Function Of Ergothioneine Research Articles

Diet-Derived Antioxidants: The Special Case of Ergothioneine.

This article reviews what is presently known about the biological roles of the diet-derived compound ergothioneine (ET). ET seems important to humans because it is rapidly taken up from the diet by a transporter largely or completely specific for ET, and once taken up it is retained within the body for weeks or months. The various possible functions of ET in vivo are explored. Much emphasis has been placed on the antioxidant properties of ET, but although these are well established in vitro, the evidence that antioxidant activity is the principal function of ET in vivo is weak. ET is not unique in this: The evidence for the antioxidant roles of vitamin C and polyphenols such as the flavonoids in vivo is also weak. By contrast, α-tocopherol has demonstrated in vivo antioxidant effects in humans.

Antioxidant Functions of Ergothioneine

The function of ergothioneine (EGT) has been in controversy since the discovery of EGT in 1909. The present common understanding is that the functions of EGT remain unclear and that EGT does not seem to provide any advantage over other antioxidants. In this study, the ultraviolet-absorption spectra of EGT were measured at various pH. Thermodynamical parameters such as the chemical structure and bond distances of EGT were derived by BMK/6-311+G**. The molecular orbital quantum parameters such as electron distribution, frontier electron density, highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and singly occupied molecular orbital (SOMO) were derived by HF/STO-3G. The results show that EGT exists as a resonance system mixing of thione state and ionic state in solution not only at physiological pH but also in a wide acidic pH range. EGT is very stable because of the resonance system, whereas the ionic state of EGT forms the metal complexes and the EGT-disulfide in the presence of divalent metal ions. The efficient delocalization of π-electrons on the imidazole ring of EGT increases the HOMO energy level and the rate constant of EGT towards hydroxyl (OH) radical. EGT has a high antioxidant activity by donating the highest active electron on the S atom of the imidazole ring of EGT to OH radical. Because OH radical reacts faster with EGT than any other biomolecules such as proteins and DNA, EGT consequently protects the biomolecules against oxidative damage due to the OH radical.

Bioavailability and antioxidant effect of ergothioneine in human blood

Edible mushrooms, such as the white button mushrooms contain several biological compounds including ergothioneine (ET). The main function of ET seems to be to protect cells from oxidative stress. Although humans cannot synthesize ET, the human body does have its own ergothioneine transporter (ETT), which allows cells, for instance red blood cells (RBCs), to take up and store ET. The existence of this transporter suggests that ET might have a physiological importance in humans. The aims of this study were to examine the availability of ET in RBCs and the protective effect of ET on oxidative stress in RBCs. The uptake of ET in RBCs was studied in vitro by treating RBCs with known concentrations of ET (0-100 µM). The amount of ET in the RBCs was determined using high performance liquid chromatography (HPLC). The protective effect of ET was studied by measuring the haemolysis in oxidative stress induced RBCs, which were pre-treated with ET in concentrations ranging from 0-100 µM. In addition, a pilot study was performed where 10,6 mg ET was administered to subjects (n = 6) through a mushroom soup. Before and three hours after consumption, blood samples were collected. The blood samples were analyzed to determine the concentration ET in the RBCs using HPLC and the protective effect of ET was studied the same way as for in vitro. Main findings were that ET was taken up in RBCs after treatment with all ET concentrations, but only after 30 minutes and two hours. Furthermore, RBCs pre-treated with ET had less haemolysis than nontreated RBCs; pre-treatment with 100 µM ET was observed to significantly (p<0,016) decrease haemolysis in comparison with the non-treated RBCs. Consumption of the mushroom soup did not result in increased ET levels in the RBCs, although three out of five subjects had less haemolysis compared to consumption of the placebo soup. However, the beneficial effects after consumption of the mushroom soup cannot be assigned to ET for sure; other unobserved compounds could be involved

Knockout of the Ergothioneine Transporter ETT in Zebrafish Results in Increased 8‐Oxoguanine Levels

Ergothioneine (ET) is expected to provide antioxidative properties due to its double‐bonded sulfur. It is acquired by plants and vertebrates solely through dietary means. However, the precise physiological function of ET remains unclear. Case‐control studies suggest an association of polymorphisms in the human ET transporter (ETT) gene with chronic inflammatory diseases like rheumatoid arthritis [Nat Genet 35, 341‐348], Crohn's disease [Nat Genet 36, 471‐475] and diabetes [Biochem J 25, 171].
 
