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

Abstract

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 (Tm) of 39.3 ± 1.0 and 37.1 ± 0.7 °C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 °C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔTm max = 8 °C, half-maximal effective ligand concentration, AC50 = 3 μM). 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 Tm of ∼16 °C, followed by MeCbl at ∼13 °C, each evaluated at 50 μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi > OHCbl > CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3 μM, which compares well with the Kd of 6 μM 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 (ΔTm max = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7 μM. 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 AC50 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.

Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism

“…In the absence of cobalamins, total scattering intensity increased exponentially above 48°C for MMADHCΔ1-12 and above 52°C for MMADHCΔ1-61, a transition point reflecting the melting temperature (T m ) for proteins [40,41]. These values are significantly higher than that reported for full length MMACHC (~40°C) [15,18]. Furthermore, the addition of 10-fold molar excesses of the four cobalamins to either MMADHC construct did not shift the transition points to a higher temperature, indicating no apparent stabilization by cobalamin.…”

“…Recent evidence suggests possible localization to the mitochondrion [14] despite its lacking a predicted mitochondrial leader sequence. MMACHC binds Cbl [15][16][17] in a base-off conformation [18].…”

Proteomics of vitamin B12 processing

“…Froese et al demonstrated that MMACHC is naturally thermolabile (T m = 39 ° C) and that some of the most frequent mutations that occur in humans exacerbate this property [ 39 ] as well as its ability to bind Cbls [ 40 ]. Studies with the bovine isoform of MMACHC revealed that the reduced form of glutathione stabilizes MMACHC, suggesting that intracellular redox control could play a role in the regulation of the protein ' s lifetime [ 41 -44 ].…”

Pathogenic Mutations Differentially Affect the Catalytic Activities of the Human B12-processing Chaperone CblC and Increase Futile Redox Cycling

“…Mutations at Arg-161 are among the most common of the missense mutations leading to the cblC disorder, and depending on the nature of the substitution, these mutations are associated with either early (R161G) or late (R161Q) onset of the disease (20). The R161Q mutant has been characterized minimally, reported to decrease the T m of CblC by 2°C (25) and the MeCbl dealkylation activity ϳ10-fold (24).…”