title: Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism.
abstract: The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patient's fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.
authors.affiliation: Division of Metabolism, Children’s Research Center (CRC), University Children’s Hospital, Zu¨ rich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Stucki
authors.forename: David
authors.initials: D
authors.lastname: Coelho
authors.forename: Terttu
authors.initials: T
authors.lastname: Suormala
authors.forename: Patricie
authors.initials: P
authors.lastname: Burda
authors.forename: Brian
authors.initials: B
authors.lastname: Fowler
authors.forename: Matthias R
authors.initials: MR
authors.lastname: Baumgartner
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015153:Blotting, Western; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006712:Homocystinuria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014805:Vitamin B 12 / Q000378:metabolism*; D014806:Vitamin B 12 Deficiency / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C527938:MMADHC protein, human; D033681:Mitochondrial Membrane Transport Proteins; D012333:RNA, Messenger; D014805:Vitamin B 12