MethodsHere we have procured, based on retroviral insertion [Genes Dev 13:2713‐24], an ETT knock‐out zebrafish (Danio rerio). The expression profile of ETT in wild‐type zebrafish was analyzed by RT‐PCR and real‐time RT‐PCR and validated by measuring ET tissue content.ResultsStrong signals were obtained with kidney, intestine, skin, brain and eye. In whole‐fish lysates of homozygous knock‐out animals, ET content (0.06 ± 0.01 ng/g wet mass; n=3) was reduced more than 1000‐fold compared to wild‐type (138 ± 61 ng/g wet mass; n = 3). Similarly, all examined organs contained virtually no ET. Carnitine and acetylcarnitine were reduced across all examined tissues of knock‐out fish by a factor of 2.0 ± 0.2, and 1.7 ± 0.1, respectively. Incubation in sublethal concentrations of Pb2+ and Cu2+ led to an increase of the lipid peroxidation markers 4‐hydroxy‐2,3‐trans‐nonenal (HNE) and malondialdehyde (MDA) in knock‐out versus wild‐type zebrafish. Comparison of unstressed fish at the level of small molecules by LC‐MS difference shading [Proc. Natl. Acad. Sci. U.S.A. 102:5256‐61] revealed a 3.8‐fold increase of 8‐oxoguanine in the skin of ETT knockout fish.ConclusionBased on this phenotype we propose that the universal and specific purpose of ET is to eliminate singlet oxygen.

Knockout of the ergothioneine transporter ETT in zebrafish results in increased 8-oxoguanine levels

Ergothioneine (ET) is a natural compound that humans and other vertebrates must absorb from dietary sources. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (human gene symbol SLC22A4) is a highly specific transporter for the uptake of ET. Here, we sought to identify and knock out ETT from zebrafish (Danio rerio) to determine the function of ET. We cloned and assayed three related proteins from zebrafish, only one of which catalyzed the uptake of ET. RT-PCR analysis revealed that the protein is strongly expressed in the skin, brain, kidney, intestine, and eye. In ETT-knockout animals generated by retroviral insertion into exon 1, ET content was reduced by more than 1000-fold compared to the wild type. Thus, ETT is the sole transporter responsible for uptake of ET into zebrafish. ETT-knockout fish did not exhibit obvious differences in morphology or behavior. In whole-fish homogenates, an increase in 4-hydroxy-2,3-trans-nonenal and malondialdehyde was observed, but only after stress caused by incubation with Pb2+ or Cu2+. Comparison of unstressed fish at the level of small molecules by LC–MS difference shading revealed a 3.8-fold increase in 8-oxoguanine (8-oxo-7,8-dihydroguanine) in the skin of ETT-knockout animals. Our knockout represents a new model for examining the consequences of complete absence of ET. Based on the phenotype observed here, we hypothesize that the specific purpose of ET could be to eliminate singlet oxygen.

