Sea anemones

Sea Anemone Pofypeptide Toxins Affecting Sodium Channels... 

Over 40 different types of polypeptide toxins have been found in marine animals (i). Many of these toxins are exquisitely selective in their actions, affecting a single process or receptor at minute concentrations. So far the sea anemone and gastropod Conus) toxins have attracted the most attention as molecular probes of ion channels. In this chapter, we discuss several approaches which are being used to investigate, at the molecular level, the interactions of the sea anemone neurotoxic polypeptides with sodium channels. 

Amino acid sequences of eleven homologous sea anemone polypeptides have been elucidated. All possess three disulfide bonds. The six half-cysteine residues always occur in the same positions (7,8). Initial studies concerning the toxin secondary and tertiary structures relied upon circular dichroism, laser Raman, and, to a lesser extent, fluorescence spectral measurements (15—18). The circular dichroism spectra of the four toxins so far examined are essentially superimpos-able and thus indicate a common secondary structure. The only peak observed, a negative ellipticity at 203 nm, largely results from a non-regular ("random")... 

Figure 1. Most cx)mmon (consensus) sequences of the two types of sea anemone toxins. Bold letters represent residues which both toxin types have in common. Letters above each sequence are nonconservative substitutions, while letters below each sequence are conservative substitutions. A nonconservative substitution was defined as one in which (a) electronic charge changed, (b) a hydrogen-bonding group was introduced or removed, (c) the molecular size of the sidechain was changed by at least 50%, or (d) the secondary structure propensity was changed drastically from b to h or vice versa (Ref.

A multivariate analytical approach has recently been applied to smaller molecules, including peptides (26,27). Recently, we have applied this approach to the sea anemone type 1 toxins. About 90% of the differences observed in... 

Figure 2. Relative toxicity (LD50 and LD qq) estimates for actiniid sea anemone toxins upon crabs (Carcimis maenas) and mice. Values for Anemonia sulcata (As) and Anthopleura xanthogrammica (Ax) toxins are from ref. 24 data for Condylactis gigantea and Phyl-lactis flosculifera toxins are unpublished (Kem). The arrows indicate that the real mouse LD q values for Cg II and Pf II must exceed the values indicated in the Ogure. Although insufficient data are presently available to quantitatively define a relationship between mammalian and crustacean toxicity, it seems that there is usually an inverse relationship, which may be approximately defined by the stipple zone.

Comparisons between these toxins allow delineation of the variability of each position in the sequence. For instance, the residues which are extremely invariant (conservative) for both types of sea anemone toxin are the half-cystines, certain glycyl residues which are expected to be involved in )9-turns, and only a few other residues - Asp 5 or 6, Arg 13 or 14, and Tryp 30 or 31 (the numbering depends upon the toxin type) — expected to be important for folding or receptor binding. Rather surprising is the variation in the residues which NMR studies (22,23) have shown are involved in formation of the four stranded )9-pleated sheet. 

Some chemical modification studies on the sea anemone toxins have unfortunately been less than rigorous in analyzing the reaction products. Consequently, results from many of these studies can only provide suggestions, rather than firm conclusions, regarding the importance of particular sidechains. Many such studies also have failed to determine if the secondary and tertiary structures of the toxin products were affected by chemical modification. 

Schweitz et al. purified four related toxins (Rpl, RpII, RpIII, and RpIV) from sea anemone Radianthus paumotensis (Rp) and studied their pharmacological properties (9). During the course of initial NMR studies, the reported sequence of RpII was found to have errors, and was redetermined (8). Subsequently Metrione et al. determined the sequence of RpIII as well (10). T e other two Rp toxin sequences are yet to be determined. Sequences of the Rp, and several other sea anemone toxins, are shown in Table I. We have used a two letter code to denote the species consistently and this notation differs from the earlier designations of Norton and Wiithrich groups. In our notation. As la and Ax I correspond to ATX la and AP-A, respectively. From alignment of the cystines in these sequences, it is clear that Rp toxins have three disulfide bonds, as do the other toxins. 

We limit the discussion to the level of secondary structures of sea anemone toxins since the tertiary structure of no other anemone toxins is known at present. Wid-mer et al. (7), on the basis of high resolution 2D-NMR results, have provided the most detailed information about the secondary structure of the A, sulcata toxin ATX la. Gooley and Norton (4-6) have partially assigned the toxins ATX I and AP-A, and compared their secondary structures. The NMR results are consistent and show that the anemone toxins all consist of a four strand antiparallel -sheet core and have no significant helical structure. 

The characteristics and possible mechanisms of action of cytolytic peptides isolated from sea anemones during the past 20 years are described. These agents fall into three categories (1)... 

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