Processing of alkylcobalamins in mammalian cells: A role for the MMACHC (cblC) gene product

Abstract

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B(12)). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [(57)Co]-AdoCbl and MeCbl analogs consisting of [(57)Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [(57)Co]-AdoCbl was converted to [(57)Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5'-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [(57)Co]-propylcobalamin to [(57)Co]-AdoCbl and [(57)Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.

Fig. 1. Processing [⁵⁷Co]-CNCbl in BAEC

Kinetics of cobalamin biosynthesis in BAEC for 6, 12, 24, 48 and 72 h. BAEC were grown in the presence of [⁵⁷Co]-CNCbl (0.2 nM final concentration; 0.1 μCi/ml culture medium) as the cobalamin source. Absorbance at 254 nm for cobalamin standards is shown in the upper chromatogram. Radioactivity for [⁵⁷Co]-cobalamins at 6, 12, 24, 48 and 72 h is shown in the bottom chromatograms.

Fig. 2. Processing [⁵⁷Co]-PrCbl in BAEC

A. Typical cobalamin profile from BAEC grown in the presence of [⁵⁷Co]-PrCbl (0.125 nM final concentration; 0.06 μCi/ml culture medium) as the cobalamin source for 48 h. Absorbance at 254 nm for cobalamin standards is shown in the lighter tracing; radioactivity for [⁵⁷Co]-cobalamins is shown in the darker tracing. B. Cobalamins extracted from conditioned medium after 48 h. The darker tracing shows that [⁵⁷Co]-PrCbl is largely intact after 48 h in the culture medium.

Fig. 3. Processing of [⁵⁷Co]-PrCbl by human normal and cblC mutant fibroblasts (WG1801, WG2176 and WG3354)

Cells were cultured in the presence of 0.125 nM [⁵⁷Co]-PrCbl (0.06 μCi/ml culture medium) for 48 h. [⁵⁷Co]-labeled cobalamins were then extracted and analyzed by HPLC as described[15]. Results represent two pooled samples per cell line. “Others” refers to cobalamins not quantitatively determined in the present study, which include glutathionylcobalamin, sulphitocobalamin and nitrocobalamin (See ref [15]).

Fig. 4. Possible mechanisms for the dealkylation of alkylcobalamins mediated by the cblC protein

Formation of the base-off conformation of the cobalamin leads to an enhanced reactivity of the upper axial ligand. Reaction 1: homolysis of the cobalt-carbon bond would generate cob(II)alamin and an alkyl radical. Reaction 2: nucleophilic displacement of the alkyl group would result in the formation of cob(I)alamin and the transfer of the alkyl carbocation to the acceptor as described by model studies [29, 30]. Reactions 3 and 4: reductive dealkylation could occur resulting in the formation of either cob(II)alamin or cob(I)alamin and the departure of the alkyl group as a carbanion or a radical, respectively.