The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12

Abstract

CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B₁₂ levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology, and activity-based metabolomics, we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human anti-microbial metabolite and immunomodulator. We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B₁₂ metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analog inhibitor of the mitochondrial B₁₂-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B₁₂ inactivation.

Keywords: C5 metabolism; CLYBL; citramalyl-CoA lyase; human genetics; itaconate; metabolism; mitochondria; systems biology; vitamin B(12).

Figure 1. Enzymatic activity and crystal structure of CLYBL

(A) Putative enzymatic activities of CLYBL. We detected (1) malate/methylmalate/citramalate synthase activity, (2) citramalyl-CoA lyase activity and (3) malyl-CoA thioesterase activity. (B) In vitro enzyme analysis of CLYBL reveals high citramalyl-CoA lyase activity. (C) Crystal structure of human CLYBL trimer. One subunit is shown in pink, magnesium ion in magenta, and the substrate mimic propionyl-CoA in cyan. The critical residue for catalytic activity Asp³²⁰ is highlighted. (D) Docking of citramalyl-CoA in the substrate binding pocket of the CLYBL structure. Free CoASH in the structure is shown in green, docked citramalyl-CoA in blue, one CLYBL subunit in pink and the neighboring subunit in gray. (E) The distances between docked citramalyl-CoA and the CLYBL residues are too close to accommodate additional carbon atoms in substrates larger than citramalyl-CoA. Three different views are shown. Labeled distances are in Angstrom (Å). The substrate-binding pocket of CLYBL is shown in surface representation.

Figure 2. CLYBL is a citramalyl-CoA lyase operating in C5 dicarboxylate catabolism

(A) Citramalyl-CoA levels in extracts from control and Clybl KO brown adipocytes. All results are means ± SD (n ≥ 3 biologic replicates) and were confirmed in independent experiments. (B) Citramalyl-CoA levels in the control brown adipocytes treated with 2 mM itaconate. (C) Schematic of the proposed CLYBL-mediated reaction in itaconate metabolism. (D) Schematic of itaconate -¹³C₅ labeling into citramalyl-CoA. (E) – (F) Relative abundances of different mass isotopomers of cellular itaconate (in E) and citramalyl-CoA (in F) from control brown adipocytes treated with either labeled and unlabeled itaconate.

Figure 3. Loss of CLYBL leads to a cell-autonomous defect in mitochondrial B₁₂ metabolism

(A) Western blot of Clybl in Clybl CRISPR KO murine brown adipocytes and Clybl KO cells expressing FLAG-tagged human CLYBL. (B) Schematic of the LC-MS-based untargeted metabolomics experiment design. (C) Volcano plot of the steady state level of metabolite ions from 3 d spent media and cellular extract from Clybl KO brown adipocytes and controls. (D) Bar graphs showing the change of the mitochondrial B₁₂ pathway metabolites in Clybl KO brown adipocytes. (E) Schematic of the mitochondrial B₁₂ metabolic pathway. (F) Coenzyme B₁₂ level in Clybl KO brown adipocytes and controls. (G) Methylmalonate (MMA) accumulation in Clybl KO cellular extract can be reversed by excess vitamin B₁₂ in the media (500 nM) or re-expressing human CLYBL that is resistant to the CRISPR reagent.

Figure 4. Enzyme activity of CLYBL is required to support mitochondrial B₁₂ in brown adipocytes

(A) Western blot of FLAG-tagged wild type human CLYBL and catalytic dead mutant CLYBL^D320A in Clybl KO cells. (B–D) Methylmalonate (MMA) accumulation (in B), coenzyme B₁₂ inhibition (in C), and citramalyl-CoA accumulation (in D) in Clybl KO cellular extract cannot be rescued by catalytically dead mutant of CLYBL.

Figure 5. Itaconyl-CoA directly inactivates coenzyme B12 through MUT

(A) Cellular methylmalonate (MMA) is accumulated in control brown adipocytes treated with 2 mM itaconate. (B) Coenzyme B₁₂ is reduced in control brown adipocytes treated with 2 mM itaconate. (C) Western blot of Clybl, Mut in double KO cells. (D–E) Loss of Mut suppresses coenzyme B₁₂ deficiency in Clybl KO cells (in D), despite similarly increased MMA level (in E), suggesting that the inhibition mechanism is via MUT activity. (F–G) Itaconyl-CoA inactivates human MUT enzyme. Purified human MUT enzyme was preloaded with coenzyme B₁₂ AdoCbl (with an absorbance maximum at 529 nm). UV-visible spectra were recorded before (red), immediately after (gray), and 20 min after (black) addition of either substrate M-CoA (in F) or itaconyl-CoA (in G). Unlike M-CoA, which elicited only minor spectral changes, itaconyl-CoA triggered a large blue shift in the absorbance maximum to 466 nm, indicating formation of inactive cob(II)alamin.

Figure 6. The immunomodulatory metabolite itaconate poisons vitamin B12 in various cell types

(A–B) Coenzyme B₁₂ is reduced by 2 mM itaconate treatment in HEK293T cell (in A), and in human B-lymphocytes (in B). Itaconyl-CoA is undetected in untreated cells, but dramatically increased in treated cells. (C) Coenzyme B₁₂ is ablated in macrophages upon 10 ng/ml LPS stimulation for 6 hr. Itaconyl-CoA is dramatically increased upon stimulation.

Figure 7. Relationship between CLYBL, itaconate and B₁₂

CLYBL functions as a citramalyl-CoA lyase, and its loss leads to an accumulation of upstream metabolites. Itaconyl-CoA serving as a substrate analog directly inhibits the MUT enzyme and rapidly inactivates the coenzyme B₁₂.