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

Metallothioneins (MTs) are predominantly low-molecular weight, Cysteine containing proteins with a high binding affinity for transition metal ions such as Cu+, Zn2+ und Cd2+. MTs play a pivotal role in metal metabolism of animals. Most MTs normally form mixed complexes with different metal ions. In terrestrial gastropods (land snails), however, metal-specific MT isoforms have evolved. One of them is a Cu-specific isoform (CuMT) which exclusively binds Cu+. This isoform is expressed in one single cell type, called rhogocyte. Rhogocytes are mollusk-specific cells found in the connective tissue and in the blood haemocoel. Significantly, they are also the expression sites of hemocyanin, which is the Cu- containing respiratory protein of mollusks. We suggested in our previous work that CuMT in snail rhogocytes may provide Cu+ to nascent hemocyanin molecules. This hypothesis is supported by the fact that in crustaceans too, Cu-specific MTs are apparently involved in Cu donation to hemocyanins, though the mechanisms of this transfer have never been fully elucidated. In the present proposal we aim at testing the hypothesis of Cu transfer from CuMT to hemocyanin in two resident land snail species (Helix pomatia und Cantareus aspersus) by exploring the underlying mechanisms. This will be achieved within the frame of a so-called DACH project under guidance and coordination by the project submitter (Dr. Reinhard Dallinger) in cooperation with a German partner (Dr. Bernhard Lieb) und with the collaboration of two Spanish researchers (Dr. Silvia Atrian und Dr. Mercé Capdevila). The research will be organized within four work packages, aiming at testing the central working hypothesis of Cu transfer from CuMT to hemocyanin by applying different methods. First, we want to generate genomic data bases to be screened by bioinformatic methods for proteins possibly involved in Cu transfer. Secondly, we want to perform Cu exchange experiments between CuMT and hemocyanin by using stable Cu isotopes in vitro and in vivo, with recombinantly expressed proteins in Cu- exposed E. coli cells. Thirdly, we want to test protein interplay between CuMT and hemocyanin by applying a yeast two-hybrid approach. Fourthly, we want to visualize potential interactions between CuMT and hemocyanin by applying imaging methods, including transmission electron microscopy, laser scanning fluorescence and luminescence microscopy. We expect from our research a better imagination of how the essential trace element copper might be allocated into hemocyanin within the snail rhogocytes. This is certainly an intriguing question of general importance in metal biochemistry, considering the so far fragmentary knowledge in this field. A particularly intriguing, yet admittedly speculative, prospect would be the potential discovery of a metallochaperonin function of snail CuMT. If true, this finding would significantly modify our view on the functional role of MTs overall, and of invertebrate MTs in particular.

Most gastropod species possess a Cu-containing respiratory protein called hemocyanin (hc), in which the oxygen molecule is bound by two Cu ions. In terrestrial helicid (pulmonate) snails, the hc molecule forms huge quaternary structures with a molecular mass of 9 million Da, consisting of two cylindrical decamers, each of them comprising 10 chain-shaped strings of hc subunits. We know that in many gastropods, these molecules are synthesized in the so-called rhogocytes which represent a mollusc-specific cell type that serves metal-homeostatic functions and stress resistance. In addition, these cells are also the expression sites of a Cu-specific Metallothionein isoform (CuMT), which is apparently dedicated to homeostatic Cu regulation. Hence it was assumed that CuMT may serve as a Cu(I) donor for the synthesis of the hc molecule. The task of the present project was to test this hypothesis using the helicid snail Cornu aspersum as a model organism. It appeared that this species possesses, apart from CuMT, three distinct hc isoform genes, called hc-N, hc-D und hc-. Interestingly, the structure of these genes is highly complex, exhibiting a multitude of conserved introns that probably play an important regulatory function during hc gene transcription. The Cu(I) of the native hc isolated from snail hemolymph can easily be removed from the molecule, resulting in its Cu-free apo-form. The apo-hc itself can subsequently be reconstituted by Cu(I) transfer through a Cu chelating agent. This reaction can only be perfomed in vitro, and does not work through mediation of the cell-specific CuMT. Our preliminary results indicate, however, that CuMT is capable of donate its Cu(I) ions to the single hc-subunits. This would make sense, since it suggests that the transfer of Cu(I) in snail rhogocyte cells may occur under physiological conditions through a step-by-step reaction during the synthesis of nascent hc-subunits in vivo, which would also allow a precise control of the Cu transfer process itself. Moreover, it appeared that the transcriptional expression of some hc isoform genes can be upregulated through stressful conditions, which possibly reflects an adaptation to an increasing oxygen demand upon stress exposure.