Clinical or ATPase domain mutations in ABCD4 disrupt the interaction between the vitamin B12-trafficking proteins ABCD4 and LMBD1

Abstract

Vitamin B (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes and result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.

An update on vitamin B12-related gene polymorphisms and B12 status

“… ATP binding cassette subfamily D member 4 ( ABCD4 ) 14q24.3 ABCD4 codes for an ABC transporter. It has been postulated that ABCD4 is involved in intracellular cobalamin processing [ 69 ], and is involved in transporting vitamin B12 from lysosomes to the cytosol. In the cytosol, vitamin B12 can be converted into methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) [ 70 ].…”

“…The ATP-binding cassette subfamily D member 4 ( ABCD4 ) gene is located on chromosome 14. This gene codes for the ABCD4 protein, which is a membrane transporter involved in transporting vitamin B12 out of lysosomes [ 69 ]. It has been shown that polymorphisms of the ABCD4 gene affect the functioning of the ABCD4 protein and the intracellular processing of vitamin B12 [ 70 ].…”

The clinical presentation of cobalamin‐related disorders: From acquired deficiencies to inborn errors of absorption and intracellular pathways

“…Within the cytosol, the MMACHC protein binds, dealkylates, and decyanides Cbl. The MMADHC protein targets the MMACHC‐Cbl complex to process Cbl to its cofactor function sites in the cytosol and/or the mitochondria.…”

Assessment of cellular cobalamin metabolism in Gaucher disease

“…The content of holo-TC in cells treated with HOCbl showed a trend similar to the reduction of marker metabolites Hcy and MMA. Canonical inborn errors of cobalamin involving the lysosome, such as cblF and cblJ [14,16,[48][49][50] and other lysosomal defects that impair cobalamin metabolism secondarily such as mutations in the rabenosyn-5 gene or defective lysosomal acidification in Alzheimer's disease [12,13], feature one or all of the classical marker metabolite trends, namely, elevated Hcy and MMA and reduced Met in cells and/or plasma. Our experimental results suggest an overall preserved metabolism of cobalamin in GD cells.…”