Actinoporins (AP) are pore-forming toxins produced by cnidarians that participate in prey capture, digestion and defense by forming transmembrane pores through specific binding to sphingomyelin-containing cell membranes. Due to their unique membrane interacting properties, they hold considerable potential for applications in diagnostics,
therapeutics and nanopore technologies. Actinoporin-like proteins (ALP) represent an evolutionary related group that also inludes conoporins from cone snails. In this master's
thesis, we recombinantly expressed and biophysically and biochemically characterized conoporins Eb2 and Eb3 from Conus ebraeus for the first time. Despite their pronounced
structural similarity to APs, we found that substitutions at key binding sites prevent specific recognition of sphingomyelin and abolish pore-forming activity. In the
Mediterranean mussel Mytilus galloprovincialis, in addition to the ALP mytiporin-1 (MYTP1), mytilectins are also present. These chimeric proteins contain lectin domain
and proaerolysin-like domain, enabling the recognition of specific glycans and the formation of β-barrel pores. To further elucidate their function, we investigated mytilectins MytiLec-3 and OrbiLec-1. We demonstrated that MytiLec-3 is hemolytic and specifically binds the glycan epitopres LacNAc, LacdiNAc and α-Gal, whereas OrbiLec-1 exhibits neither glycan-binding nor hemolytic activity. Using cryo-electron microscopy, we experimentally confirmed the presence of a nonameric MytiLec-3 pore in native erythrocyte membranes, which we had previously predicted using AlphaFold3. Our findings reveal the functional diversity of ALPs and mytilectins and highlight their
potential applications in diagnostics, biosensing and pharmaceutical research.
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