Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment.

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9 weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment.

Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder.

Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (T(m)) of 39.3+/-1.0 and 37.1+/-0.7 degrees C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 degrees C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (Delta T(m max)=8 degrees C, half-maximal effective ligand concentration, AC(50)=3 microM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in T(m) of approximately 16 degrees C, followed by MeCbl at approximately 13 degrees C, each evaluated at 50 microM cofactor. The other cobalamins stabilized in the order (CN)(2)Cbi>OHCbl>CNCbl. Interestingly, the AC(50)'s for AdoCbl, MeCbl, (CN)(2)Cbi and OHCbl were similar and ranged from 1-3 microM, which compares well with the K(d) of 6 microM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (Delta T(m max)=4 degrees C). The dose-response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC(50) of 7 microM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC(50) for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.

Mechanism of vitamin B12-responsiveness in cblC methylmalonic aciduria with homocystinuria.

Patients with the cblC vitamin B(12) (cobalamin, cbl) disorder are defective in the intracellular synthesis of adenosylcobalamin and methylcobalamin and have combined homocystinuria and methylmalonic aciduria. While other vitamin B(12) disorders are treatable with high dose cyanocobalamin (CNCbl) or hydroxocobalamin (OHCbl), cblC patients respond well to OHCbl but not to CNCbl. Patient mutations were introduced into recombinant MMACHC (cblC) protein and the binding of CNCbl and OHCbl was examined. Three mutations were analyzed: G147D, associated with early onset, vitamin B(12) unresponsive disease; R161Q, associated with late onset disease that is highly responsive to OHCbl; and H122A, selected to test the hypothesis that H122 is central to a proposed vitamin B(12) binding motif on MMACHC. We report here that wild-type MMACHC binds both OHCbl and CNCbl with similar, tight affinity (K(d)=5.7 microM). We also report that MMACHC binds CNCbl in the base-off form, with the dimethylbenzimidazole (DMB) base of cobalamin displaced from coordination with the cobalt. In this form, wild-type MMACHC is able to reductively decyanate CNCbl to cob(II)alamin requiring only the presence of NADPH and FAD. We demonstrate that MMACHC with the G147D mutation is unable to bind either CNCbl or OHCbl, providing a straight forward explanation for the absence of response to either vitamin form. However, we show that MMACHC containing the R161Q mutation binds OHCbl with wild-type affinity, but is disturbed in binding CNCbl and has impaired decyanation. Finally, we show that H122A has reduced binding, but like R161Q, it binds OHCbl more tightly than CNCbl, suggesting that this histidine is not absolutely required for binding. These studies suggest that the ability of mutant MMACHC to respond to vitamin therapy depends on its ability to bind the vitamin with significant affinity, and for CNCbl, also on its ability to bind in the base-off form to facilitate reductive decyanation. These studies emphasize the continued use of OHCbl with cblC patients for maximum therapeutic effect.

Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli.

In Escherichia coli, a four-gene operon, sbm-ygfD-ygfG-ygfH, has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro [Haller T, Buckel T, Retey J, Gerlt JA. Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 2000;39:4622-4629]. However, the operon was thought to be silent in vivo, illustrated by the eponym describing its first gene, "sleeping beauty mutase" (methylmalonyl-CoA mutase, MCM). Of the four genes described, only ygfD could not be assigned a function. In this study, we have evaluated the functional integrity of YgfD and Sbm and show that, indeed, both proteins are expressed in E. coli and that YgfD has GTPase activity. We show that YgfD and Sbm can be co-immunoprecipitated from E. coli extracts using antibody to either protein, demonstrating in vivo interaction, a result confirmed using a strain deleted for ygfD. We show further that, in vitro, purified His-tagged YgfD and Sbm behave as a monomer and dimer, respectively, and that they form a multi-subunit complex that is dependent on pre-incubation of YgfD with non-hydrolysable GTP, an outcome that was not affected by the state of Sbm, as holo- or apoenzyme. These studies reinforce a role for the in vivo interaction of YgfD and Sbm.

Restricted role for methionine synthase reductase defined by subcellular localization.

Methionine synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of methionine synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target the cytosol where methionine synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target mitochondria was also suggested by the work of Leal et al. [N.A. Leal, H. Olteanu, R. Banerjee, T.A. Bobik, Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase, J. Biol. Chem. 279 (2004) 47536-47542.] who showed that it can act as the reducing enzyme in combination with MMAB (ATP:Cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol, but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin.