Biochemistry, function, and deficiency of vitamin B₁₂ in Caenorhabditis elegans

Abstract

Caenorhabditis elegans is a nematode that has been widely used as an animal for investigation of diverse biological phenomena. Vitamin B₁₂ is essential for the growth of this worm, which contains two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase. A full complement of gene homologs encoding the enzymes associated with the mammalian intercellular metabolic processes of vitamin B₁₂ is identified in the genome of C. elegans. However, this worm has no orthologs of the vitamin B₁₂-binders that participate in human intestinal absorption and blood circulation. When the worm is treated with a vitamin B₁₂-deficient diet for five generations (15 days), it readily develops vitamin B₁₂ deficiency, which induces worm phenotypes (infertility, delayed growth, and shorter lifespan) that resemble the symptoms of mammalian vitamin B₁₂ deficiency. Such phenotypes associated with vitamin B₁₂ deficiency were readily induced in the worm.

Introduction

Vitamin B₁₂ is well known as a corrinoid compound containing a corrin macrocycle in their structure. Vitamin B₁₂ is usually presented as cyanocobalamin (CN-B₁₂). This unnatural form of cobalamin is more chemically stable than naturally occurring cobalamins.¹ However, in this review, the term vitamin B₁₂ (B₁₂) refers to cobalamin compounds having B₁₂ activity. CN-B₁₂ is usually used for human dietary supplements and the CN-B₁₂ taken up by living cells is synthesized into the coenzyme forms of cobalamin,² methylcobalamin (CH₃-B₁₂), and 5′-deoxyadenosylcobalamin (AdoB₁₂). Methionine synthase (MTR) (EC 2.1.1.13) contains CH₃-B₁₂ as a prosthetic group and functions in methionine biosynthesis.² AdoB₁₂ functions as a coenzyme for methylmalonyl-CoA mutase (MCM) (EC 5.4.99.2) catalyzing the isomerization of R-methylmalonyl-CoA to succinyl-CoA in the catabolism of amino acid and odd-chain fatty acid in mammalian cells³ (Figure 1).

Figure 1.

Structural formula of vitamin B₁₂ and partial structures of vitamin B₁₂ compounds. The partial structures of vitamin B₁₂ compounds show only the portions of the molecules that differ from vitamin B₁₂. 1: 5′-deoxyadenosylcobalamin; 2: methylcobalamin; 3: hydroxocobalamin; 4: cyanocobalamin or vitamin B₁₂

Only certain bacteria have the de novo biosynthetic pathway of B₁₂, which is accumulated in the bodies of predatory animals located higher in the food chain.⁴ Thus, B₁₂ is the only vitamin that does not exist in plant-derived foods. In particular, the best dietary sources of B₁₂ include meat, milk, fish, and shellfish.⁵ The recommended dietary allowance of B₁₂ is 2.4 µg per day for adults.^6,7 People with atrophic gastritis developing low stomach acid output easily present the food protein-bound B₁₂ malabsorption, which prevails in elderly people.⁸ Thus, strict vegetarians and elderly people have an increased risk of developing B₁₂ deficiency.^6,9 Megaloblastic anemia and neuropathy are well-known symptoms of B₁₂ deficiency,⁶ but the molecular mechanism underlying such diseases is still poorly understood.¹⁰

Caenorhabditis elegans, consisting of 959 somatic cells, shows a very short life cycle and has the ability to change its locomotor behavior, reproductive rate, and lifespan.¹¹ Caenorhabditis elegans conserves many molecular processes and cellular metabolisms of mammals, and many genes responsible for human disease are also present in the worm.¹² Therefore, C. elegans has been extensively used as a model animal for understanding various biological phenomena.¹¹

Recently, C. elegans has been used to gain a new understanding between physiological events and diets, which contain nutrients and other bioactive compounds.^13,14 Different diets significantly affect various gene expressions and metabolic pathways in worms.¹⁵ Dietary B₁₂ is identified as a potent modulator in C. elegans.¹³ Our study¹⁶ indicates that C. elegans essentially requires B₁₂ for growth, and dietary B₁₂ deficiency results in infertility, retardation of growth, and reduced lifespan. Such B₁₂-deficient phenotypes are reported in mammals. Thus, the worm appears to be a highly suitable model animal for elucidating the mechanisms of nutrient-related diseases.

In the minireview, we describe up-to-date information on the physiological functions of B₁₂ and on the disordered biological processes due to B₁₂ deficiency in C. elegans.

Vitamin B₁₂ requirements

Like all animals, C. elegans has to eat to obtain nutrients for development, growth, and reproduction. Although Escherichia coli OP50 is considered best suitable as a bacterial diet, C. elegans has been grown using many other bacteria.¹⁵ Although C. elegans can be grown well by feeding different bacterial diets, its physiological properties are significantly affected by different diets.¹⁵ Although E. coli OP50 does not differ significantly from Comamonas sp. DA1877 in macronutrient levels (carbohydrates, protein, and fat), feeding with Comamonas sp. DA1877 shows faster development in worms than with E. coli OP50.¹⁴ When Watson et al.¹³ compared E. coli OP50 and Comamonas sp. DA1877 diets to identify biologically active compounds affecting the worm’s gene expression and physiology, B₁₂ was identified as the major compound provided by Comamonas DA1877. The B₁₂ level in the Comamonas DA1877 diet is higher than that in the E. coli OP50 diet. Our unpublished data showed that E. coli OP50 contained 11.1 µg of B₁₂ per gram of wet weight of cells when the bacterium was grown in Luria-Bertani medium. To clarify B₁₂ requirements for the growth of C. elegans, the worm was grown on B₁₂-sufficient M9 agar plate and fed with B₁₂-deficient E. coli OP50 (0.9 µg of B₁₂ per gram of wet weight of cells). Worms absorbed a sufficient amount of free B₁₂ from the B₁₂-supplemented agar medium.¹⁶ Control worm eggs were transferred onto the B₁₂-deficient M9 agar plate and fed with the B₁₂-deficient E. coli OP50. The eggs hatched and then worms were grown until adults (these worms represent the F1 generation). When this process was repeated for five generations, the cellular B₁₂ level of the worms was significantly reduced over four generations. The B₁₂ level of the fifth generation worms is only 4% of that in the B₁₂-sufficient worms (138 ± 51 ng of B₁₂ per gram of wet weight).¹⁶ The methylmalonic acid (MMA) and homocysteine (Hcy), which are usually used as the indices of B₁₂ deficiency, are significantly increased because holo-MCM and -MTR activities are significantly decreased in the worms of the fifth generation grown under B₁₂ deficiency conditions.¹⁶ These results demonstrate that C. elegans absolutely requires B₁₂ for growth and develops severe B₁₂ deficiency when it is grown under B₁₂ deficiency conditions for five generations. Such changes in B₁₂-dependent enzyme activities are reported to occur in mammals^17,18; a B₁₂-deficient animal can be produced in only 15 days (five generations) using the nematode model.

Intestinal absorption and body circulation

Humans have a complicated system for intestinal absorption of B₁₂.¹⁹ Dietary B₁₂ reaches the stomach and is liberated from food protein by stomach acid and protease. Salivary protein haptocorrin binds with B₁₂ and transports it through the stomach and into the small intestine. The protein moiety of the B₁₂ complex is digested by pancreatic proteases and then free B₁₂ is formed. The liberated B₁₂ attached to the gastric protein intrinsic factor (IF). The B₁₂–IF complex is internalized into mucosal cells of the terminal ileum by receptor-mediated endocytosis. After the protein moiety of the B₁₂–IF complex is digested in the mucosal cells, released B₁₂ binds to transcobalamin II (TC) and moves into the blood circulation.

Caenorhabditis elegans is not a suitable model animal to elucidate the mechanisms of megaloblastic anemia and disorders of B₁₂ absorption and transport as human diseases caused by B₁₂ deficiency because the blood corpuscle systems and the three B₁₂ transporters (haptocorrin, IF, and TC) are not found in this worm.

