Stichodactyla helianthus Neurotoxins

Definition
The Caribbean sea anemone Stichodactyla helianthus produces a 48-amino acid residue neurotoxin (ShI) that is employed for defence and the capture of prey. This small protein, which is much more active against crustacea than mammals, binds to the neuronal voltage-gated sodium channel, thereby slowing down channel inactivation and delaying the repolarization phase of the action potential 1.

Related Peptides
ShI resembles a group of proteins from sea anemones of the genera Anthopleura and Anemonia which act on nerve and striated muscle sodium channels from mammals as well as crustacea. However, it appears that ShI, together with a series of four homologous proteins from the anemones Heteractis macrodactylus and H. paumotensis, form a separate group distinguishable from the actiniid (i.e. Anthopleura and Anemonia) proteins on the basis of their different amino acid sequences, lack of immunological cross-reactivity, and their inability to prevent binding of the actiniid proteins to the sodium channel 1.

Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to isoforms of the same hemolysin. The spectroscopic measurements show a close similarity in the secondary structure content, conformation and stability of both toxins 2.

Discovery
The polypeptide ShI is selectively toxic to crustaceans was isolated from the aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, by Kem et al., in 1989 3.

Structural Characteristics
The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. The structure consists of a core of twisted, four-stranded, antiparallel beta- sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution 1.

Mode of Action
Expression of the two lymphocyte potassium channels, the voltage-gated channel Kv1.3 and the calcium activated channel IKCa1, changes during differentiation of human T cells. ShK, the 35-residue polypeptide toxin from Stichodactyla helianthus, blocks Kv1.3 implicated in the pathogenesis of experimental autoimmune encephalomyelitis, at low pM concentrations. ShK adopts a central helix-kink-helix fold, and alanine-scanning and other mutagenesis studies have defined its channel-binding surface. Models have been developed of how this toxin effects K+channel blockade and how its docking configuration might differ in ShK-Dap22, which contains a single side chain substitution that confers specificity for Kv1.3 blockade 4.

Functions
This ShK analog containing a fluorescein-6-carboxyl group attached through an {2-[2-amino-ethoxy]-ethoxy}-acetic acid (AEEAc) linker to the a-amino group of Arg1, selectively blocked the voltage gated T-lymphocyte K+ Kv1.3 channels implicated in the pathogenesis of experimental autoimmune encephalomyelitis. It potently blocked Kv1.3 channels at pM concentrations and exhibited >80 fold specificity for Kv1.3 over Kv1.1 and other Kv channels. In flow cytometry experiments, chronically activated rat and human T-lympocytes with >600 Kv1.3 channels/cell were readily distinguished from resting and acutely activated T-lymphocytes with lower Kv1.3 channel numbers. 6-FAM-AEEAc-Stichodactyla helianthus neurotoxin (ShK) may therefore be a useful tool to detect the presence of T-lymphocytes with high expression of Kv1.3 channels in normal and diseased tissues 5.

References
1. Fogh RH, Kem WR, Norton RS (1990). Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study. J. Biol. Chem., 265(22):13016-13028..
2. Martinez D, Campos AM, Pazos F, Alvarez C, Lanio ME, Casallanovo F, Schreier S, Salinas RK, Vergara C, Lissi E (2001). Properties of St I and St II, two isotoxins isolated from Stichodactyla helianthus: a comparison. Toxicon., 39(10):1547-1560.
3. Kem WR, Parten B, Pennington MW, Price DA, Dunn BM (1989). Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus. Biochemistry, 28(8): 3483-3489.
4. Norton RS, Pennington MW, Wulff H (2004). Potassium channel blockade by the sea anemone toxin ShK for the treatment of multiple sclerosis and other autoimmune diseases. Curr Med Che., 11(23):3041-3052.
5. Beeton C, Wulff H, Singh S, Botsko S, Crossley G, Gutman GA, Cahalan MD, Pennington M, Chandy KG (2003). A Novel Fluorescent Toxin to Detect and Investigate Kv1.3 Channel Up-regulation in Chronically Activated T Lymphocytes.. J. Biol. Chem., 278(11):928-9937.