Cell-specific interactions of snail Cu metallothionein with hemocyanin: A metallomic approach

• Applicant: Professor Dr. Bernhard Lieb
• Subject Area: Animal Physiology and Biochemistry
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 320276532

Metallothioneins (MTs) constitute a family of cysteine-rich metal binding proteins with a high affinity for transition metal ions. They inactivate toxic metals and help to maintain homeostatic balance and distribution of essential trace elements within the cell. In terrestrial snails, a Cu-specific MT isoform (CuMT) is expressed in the so-called rhogocytes. These are particular cells found within Mollusca. Rhogocytes are also the expression sites of hemocyanins (Hcs), the snails` Cu-carrying respiratory proteins. The cell-specific coexpression of CuMT with a respiratory Cu-protein suggests that in these animals, CuMT may serve as a Cu donator to nascent Hc molecules. This is supported by findings that in arthropods too, Cuspecific MT isoforms can deliver Cu to arthropod Hcs. In the present proposal, we want to test this hypothesis in the two snail species Helix pomatia and Cantareus aspersus by applying a metallomics approach. To this aim, we have assembled a networking team of researchers whose combined expertise and skills lie in the fields of toxicology, animal physiology, cell biology, biochemistry and bioinorganic chemistry, genomics, bioinformatics and molecular biology. The research activities will be structured along four work packages (WP) dedicated to different aspects of metal interactions between CuMT, Cu binding proteins and ligands, and snail Hc. (1) In WP 1, we will generate genomic and transcriptomic data bases to be screened for the presence of proteins relevant to our proposal (with all MT and Hc isoforms present in these two species, and with other potential protein candidates that may be involved in Cu transfer to nascent Hc molecules). (2) In WP 2, we will perform Cu transfer experiments from snail CuMT to Hc using Cu isotope salts, by performing (i) exchange experiments in natively isolated snail CuMT and apo-Hc mixtures in vitro, and (ii) recombinant expression of CuMT and Hc in E. coli cells exposed to Cu isotope salts in vivo. (3) In WP 3 we will screen for potential protein-protein interactions between snail CuMT and Hc by applying a yeast two-hybrid approach. (4) In WP 4, we will visualize potential CuMT-Hc interactions by imaging methods, using (i) TEM and ESI / EELS to screen for Cu distribution in rhogocyte compartments, (ii) LSM for CuMT luminescence, CuMT and Hc mRNA fluorescence in situ hybridization, and (iii) TEM and Western blotting for CuMT / Hc co-localization. We expect from our studies a better understanding of how Cu may be allocated to an important Cu-containing invertebrate protein, possibly by the involvement of a CuMT. A particularly intriguing, yet admittedly speculative, prospect would be the potential discovery of a chaperonin (and thus enzyme-like) function of snail CuMT. If true, this finding would dramatically change our view on the functional significance of MTs overall.

• DFG Programme: Research Grants
• International Connection: Austria
• Co-Investigator: Professor Dr. Reinhard Dallinger