[
Metallomics,
2018]
Four highly similar genes (W08E12.2, W08E12.3, W08E12.4 and W08E12.5) which are consecutively aligned on chromosome IV of the C. elegans genome are predicted to code for small (120-141aa) yet cysteine rich (18-19 cysteines) proteins. Cloning and sequencing of the genomic regions of the isoforms confirmed the presence and order of all genes. The generation of transgenic worms strains with an integrated single copy or extrachromosomal multi-copy PW08E12.3;W08E12.4::GFP uncovered that W08E12.3 and W08E12.4 are constitutively expressed in the pharynx and significantly induced in worms exposed to 100 M Zn. Knockdown by RNAi did not have a marked consequence on reproductive performance nor was a Zn-dependent effect on nematode growth observed. However, RNAi of these genes led to an accumulation of Zn in the intestinal cells. W08E12.3 was recombinantly expressed in E. coli and the purified protein was shown to be able to bind up to 6.5 Zn molecules at neutral pH. Zn-binding was acid-labile and the apo protein was observed at pH < 4.3. This characterization suggests W08E12.2, W08E12.3, W08E12.4 and W08E12.5 belong to a family of putative Metalloproteins which, akin to metallothioneins, may play an important role in Zn-sensing, homeostasis and/or detoxification.
[
International Worm Meeting,
2013]
Microarray experiments performed with metallothionein (MT) double knockouts revealed that the transcription of a hitherto uncharacterised family of genes is transcriptionally activated upon metal exposure in an MT null background. The four isomers (W08E12.2, W08E12.3, W08E12.4 and W08E12.5) are consecutively aligned on chromosome IV and encode cysteine rich (13%) proteins.W08E12.2 - W08E12.5 are ³ 90% similar in their coding region and the promoters of W08E12.3 and W08E12.4 display an identity of 100%. Cloning and sequencing of the genomic region of the isomers have confirmed the presence and order of W08E12.2, W08E12.3 and W08E12.5 within the worm genome, however the presence of W08E12.4 still needs to be confirmed. Bioinformatic screening of the isomers predicted the presence of putative metal binding sites within the respective promoter regions. Transgenic-GFP tagged worms revealed the constitutive expression of PW08E12.3::GFP in the pharyngeal region which is induced by metals in the following order: Cd>Zn>Cu. The coding regions were subcloned into an expression vector (pTXB) to allow the in-frame expression of the proteins fused to an affinity tag (intein-CBD domain) thus enabling a single-step purification. SDS-PAGE and Western Blot analysis have confirmed the successful expression of the proteins and subsequent characterisation will provide insight into the structure, function and metal binding characteristics. Taken together, we are addressing the role of an uncharacterised family of genes in metal homeostasis or xenobiotic detoxification which in turn will add to ongoing efforts to develop enhanced biomarkers for metal toxicosis.