Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC
Graphical abstract
Introduction
Cobalamin (Cbl) is an essential micronutrient that is exclusively synthesized by some groups of bacteria and archaea [1]. Structurally, cobalamins are tetrapyrroles coordinating a cobalt center. The corrin ring presents seven acetamide and propionamide side chains and ligand 5-dimethylbenzimidazole via a phosphoester linkage (Fig. 1). The Co center can be coordinated axially by a 5-dimethylbenzimidazole moiety in the 5th coordination position (α-ligand) and by a number of possible chemical groups in the 6th coordination position (β-ligand). Based on whether the 5-dimethylbenzimidazole moiety is bound to the cobalt center via a N-atom or unbound, cobalamins exist in two configurations, namely, base-on and base-off, respectively. The upper or 6th coordination position can be occupied by a variety of biologically relevant as well as synthetic ligands via Co–C, Co–N, Co–O and Co–S bonds [[2], [3], [4], [5], [6]]. Likewise, biologically relevant Cbl forms exploit all three possible oxidation states of the cobalt center to give cob (III)alamin, cob(II)alamin and cob(I)alamin [5,7]. The interaction of Cbls with sulfur compounds represents an important part of their redox and coordination chemistry; for example, reactions with thiols not only produce stable cob(III)alamin complexes, but also increase reactivity of the corresponding cob(II)alamin species [5,7].
Humans obtain B12 consumption of animal food sources. Because B12 does not occur in the plant kingdom, individuals who adhere to plant-based nutrition must rely on supplemental forms of B12. Cobalamin is required to drive the enzymatic reactions of cytosolic methionine synthase (MS) and of mitochondrial methylmalonyl-CoA mutase (MUT) [8]. In the catalytic cycle of MS, homocysteine is methylated by methylcobalamin (MeCbl) to form the amino acid methionine. Cob(I)alamin, the other product of this reaction, is then remethylated by N5-methyltetrahydrofolate, to regenerate MeCbl and tetrahydrofolate. Occasional unwanted oxidation of cob(I)alamin to form cob(II)alamin is repaired by the enzyme methionine synthase reductase, thus returning the Cbl moiety into the catalytic cycle of MS as cob(I)alamin. The reaction driven by MUT comprises cycling of adenosylcobalamin (AdoCbl) and cob(II)alamin for the isomerization of methylmalonyl-CoA into succinyl-CoA. Failed reduction of cob(II)alamin to cob(I)alamin leads to the formation of aquacobalamin (H2OCbl). Prior to delivery to MS and MUT dietary cobalamins undergo chemical processing by the processing enzyme CblC (a.k.a. MMACHC) [8,9] and partition into cytosol and mitochondrion orchestrated by interactions of CblC with the adaptor protein CblD (MMADHC) [[10], [11], [12]]. Evidence for multi-partite protein complexes involving CblC, CblD and recipient protein MS has also been reported [13].
As expected from the above described metabolism of vitamin B12, the major Cbl forms found in foods are MeCbl, AdoCbl and H2OCbl [[14], [15], [16], [17]]. Cyanocobalamin (CNCbl), the most inexpensive commercial form of vitamin B12 occurs in ocean water [18] but it is absent in human tissue and fluids unless previously taken as an oral supplement or by intramuscular injection. Mutations in the cblC gene (MMACHC) disrupt the cellular processing of dietary cobalamins. This pathology is known as the cblC disease and is characterized by combined homocystinuria with methylmalonic aciduria. The cblC disease is the most common inherited disorder of intracellular cobalamin metabolism. Current treatment with hydroxycobalamin does unfortunately not prevent from the development of clinical symptoms. The cblC disorder has a spectrum of severity with two distinct phenotypic forms: early and late onset. Early-onset patients present with intrauterine growth retardation (IUGR), microcephaly, failure to thrive, developmental delay, hypotonia, progressive retinopathy, and maculopathy [19]. Late-onset CblC patients usually present with extrapyramidal and neuropsychiatric symptoms in any decade of life [20]. In some countries cblC disease is also part of newborn screening programs. An intrauterine treatment is discussed in order to prevent the early onset phenotype [21].
Early work suggested that the thiolatocobalamin glutathionylcobalamin (GSCbl), and sulfitocobalamin (SO3Cbl) also occur naturally, and that they may represent reaction intermediates in the intracellular processing of dietary cobalamins [22]. Comprehensive profiling of cobalamins in mammalian fluids and cells using [57Co]-cyanocobalamin as a precursor demonstrated that GSCbl and SO3Cbl are indeed part of the endogenous cobalamin pool [14,23]. Work performed with the cobalamin processing enzyme CblC later demonstrated that glutathione (GSH) serves as the nucleophile for the dealkylation of MeCbl and AdoCbl as well as for the decyanation of CNCbl, but these reactions occur without formation of a GSCbl intermediate [24,25]. The enzyme CblC catalyzes a reaction that is from the point of view of pH considerations and redox potentials, otherwise unattainable under the conditions of the biological milieu. Firstly, dealkylation of MeCbl and AdoCbl requires weakening of the Co–C bond, which is achieved by induction of the base-off configuration of the α-ligand thus leading to a trans-weakening effect on the β-ligand [26,27]. Because the pKa for the base-on to base-off transitions of biologically relevant Cbls MeCbl, AdoCbl and H2OCbl are 2.90 [28], 3.50 [5], and −2.40 [29], respectively, this change in configuration requires enzyme-mediated distortion of the α-ligand. Secondly, disruption of the Co–C bond most typically requires reduction of the cobalt center, which in biologically relevant cobalamins means a thermodynamic barrier of approximately 1 V [5]. This requires protein-mediated reduction as none of the known intracellular reductants could reach this thermodynamic threshold. In humans, CblC is responsible for the induction of the base-off conformation of cobalamins and glutathione-mediated rupture of the Co–C via nucleophilic attack and reductive elimination [24]. Recent mechanistic studies with non-mammalian variants of CblC demonstrated that GSCbl does form as a stable reaction intermediate in reactions of MeCbl dealkylation by GSH [30]. This suggests that the reactivity of cobalamins with glutathione to form the stable thiolatocobalamin GSCbl has changed through evolution to adjust to nutritional demands or perhaps, to environmental availability of the micronutrient. In comparison to alkylcobalamins, the reduction of thiolatocobalamins requires large negative potentials [31]. At the same time, the Co(III)–S bond is more reactive than the Co(III)–C bond; for example, GSCbl can be readily reduced by selenocysteine [32] whereas its reaction with MeCbl is substantially slower (unpublished). Conversion of the thiolatocobalamins from the base-on to the base-off configuration might destabilize the Co(III)–S bond toward the Co(II)–S state, as suggested by the lower stability of the nucleotide-free Cbl analogue glutathionylcobamide compared to GSCbl [33].
