Abstract
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 μM 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.
Highlights
C. elegans metallothionein and phytochelatin synthase mutants are surprisingly resilient when challenged with a toxic load of heavy metals
The PCs form complexes with toxic metals in the cytosol of the cell which are transported into the vacuole, protecting the organism from heavy metal toxicosis[18] and the biosynthesis of PCs is autoregulated by this metal chelation.[19]
The abundance and conservation of cysteine residues within W08E12.2, W08E12.3, W08E12.4 and W08E12.5 suggests that this family may be involved in the binding of heavy metals, a notion this paper sets out to explore in more detail
Summary
C. elegans metallothionein and phytochelatin synthase mutants are surprisingly resilient when challenged with a toxic load of heavy metals. Two prominent pathways involved in this challenge are the phytochelatins (PCs)/phytochelatin synthase ( pcs-1)[8,9,10,11] and the MTs (in C. elegans nomenclature MTs are referred to as MTLs).[12,13,14,15,16] PCs are a family of metal-inducible thiol-rich peptides that are synthesised enzymatically and play a prominent role in the detoxification of heavy metals by acting as chelators.[17] The PCs form complexes with toxic metals in the cytosol of the cell which are transported into the vacuole, protecting the organism from heavy metal toxicosis[18] and the biosynthesis of PCs is autoregulated by this metal chelation.[19] Unlike PCs, expression of metallothionein is driven by transcriptional activation and the resultant proteins bind metals and act as antioxidants.[20] C. elegans possesses two metallothionein (MT) genes, mtl-1 and mtl-2, which have been shown to play an important role in the protection from metal toxicity. The abundance and conservation of cysteine residues within W08E12.2, W08E12.3, W08E12.4 and W08E12.5 suggests that this family may be involved in the binding of heavy metals, a notion this paper sets out to explore in more detail
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