Intracellular metabolisms and functions

In human cells, the B₁₂–TC complex is internalized by the receptor-mediated endocytosis.²⁰ The protein moiety of the complex is digested by the action of lysosomal proteases and then B₁₂ is released from the complex.²¹ The released B₁₂ is transferred to the cytosol by the actions of LMBD1, a lipocalin-interacting membrane receptor,²² and ABCD4, an ATP-binding cassette transporter protein.²³ In the cytosol, the cobalt atom of B₁₂ is reduced by methylmalonic aciduria and homocystinuria type C protein (MMACHC or CblC), which catalyzes the reductive decyanation of CN-B₁₂ as dietary B₁₂²⁴ and intracellular delivery of B₁₂²⁵ is converted into CH₃-B₁₂, a coenzyme for MTR in the cytosol, or transferred into the mitochondria, and then AdoB₁₂ is synthesized and functions as the coenzyme for MCM. In the enzymatic reaction of MTR, CH₃–B₁₂ serves as acceptor of the methyl group in the catalytic cycle between cob(I)alamin and CH₃–B₁₂. Methionine synthase reductase (MTRR) catalyzes the reductive reactivation of inactive MTR bound to oxidized cob(II)alamin to maintain its active form using S-adenosylmethionine (SAM) as a methyl donor.²⁶ In the mitochondria, the methylmalonic aciduria type A protein (MMAA) binds to the MCM protein to prevent MCM inactivation as a chaperone.²⁷ ATP:cob(I)alamin adenosyltransferase (MMAB) catalyzes the adenosyl transfer reaction from ATP to cob(I)alamin in the AdoB₁₂ biosynthesis.²⁸ The MMAB protein also functions as a chaperone involved in the transfer of the formed AdoB₁₂ to the apo-MCM protein.²⁹

Using bioinformatics, a full complement of homologs of the mammalian B₁₂-related metabolic pathways has been identified in the genome of C. elegans, including propionyl-CoA carboxylase (pcca-1, pccb-1), methylmalonyl-CoA epimerase (mce-1), MMACHC (cblc-1), MMAA (mmaa-1), MMAB (mmab-1), MCM (mmcm-1), MTR (metr-1), MTRR (mtrr-1), and SAM synthase (sams-1) (Table 1).

Table 1.

Mammalian homologs of members of the Caenorhabditis elegans B₁₂-related metabolic pathways deduced from the genome sequence

Protein name	Homo sapiens	Caenorhabditis elegans	BLAST score	Reference
Propionyl-CoA carboxylase alpha chain	PCCA	pcca-1	1.20 E-214	30
Propionyl-CoA carboxylase beta chain	PCCB	pccb-1	4.50 E-213	31
Methylmalonyl-CoA epimerase	MCE	mce-1	2.50 E-45	32
Methylmalonyl-CoA mutase	MCM	mmcm-1	9.91 E-280	33
Methylmalonic aciduria type A protein	MMAA	mmaa-1	2.60 E-89	34
Methylmalonic aciduria type B protein	MMAB	mmab-1	4.80 E-35	33
Methylmalonic aciduria and homocystinuria type C protein	MMACHC	cblc-1	6.20 E-26	35
Methionine synthase	MTR	metr-1	0	36
Methionine synthase reductase	MTRR	mtrr-1	1.40 E-74	30
S-adenosylmethionine synthase	MAT	sams-1	1.50 E-148	37

Chandler et al.³³ characterized the AdoB₁₂-dependent methymalonlyl-CoA pathway in the tricarboxylic acid (TCA) cycle. RNA interference (RNAi) experiments indicate that the decreased mmcm-1, mmab-1, and mmaa-1 gene expressions result in increased MMA. Worm deletion mutants mmab-1, mmcm-1, and mce-1 decrease the incorporation of propionate into macromolecular compounds. These results show occurrence of AdoB₁₂-dependent methymalonlyl-CoA metabolic pathway in worms. The worm mce-1 deletion mutant demonstrates that the flux through the methylmalonyl-CoA pathway is significantly reduced because of the defect in the epimerase reaction but the mce-1 deletion shows no significantly phenotypic change.

The worm’s methylmalonyl-CoA epimerase, a 15 kDa protein, was shown to be significantly expressed in body-wall muscles, and its subcellular pattern of expression suggests the mitochondrial localization of the enzyme.³⁸

The methionine synthetic pathway is localized in the cytosolic fraction of mammalian cells.³⁹ In C. elegans, MTR, MTRR, and SAM synthase are likely to be cytosolic enzymes, considering their involvement in methionine metabolism. Human MMACHC (or CblC) exhibits reduced glutathione-dependent alkyltransferase activity and flavin-dependent reductive decyanation activity with CN-B₁₂.^25,40 The C. elegans MMACHC, which shares 36% identity and 53% similarity with the human enzyme, catalyzes the decyanation reaction of CN-B₁₂ by the addition of reduced glutathione, an activity that has been overlooked previously with human MMACHC.³⁵ The decyanation product, cob(II)alamin, is stabilized by worm MMACHC. Worm and human MMACHC activities show different susceptibilities to oxygen.