Ergothioneine protects Streptomyces coelicolor A3(2) from oxidative stresses

Thiol compounds with low-molecular weight, such as glutathione, mycothiol (MSH), bacillithiol, and ergothioneine (ERG), are known to protect microorganisms from oxidative stresses. Mycobacteria and actinobacteria utilize both MSH and ERG. The biological functions of MSH in mycobacteria have been extensively studied by genetic and biochemical studies, which have suggested it has critical roles for detoxification in cells. In contrast, the biological functions of ERG remain ambiguous because its biosynthetic genes were only recently identified in Mycobacterium avium. In this study, we constructed mutants of Streptomyces coelicolor A3(2), in which either the MSH or ERG biosynthetic gene was disrupted, and examined their phenotypes. A mshC (SCO1663)-disruptant completely lost MSH productivity. In contrast, a disruptant of the egtA gene (SCO0910) encoding γ-glutamyl-cysteine synthetase unexpectedly retained reduced productivity of ERG, probably because of the use of l-cysteine instead of γ-glutamyl-cysteine. Both disruptants showed delayed growth at the late logarithmic phase and were more susceptible to hydrogen peroxide and cumene hydroperoxide than the parental strain. Interestingly, the ERG-disruptant, which still kept reduced ERG productivity, was more susceptible. Furthermore, the ERG-disruptant accumulated 5-fold more MSH than the parental strain. In contrast, the amount of ERG was almost the same between the MSH-disruptant and the parental strain. Taken together, our results suggest that ERG is more important than MSH in S.coelicolor A3(2).

Mechanism of protection by natural antioxidant ergothioneine against copper-induced oxidative damage to DNA and protein

Ergothioneine is a naturally occurring amino acid found in up to millimolar concentrations in several biological tissues and fluids. The biological functions of ergothioneine are not completely understood. In this study, the role of ergothioneine in copper-induced oxidative damage to DNA and proteins was investigated using the following two copper-containing systems: Cu(II) with ascorbate and Cu(II) with H2O2. Strand breakage and protein carbonyl formation were used as indicators of oxidative damage to DNA and bovine serum albumin, respectively. Ergothioneine provided strong, dose-dependent protection against oxidation of DNA and proteins in both copper-containing systems. By contrast, only limited protection was observed with the hydroxyl radical scavengers dimethyl sulfoxide and mannitol, even with the scavengers at concentrations as high as 100 mmol/L. UV-visible spectroscopy and low-temperature electron spin resonance spectroscopy showed ergothioneine significantly inhibited copper-catalyzed oxidation of ascorbate and competed effectively with histidine and 1,10-phenanthroline for binding of Cu(I) but not Cu(II). In conclusion, ergothioneine is a potent, natural, sulfur-containing antioxidant that prevents copper-dependent oxidative damage to biological macromolecules by forming a redox-inactive ergothioneine-copper complex.

Discovery of the ergothioneine transporter.

Variants of the SLC22A4 gene are associated with susceptibility to rheumatoid arthritis and Crohn's disease. SLC22A4 codes for an integral membrane protein, OCTN1, that has been presumed to carry organic cations like tetraethylammonium across the plasma membrane. Here, we show that the key substrate of this transporter is in fact ergothioneine (ET). Human OCTN1 was expressed in 293 cells. A substrate lead, stachydrine (alias proline betaine), was identified by liquid chromatography MS difference shading, a new substrate search strategy. Analysis of transport efficiency of stachydrine-related solutes, affinity, and Na+ dependence indicates that the physiological substrate is ET. Efficiency of transport of ET was as high as 195 microl per min per mg of protein. By contrast, the carnitine transporter OCTN2 from rat did not transport ET at all. Because ET is transported >100 times more efficiently than tetraethylammonium and carnitine, we propose the functional name ETT (ET transporter) instead of OCTN1. ET, all of which is absorbed from food, is an intracellular antioxidant with metal ion affinity. Its particular purpose is unresolved. Cells with expression of ETT accumulate ET to high levels and avidly retain it. By contrast, cells lacking ETT do not accumulate ET, because their plasma membrane is virtually impermeable for this compound. The real-time PCR expression profile of human ETT, with strong expression in CD71+ cells, is consistent with a pivotal function of ET in erythrocytes. Moreover, prominent expression of ETT in monocytes and SLC22A4 polymorphism associations suggest a protective role of ET in chronic inflammatory disorders.