Laetiporus sulphureus lectin (LSL) and CC1G_11805 (coprinolysin) are pore forming proteins under the aerolysin-ETX/MTX-2 superfamily. These fungal lectins are structurally homologous, when observing the pore forming domain. The receptor binding domain differs between the two and confers their target specificity. LSL is haemolytic, whereas CC1G_11805 is insecticidal. However, the pore formation mechanism remains unknown. Characterisation of these PFPs brings us a firmer understanding to the proteins function and structure. Although, pore characterisation has been trialled against coprinolysin with no success.
This study utilises a chimeric approach, where chimeric constructs of the PFPs were designed where the N-terminus RBD of both proteins were switched with each other, creating two chimeras: Chimera 1[N-terminal LSL/C-terminal coprinolysin] and Chimera 2[N-terminal coprinolysin/C-terminal LSL].
These proteins were expressed recombinantly in E. coli, purified and the activity was assessed via functional assays. Haemolytic and dye release assays were used involving red blood cells and liposomes containing carboxy fluorescein dye, respectively.
It was found that both chimeras could be expressed and purified but furthermore Chimera 1 could recapitulate haemolytic activity, killing red blood cells. Interestingly, chimera 1 was also able to perforate liposomes when modified with a sugar to bind to, resulting in release of fluorescence. This provides evidence that the pore forming domain of coprinolysin works in the same mechanism as LSL as it now has haemolytic activity, whereas previously it was only insecticidal. Moreover, evidence does suggest no proteolytic event is required for activation.
This indicates that chimeric pore forming proteins can be designed, showing indirect evidence that coprinolysin is a PFP, this indicates that function of PFD is not dependent on presence of its native RBD. Furthermore, this has a strong implication towards agribusiness, where we could potentially manipulate PFP RBDs to specifically target glycans in pests and insects.