The nematode Caenorhabditis elegans expresses two metallothioneins (MTs), CeMT‐1 and CeMT‐2, that are believed to be key players in the protection against metal toxicity. In this study, both isoforms were expressed in vitro in the presence of either Zn(II) or Cd(II). Metal binding stoichiometries and affinities were determined by ESI‐MS and NMR, respectively. Both isoforms had equal zinc binding ability, but differed in their cadmium binding behaviour, with higher affinity found for CeMT‐2. In addition, wild‐type C. elegans, single MT knockouts and a double MT knockout allele were exposed to zinc (340 μm) or cadmium (25 μm) to investigate effects in vivo. Zinc levels were significantly increased in all knockout strains, but were most pronounced in the CeMT‐1 knockout, mtl‐1 (tm1770), while cadmium accumulation was highest in the CeMT‐2 knockout, mtl‐2 (gk125) and the double knockout mtl‐1;mtl‐2  (zs1). In addition, metal speciation was assessed by X‐ray absorption fine‐structure spectroscopy. This showed that O‐donating, probably phosphate‐rich, ligands play a dominant role in maintaining the physiological concentration of zinc, independently of metallothionein status. In contrast, cadmium was shown to coordinate with thiol groups, and the cadmium speciation of the wild‐type and the CeMT‐2 knockout strain was distinctly different to the CeMT‐1 and double knockouts. Taken together, and supported by a simple model calculation, these findings show for the first time that the two MT isoforms have differential affinities towards Cd(II) and Zn(II) at a cellular level, and this is reflected at the protein level. This suggests that the two MT isoforms have distinct in vivo roles.

The genome of the nematode Caenorhabditis elegans encodes for two multifunctional metal binding metallothioneins (MTs), CeMT-1 and CeMT-2. Here we applied qPCR to identify a transcriptional up-regulation following the exposure to free radical generators (ROS) paraquat or hydrogen peroxide, a trend that was confirmed with Pmtl::GFP expressing alleles. The deletion of the MT loci resulted in an elevation of in vivo levels of hydrogen peroxide and exposure to ROS caused a reduction in total egg production, growth and life span in wild type nematodes, effects that were particularly pronounced in the CeMT-2 and double knockout. Moreover, in vitro incubation of recombinant MTs with hydrogen peroxide demonstrated the presence of direct oxidation of the CeMTs, with zinc released from both isoforms and concomitant formation of intra-molecular disulfides. Finally, metabolic profiling (metabolomic) analysis of wild type and MT knockouts in the presence/absence of oxidative stressors, confirmed the overall trend described by the other experiments, and identified 2-aminoadipate as a potential novel small-molecule marker of oxidative stress. In summary, this study highlights that C. elegans metallothioneins scavenge and protect against reactive oxygen species and potentially against oxidative stress, with CeMT-2 being more effective than CeMT-1.

In vitro evidence for the isoform-specific partitioning of cadmium and zinc ions between the two C. elegans metallothioneins is presented. This observation is discussed in terms of isoform-specific affinities towards zinc and cadmium and the implications of our study on the in vivo roles of C. elegans metallothioneins.

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