Molecular Cloning and Characterization of a Hemolytic Lectin from Venom of the Stinging Sea Nettle, Chrysaora quinquecirrha

I have identified a novel pore-forming toxin (PFT) in the venom of the Sea Nettle (Chrysaora quinquicherra), a protein I have named Chrysaoralin. This protein is discharged from specialized organelles called cnidocysts (nematocysts) found primarily in the tentacles of this jellyfish. Chrysaoralin was first identified by Nextgen sequencing (RNA-Seq) of libraries made from mRNA isolated from tentacles of mature medusa collected from Barnegat Bay, NJ. The full-length of the Chrysaoralin gene is 1365 bp, encoding a protein of 454 AA (50.695 kD; pI = 6.58). The SignalP 4.1 algorithm (http://www.cbs.dtu.dk/services/SignalP/) predicts a signal peptide of 22 AA. The mature protein (minus the putative signal peptide) is 432 AA (48.321 kD; pI = 6.58). This protein shows strong homology (66%) to a hemolytic lectin from the sea cucumber, Cucumaria echinata (Phylum Echinodermata). In support of this fact hemolytic activity was detected in the purified nematocyst preparations, which demonstrates sensitivity to both boiling and Proteinase K digestion, suggesting this activity is proteinaceous. The RNASeq data was verified by generating PCR amplicons using 9 sets of primers that span the full gene. Genomic sequences from both Barnegat Bay and Chesapeake Bay Chrysaoralin were intron-less. I also modified and subcloned the full-length Chrysaoralin gene into a pET SUMO expression vector and transformed into E. coli. Future expression of this recombinant protein in E. coli may further the understanding of the physiological role of Chrysaoralin in human envenomations