Isolation of a Nav channel blocking polypeptide from Cyanea capillata medusae - a neurotoxin contained in fishing tentacle isorhizas.

Jellyfish are efficient predators which prey on crabs, fish larvae, and small fish. Their venoms consist of various toxins including neurotoxins that paralyse prey organisms immediately. One possible mode of action of neurotoxins is the blockage of voltage-gated sodium (Na(v)) channels. A novel polypeptide with Na(v) channel blocking activity was isolated from the northern Scyphozoa Cyanea capillata (L., 1758). For that purpose, a bioactivity-guided multidimensional liquid chromatographic purification method has been developed. A neurotoxic activity of resulting chromatographic fractions was demonstrated by a bioassay, which based on the mouse neuroblastoma cell line Neuro2A. The purification process yielded one fraction containing a single polypeptide with proven activity. The molecular weight of 8.22 kDa was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF MS). Utilising Laser Microdissection and Pressure Catapulting (LMPC) for the separation of different nematocyst types in combination with direct MALDI-ToF MS analysis of the intact capsules, the neurotoxin was found to be present in all types of fishing tentacle isorhizas (A-isorhizas, a-isorhizas, O-isorhizas) of C. capillata medusae.

A novel proteinaceous cytotoxin from the northern Scyphozoa Cyanea capillata (L.) with structural homology to cubozoan haemolysins.

It is well known that jellyfish are producers of complex mixtures of proteinaceous toxins for prey capture and defence. Nevertheless, studies on boreal scyphozoans concerning venom composition and toxic effects are rare. Here the isolation of a novel cytotoxic protein from the fishing tentacle venom of Cyanea capillata (L. 1758) using bioactivity-guided, multidimensional liquid chromatography is described. The crude venom was purified utilising preparative size-exclusion, ion-exchange, and reversed-phase chromatography. The cytotoxicity of resulting chromatographic fractions has been proven by a dye-uptake assay with the human hepatocyte cell line HepG2. The final purification step yielded, among other fractions, a fraction containing a single protein (named CcTX-1) with a molecular weight of its main isoform of 31.17 kDa The purification process leads to an increased cytotoxic activity per protein equivalents and the finally isolated CcTX-1 caused a nearly total loss of cell viability at a protein concentration of 1.3 µg mL⁻¹ corresponding to 0.4 µg/105 cells. De novo sequencing of CcTX-1 was conducted after enzymatic digestion and subsequent matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-ToF/ToF MS/MS). The obtained sequence data provide an approximate 85% description of the amino acid sequence. This sequence information partially matched that of two known haemolytic proteins of two cubozoan species: CaTX-1 from Carybdea alata Reynaud, 1830 and CrTX-1 from Carybdea rastonii Haacke, 1886.