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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 a...

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Autores principales: Froese, D.S., Healy, S., McDonald, M., Kochan, G., Oppermann, U., Niesen, F.H., Gravel, R.A.
Formato: Texto
Lenguaje:English
Publicado: Academic Press 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923755/
https://www.ncbi.nlm.nih.gov/pubmed/20219402
http://dx.doi.org/10.1016/j.ymgme.2010.02.005
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author Froese, D.S.
Healy, S.
McDonald, M.
Kochan, G.
Oppermann, U.
Niesen, F.H.
Gravel, R.A.
author_facet Froese, D.S.
Healy, S.
McDonald, M.
Kochan, G.
Oppermann, U.
Niesen, F.H.
Gravel, R.A.
author_sort Froese, D.S.
collection PubMed
description 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 °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 (ΔT(m max) = 8 °C, half-maximal effective ligand concentration, AC(50) = 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 T(m) of ∼16 °C, followed by MeCbl at ∼13 °C, each evaluated at 50 μM 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 μM, which compares well with the K(d) 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 (ΔT(m max) = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC(50) 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 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.
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spelling pubmed-29237552010-09-08 Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder Froese, D.S. Healy, S. McDonald, M. Kochan, G. Oppermann, U. Niesen, F.H. Gravel, R.A. Mol Genet Metab Article 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 °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 (ΔT(m max) = 8 °C, half-maximal effective ligand concentration, AC(50) = 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 T(m) of ∼16 °C, followed by MeCbl at ∼13 °C, each evaluated at 50 μM 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 μM, which compares well with the K(d) 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 (ΔT(m max) = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC(50) 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 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. Academic Press 2010-05 /pmc/articles/PMC2923755/ /pubmed/20219402 http://dx.doi.org/10.1016/j.ymgme.2010.02.005 Text en © 2010 Elsevier Inc. https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Article
Froese, D.S.
Healy, S.
McDonald, M.
Kochan, G.
Oppermann, U.
Niesen, F.H.
Gravel, R.A.
Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder
title Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder
title_full Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder
title_fullStr Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder
title_full_unstemmed Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder
title_short Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder
title_sort thermolability of mutant mmachc protein in the vitamin b12-responsive cblc disorder
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923755/
https://www.ncbi.nlm.nih.gov/pubmed/20219402
http://dx.doi.org/10.1016/j.ymgme.2010.02.005
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