Dethiolation of GSCbl by CblC in the presence of GSH yields the catalytically active cob(II)alamin and cob(I)alamin when reactions are performed under anaerobic conditions reactions [[34], [35], [36]]. Based on these findings, we hypothesized that the Co–S bond enables a more facile conversion of base-on to base-off conformation of the Cbl substrate, thereby decreasing thermodynamic barriers of redox potential that are necessary for the removal of the β-axial ligand. Herein, we describe the synthesis and spectroscopic characterization of two novel thiolatocobalamins, CyaCbl and MpgCbl. The two cobalamins were synthesized in pure form and exhibited spectral properties similar to that of other thiolatocobalamins. CyaCbl and MpgCbl bound to human CblC inducing their respective base-off configurations, and served as substrates for dethiolation in reactions driven with GSH under physiologically relevant conditions. Under aerobic conditions, this reaction yielded H2OCbl as the only cobalamin species. The new thiolatocobalamins CyaCbl and MpgCbl repaired the activity of human pathogenic variants R161G and R161Q, responsible for early and late onset cblC disease in humans, respectively. Our study enriches the repertoire of thiolatocobalamins with biological activity that are suitable for the repair of pathogenic variants of CblC that lead to combined homocystinuria and methylmalonic aciduria in humans. These new compounds may represent an effective treatment alternative.
Section snippets
General procedures and chemicals
Hydroxocobalamin hydrochloride (Sigma, FW 1382.82, product Nr. 95200-1G), cysteamine (Sigma, FW 113.61, product Nr. 30078-25G) and N-(2-mercaptopropionyl)-glycine (Mpg, also known as Tiopronin, FW 163.19, Sigma product Nr. M6635-5G) were used without further purification. Water was purified using a PureLab classic water purification system from ELGA. Milli Q water was used for all reactions. Water (product Nr. 39253) and methanol (product Nr. 34966) for mass spectrometry were purchased from
MpgCbl
MpgCbl was synthesized by addition of an aqueous solution of Mpg (1.9 mol equiv., phosphate buffer, pH 7.2) to an aqueous solution of H2OCbl, in accordance to a previously reported procedure to synthesize other thiolatocobalamins [38]. Formation of MpgCbl was followed by UV–visible spectroscopy, as H2OCbl and thiolatocobalamins have clearly different UV–visible spectra.
CyaCbl
Preliminary tests showed that Cya and H2OCbl react much faster under mild acidic conditions, a finding that is in agreement
Discussion
Evidence of the formation of complexes between thiols and aquacobalamin date back to the early 1960’s [48,49]. The reaction of cysteamine with aquacobalamin was first described by Cavallini et al. [41]. Paucity in further investigation of this reaction partly motivated us to explore the biological properties and potential pharmacological utility of cysteaminylcobalamin and the new derivative mercaptopropionylglycylcobalamin. Several studies on the reaction mechanism of the CblC protein
Conclusions
We synthesized two novel thiolatocobalamins that are stable under physiologically relevant conditions. The two novel thiolatocobalamins bound to wild type and pathogenic variants of human CblC inducing blue-shifted spectra consistent with the formation of base-off cobalamin species. Both GSCbl and new thiolatocobalamins CyaCbl and MpgCbl support dethiolation reactions by human wild type CblC and repair the enzymatic activity of pathogenic variants R161G and R161Q. Furthermore, MpgCbl and CyaCbl
Author contributions
V.W., S.M., A.J.E., S.B., M.K., S.T. and L.H. performed experiments and analyzed data. I.D., S.V.M., D.W.J., U.S. and L.H. analyzed data and critically reviewed the manuscript. V.W. wrote the first draft of the manuscript. I.A.D., S.V.M., D.W.J, U.S. and L.H. wrote and edited the manuscript. L.H. conceived the study and supervised all facets of the experimental work.
Funding
This study was supported with intramural funds from the Department of General Pediatrics, Adolescent Medicine and Neonatology, Faculty of Medicine, Medical Center, University of Freiburg, Germany.
Availability of data and materials
The data and materials used in this study are available from the corresponding author on reasonable request.
Ethical approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Disclosures
The novel cobalamins CyaCbl and MpgCbl are part of a patent application (L.H., filed by University of Freiburg, International Application Nr. PCT/EP2018/063597, Publication Nr. WO/2018/215578).
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgements
The authors are grateful to Katharina Klotz for excellent technical support.
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