co-C bond by the thiolate anion of glutathione.
In Specific Aim 3, I investigated the protein changes that accompany functional cobalamin deficiency in humans. The proteome of normal and cblC mutant fibroblasts was quantitatively examined by two-dimensional difference in-gel electrophoresis and mass spectrometry. Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. The effect of hydroxycobalamin (HOCbl) supplementation on the proteome of normal and cblC fibroblasts was also examined. A bioinformatics analysis of the differentially expressed proteins was conducted using the Ingenuity Pathway Analysis® software. The in silico analysis established strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases. Supplementation of the cell cultures with HOCbl did not restore the cblC proteome to the patterns of expression observed in the normal cell line. Our findings concur with the clinical manifestations of the cblC disorder, and the poor response of severely-ill patients to therapeutic doses of HOCbl.