Detailed properties of the remaining enzymes and proteins that are responsible for the intracellular metabolic processes of B₁₂ have not been characterized in C. elegans. The intracellular metabolisms and functions of B₁₂ in C. elegans are postulated in Figure 2 based on the above information.

Figure 2.

Postulated intracellular metabolism and function of vitamin B₁₂. SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; THF: tetrahydrofolate; TCA: tricarboxylic acid. The other abbreviations used are as shown in Table 1

Vitamin B₁₂ deficiency

The effects of B₁₂ deficiency on MCM and MTR activities in C. elegans have been evaluated. Holo-MTR activity rapidly decreased in a cell homogenate of worms treated with the B₁₂ deficient medium for one generation, but holo-MCM activity was not altered until the fourth generation. The result suggests that MTR is more susceptible to cellular B₁₂ levels than MCM.¹⁶ Hepatic MTR activity is mostly derived from the holo-enzyme in B₁₂-sufficient or -deficient rats.¹⁷ Although MTR protein levels in liver homogenates of B₁₂-deficient rats are significantly reduced compared with those of B₁₂-sufficient rats, the hepatic B₁₂ level does not affect the level of MTR-encoding mRNA.¹⁷ Thus, the apo-MTR is very unstable. Determination of the changes in MTR protein and mRNA (metr-1) levels in B₁₂-deficient worms has shown that the level of mRNA was not altered; however, the level of MTR protein in B₁₂-deficient worms was decreased to approximately 32% compared with that in B₁₂-sufficient worms.⁴¹ Considering that a trace band of MTR was detected in the liver homogenates of B₁₂-deficient rats, this result suggests that the worm apo-enzyme is considerably stable. MTR is important for resynthesizing methionine and metabolizing 5-methyltetrahydrofolate. Tetrahydrofolate generated by the action of MTR is converted to 5,10-methylenetetrahydrofolate, which is crucial for the biosynthesis of thymidine that occurs exclusively in DNA.⁴² Moreover, methionine is converted to SAM, which donates a methyl group to certain compounds in various methyl transfer reactions. Hcy acquires a methyl group from either 5-methyltetrahydrofolate or betaine to form methionine. Both synthetic pathways of methionine play important roles in mammals.⁴³ Betaine acts as a methyl group donor for reactions catalyzed by betaine homocysteine methyltransferase (EC 2.1.1.5) to form dimethylglycine.⁴⁴ Administration of betaine leads to the normalization of homocystinuria and clinical improvement of B₁₂ deficiency symptom.⁴⁵ However, bioinformatics indicate that C. elegans has no ortholog of betaine homocysteine methyltransferase. Moreover, a high dose of betaine arrests larval development of the nematode.⁴⁶ MCM activity of the apo-enzyme was significantly increased during B₁₂ deficiency. Even in B₁₂-sufficient rats, more than 95% of MCM activity was derived from the apo-enzyme.¹⁸ Thus, holo-MCM activity was very low during B₁₂ deficiency and then significant accumulation of MMA occurs in the cells.⁴⁷ MMA is a potent inhibitor of succinate dehydrogenase (EC 1.3.99.1) that participates in both the TCA cycle and respiratory chain.⁴⁸ Thus, MMA increase due to B₁₂ deficiency blocks the mitochondrial respiration and consequently disrupts various metabolic pathways.⁴⁸

However, B₁₂ deficiency did not alter the levels of mRNAs encoding proteins that participate in worm intracellular B₁₂ metabolism, including MCM (mmcm-1), MMAA (mmaa-1), MMACHC (cblc-1), and MTRR (mtrr-1). However, the level of the mRNAs encoding MMAB (mmab-1) increased significantly.⁴¹

Our results indicate that C. elegans essentially required B₁₂ for growth. A B₁₂-deficient animal was readily produced in only 15 days using this nematode. Various phenotypes, such as infertility, delayed growth, and shorter lifespan, are observed during B₁₂ deficiency. The nematode has been used as a model animal for elucidating the biological phenomena of fertilization,⁴⁹ embryogenesis,⁵⁰ nervous system,⁵¹ and learning and memory.⁵² In addition, C. elegans may become a new tool for human B₁₂ deficiency diseases, such as fertility disorder^53,54 and nerve⁵⁵ and cognitive dysfunctions.⁵⁶

Author Contributions

The authors contributed equally to preparing the manuscript and have approved the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.