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Different combinations of insect Na,K-ATPase α- and β-subunits enable fine-tuned adaptation to host plant toxins and tissue specific needs

Citation

Winter, Marlena et al. (2021), Different combinations of insect Na,K-ATPase α- and β-subunits enable fine-tuned adaptation to host plant toxins and tissue specific needs, Dryad, Dataset, https://doi.org/10.5061/dryad.j0zpc86dg

Abstract

Background

Cardiac glycosides are known to fatally inhibit the Na,K-ATPase throughout the animal kingdom. Several animals, however, evolved target-site insensitivity by substitutions in the otherwise highly conserved cardiac glycoside binding pocket located on the Na,K-ATPase α-subunit. The minimal functional enzyme consist of an α- and a β-subunit, the latter considered mainly as a chaperone responsible for correct folding and membrane integration. We here analyze resistance to cardiac glycosides and kinetic properties of different Na,K-ATPase α/β-combinations of the large milkweed bug, Oncopeltus fasciatus. These insects have adapted to high concentrations of cardiac glycosides in their food plants via several rounds of Na,K-ATPase gene duplications followed by differential resistance conferring substitutions and subfunctionalization of the enzymes.

Results

To investigate their characteristics we expressed nine combinations of O. fasciatus Na,K-ATPase α/β-sunbunits (three each) in Sf9 cells and tested them with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a commonly used toxin. Differences in the number and identity of amino acid substitutions in the cardiac glycoside binding site resulted in large differences in activity and toxin resistance of the three α-subunits. The enzymes’ kinetics were also influenced by the β-subunits leading to increased activities (αCβ3) or altered resistances. The host plant toxin calotropin proved to be a much more potent inhibitor than ouabain for the phylogenetically oldest αC based enzymes. This effect was compensated for in the αB and αA based enzymes with αAβ1 having higher resistance against calotropin than against ouabain.

Conclusion

The originally higher inhibitory potency of the host compound calotropin supports a coevolutionary escalation of plant defenses and herbivore tolerance mechanisms. For the bugs the possession of multiple paralogs improved adaptation to plant toxins in a stepwise manner and mitigates pleiotropic effects by a compromise between ion pumping activity and resistance.

Methods

Nine different Na,K-ATPase α/β-subunit combinations of O. fasciatus were expressed heterologously in Sf9 cells using the baculovirus expression system (Dalla & Dobler 2016, Evolution 70:2767–77). Enzyme activities were determined by photometric measurements of inorganic phosphate released from enzymatic ATP hydrolysis while subtracting background ATPase activity following previously described methods (Dalla et al. 2017, Insect Biochem Mol Biol 89:43–50; Petschenka et al. 2013, Evolution 67:2753–61). Based on previous results (Dalla et al. 2017) the buffer regime used was 50 mM NaCl, 4 mM MgCl2, 50 mM imidazol, 20 mM KCl, pH 7.4. The inhibition of the different Na,K-ATPases was tested under increasing ouabain and calotropin concentrations and expressed as percentage activity of a non-inhibited control. For each α/β-subunit construct technical duplicates of three biological replicates of recombinant proteins isolated from independently transfected Sf9 cells were tested.

Funding

Deutsche Forschungsgemeinschaft, Award: Do527/5-3 & Do527/10-1

John Templeton Foundation, Award: ID #41855

Nachwuchsförderung Universität Hamburg, Award: PhD Stipend to Marlena Winter

Nachwuchsförderung Universität Hamburg, Award: PhD Stipend to Marlena Winter