The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

Abstract

Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ~116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.

Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases

“…When first identified, it was speculated, based on weak primary sequence homology, to be an ATPase and/or to bind cobalamin (11). However, subsequent biochemical studies revealed that human CblD alone is incapable of binding cobalamin or hydrolyzing ATP (22,23). So far, no ligand binding or enzymatic activity has been demonstrated for CblD.…”

Clinical, Biochemical, and Molecular Presentation in a Patient with the cblD-Homocystinuria Inborn Error of Cobalamin Metabolism

“…It does not appear to bind cobalamin. The MMADHC protein is present both in the cytoplasm, where it binds the cobalamin chaperone protein MMACHC (mutated in the cblC inborn error) and in the mitochondria (Deme et al 2012;Gherasim et al 2013;Mah et al 2013). It is not understood how the protein directs cobalamin to different cellular compartments.…”

Hampered Vitamin B12 Metabolism in Gaucher Disease?

“…The protein binder TC undergoes degradation in the lysosome, liberating vitamin B 12 that is subsequently exported out of this compartment using the transporters LMBR1 Domain Containing 1 (LMBRD1) 47,48 and ATP Binding Cassette Subfamily D Member 4 (ABCD4). 49,50 Once in the cytosol, newly internalized vitamin B 12 undergoes processing and trafficking by proteins, CblC [51][52][53][54][55][56][57] and CblD, [58][59][60][61][62][63] respectively, to finally reach acceptor proteins, MS in the cytosol and MCM in the mitochondrion.…”