B12 and glutathione

Cobalamin processing

In 2020 Ruma Banerjee’s group at the University of Michigan Medical Center published an excellent review on the role of thiols in B12/cobalamin (Cbl) processing.

Sometimes the use of gene names for proteins gets confusing

• MMSDHC is the gene name of CblD is the name of a gene that codes for a protein called Cobalamin trafficking protein Cbl.
• MMACHC (the gene) codes for a protein Cyanocobalamin reductase which catalyzes the reductive decyanation of cyanocob(III)alamin (cyanocobalamin, CNCbl) to yield cob(II)alamin and cyanide, using FAD or FMN as cofactors and NADPH as a co-substrate. Here the “(II)” seems to refer to the oxidation state of cobalt. The balloon with a dot as an unpaired electron available to bond with other substrates.

The goal of our chaperone proteins is to remove the CN- group and replace it with something else. CblC removes the upper axial ligand via glutathione (GSH)-dependent dealkylation of alkyl-cobalamin. A previous report from the Banerjee group cited in the review [1] gives an “equation 1

R-Cob(III)alamin + GS^– → GSR + Cob(I)alamin → Cob(I) +e^– → Cob(II)alamin

The following reaction was mentioned in another Banerjee group previous report cited in the review [1]

GS-Cobalamin + GSH → GSSG + Cob(I)alamin +H⁺

In a personal communication Dr Manergee stated that “Cob(I) is highly reactive. An excess of GSSG would not be needed to drive the reverse reaction.” If there is an excess of GSSG and metabolic acidosis, would the probability of the reverse direction be greater? If so, how would this impact the amount of metabolically active forms of Cbl? With this introduction, we may examine a report examining various cobalamin species as they relate to age, autism, and schizophrenia.

Brain Levels B12 in Aging, Autism and Schizophrenia [2]

This was a multi-center study that used hplc to extract cobalamin (Cbl) isoforms from the frontal cortex of human brains in “brain banks”. Cbl isoforms were identified by mass spectrometry. Zhang and coauthors introduced their study with a pathway diagram that incuded, EAAT3 that is also know as the excitatory amino acid (glutamate and aspartate) transporter that exchanges K⁺ and Na⁺ for glu and asp. It is also a transporter for cysteine. These cysteines may beused to form glutathione.

The cobalamin glutathione pathway intersection overview

Glutamate-cysteine ligase is not shown in this cartoon.

As a review

• Cyano Cbl is the inactive species.
• MeCbl is a cofactor for folate-dependent methylation of HCY to methionine by methionine synthase (MS) in the cytoplasm. , whose activity supports a large number of methylation reactions, including DNA methylation, as well as dopamine-stimulated phospholipid methylation, carried out by the D4 dopamine receptor (D4R).
• AdoCbl is required for the conversion of methylmalonylCoA to succinylCoA by methylmalonyl CoA mutase in mitochondria
• OHCbl, or hydroxy cobalamin is the isoform found in dietary supplements. It seems to be the sameas H₂O-Cbl of the Banerjee review,
• EAAT3 is the excitatory amino acid transporter.
• Transsulfuration of HCY via cystathionine is restricted in human brain, increasing the importance of growth factor-dependent cysteine uptake by EAAT3. [2]

Cbl changes as we age

• Total Cbl concentration in the frontal cortex decreases with age.
• Active Cbl forms are affected more than the inactive forms.
• There’s an inverse correlation between the active form MeCbl with age.

Cbl in autism and schizophrenia.

Note that the hydroxy Cbl is higher in ASD and schizophrenia than the controls. From the Banerjee review H₂OCbl is somewhat further in terms of processing to the active forms MeCbl and Ado Cbl than is GSCbl. [1] Nothing is spelled out in really simple terms.

Thiol Status

Thiol status is defined as the percent of the control.

Homocysteine is significantly higher and methionine and cystathionine much lower.

In these aged and ASD brains we are seeing more or less what we would predict based on the pathways defined in the introduction. [2]

Methionionine Synthase activity

Postmortem brain samples were homogenized in a lysis buffer and mixed with a phosphate buffer stock also containing HCY, DTT, SAM, adding either 10 μl water or 10 μl of 5 mM OHCbl . It is assumed that DTT, dithiothreitol, is a substitute for glutahione.

GCLM knockout mice studies

Glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Here P40 and P90 refer to the age of the mice in days. Loss of GCL would be expected to decrease GSH levels, but Cys would be predicted to increase. Homocysteine is also decreased. All forms of Cbl are decreased, not just the active forms.

For another post

This post has explored the relationship between glutathione and active forms of B12/cobalamin/Cbl. Knocking out an enzyme responsible for the synthesis of glutathione has revealed more sweeping changes in the Cbl status. Something else is going on that will be explored in the next post. If you are reading this and wondering if you are getting enough cobalamin in your plant based diet, you might also be wondering about sulfur containing amino acids discussed in this post like methionine and cysteine. One or more Wikipedia authors have assembled a page of plant sources of amino acids. Soybeans appear to be the winner for cysteine+methionine for common foods. Dried seaweed has around 50% more. The Wikipedia author cites a Swedish language source of amino acids in common and not so common raw foods. The Swedish source cites a United States Dairy Association that is not easily found on the Internet. In a limited study from the Netherlands comparing the amino acid of common foods with that of human muscle, brown rice and wheat have more cysteine than eggs and whey. [3] Brown rice, corn, and potato are the best sources of methionine. [3]

References

1. Li, Z., Mascarenhas, R., Twahir, U.T., Kallon, A.,Deb, A., Yaw, M., Penner-Hahn, J., Koutmos, M., Warncke, K., and Banerjee, R. (2020b). An interprotein Co-S coordination complex in the B12-trafficking pathway. J. Am. Chem. Soc. 142,16334–16345 PMC free article
2. Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, Do KQ, Deth RC. Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One. 2016 Jan 22;11(1):e0146797. PMC free article
3. Gorissen SHM, Crombag JJR, Senden JMG, Waterval WAH, Bierau J, Verdijk LB, van Loon LJC. Protein content and amino acid composition of commercially available plant-based protein isolates. Amino Acids. 2018 Dec;50(12):1685-1695. PMC free article