Phylogenetic survey of soluble saxitoxin-binding activity in pursuit of the function and molecular evolution of saxiphilin, a relative of transferrin " # LYNDON E. LLEWELLYN , PETER M. BELL # $ EDWARD G. MOCZYDLOWSKI *, " Australian Institute of Marine Science, PMB 3, ToWnsille MC, Queensland 4810, Australia # * Department of Pharmacolog, Yale Uniersit School of Medicine, 333 Cedar Street, NeW Haen, CT 06520–8066, USA (EdwardjMoczydlowski!Yale.edu) $ Department of Cellular and Molecular Phsiolog, Yale Uniersit School of Medicine, NeW Haen, CT 06520, USA SUMMARY Saxiphilin is a soluble protein of unknown function which binds the neurotoxin, saxitoxin (STX), with high affinity. Molecular characterization of saxiphilin from the North American bullfrog, Rana catesbeiana, has previously shown that it is a member of the transferrin family. In this study we surveyed various animal species to investigate the phylogenetic distribution of saxiphilin, as detected by the presence of $ soluble [ H]STX binding activity in plasma, haemolymph or tissue extracts. We found that saxiphilin activity is readily detectable in a wide variety of arthropods, fish, amphibians, and reptiles. The $ pharmacological characteristics of [ H]STX binding activity in phylogenetically diverse species indicates that a protein homologous to bullfrog saxiphilin is likely to be constitutively expressed in many ectothermic animals. The results suggest that the saxiphilin gene is evolutionarily as old as an ancestral $ gene encoding bilobed transferrin, an Fe +-binding and transport protein which has been identified in several arthropods and all the vertebrates which have been studied. site on Na+ channels has been localized to residues 1. INTRODUCTION within a conserved sequence motif in four homologous The neurotoxin, saxitoxin (STX), and a large array of domains of the α-subunit, which forms part of the ion- STX derivatives are produced by certain species of selective pore (Terlau et al. 1991). dinoflagellates in the marine environment and cyano- While the molecular pharmacology of STX with bacteria in the freshwater ecosystem (Carmichael et al. respect to Na+ channels is well characterized, our 1990; Hall et al. 1990; Schantz 1986; Shimizu 1996). group has been attempting to uncover the biological Consumption of toxic phytoplankton by filter-feeding significance of a different high-affinity binding site for organisms results in the accumulation and dispersal of STX, which is located on a soluble protein named STX through the food chain to animals which have saxiphilin. This site was first recognized due to the $ been reported to include an ascidian, annelids, mol- finding of high-affinity binding activity for [ H]STX in luscs, crabs, fish, and ultimately mammals (Anderson tissue extracts and plasma of frogs and toads (Doyle et & White 1992; Geraci et al. 1989; Gessner et al. 1996; al. 1982; Mahar et al. 1991). A component exhibiting $ Llewellyn & Endean 1989; Nagashima et al. 1984; soluble [ H]STX binding activity was purified from Yasumoto et al. 1986). The human intoxication plasma of the bullfrog, Rana catesbeiana, and found to syndrome of paralysis and death resulting from correspond to a 91 kDa protein related to the trans- $ unwitting consumption of STX-contaminated shellfish ferrin family of Fe +-binding proteins (Li & Moczyd- is commonly known as ‘paralytic shellfish poisoning’ lowski 1991). Structural similarity of saxiphilin to (PSP). The neurotoxicity of STX is due to potent transferrin is indicated by a high level of sequence blockade of voltage-sensitive Na+ channels that me- similarity; e.g. 51% identity to Xenopus laeis trans- diate nerve and muscle action potentials (Ritchie & ferrin and 44% identity to human serum transferrin Rogart 1977). STX exerts half-maximal blockage of (Morabito & Moczydlowski 1994). Serum transferrin Na+ current at a concentration of 2–100 nM of STX, and lactoferrin have a bilobed structure owing to the depending on the particular Na+ channel isoform (Guo presence of two internally homologous domains of et al. 1987). At the molecular level, the STX binding ca. 340 residues, the N-lobe and the C-lobe, that each $+ −#! contain a high affinity site for Fe (KD ca.10 M) − and the synergistic anion cofactor, HCO$ (Baker & * Author and address for correspondence. Lindley 1992). Bullfrog saxiphilin has the same internal Proc. R. Soc. Lond. B (1997) 264, 891–902 891 " 1997 The Royal Society Printed in Great Britain 892 L. E. Llewellyn and others Phlogenetic sure of saxiphilin-like actiit duplication as the transferrins, but has substitutions in toxication from microbial sources, it ought to be $ nine out of ten highly conserved Fe +-site residues, commonly expressed by animal species inhabiting $ thereby accounting for its lack of demonstrable Fe +- ecosystems known to harbour STX-producing phyto- binding activity (Li et al. 1993; Morabito & Moczyd- plankton. Unexpectedly, we found putative saxiphilin- lowski 1994). The single high-affinity binding site for like activity in terrestrial and aquatic arthropods and STX (KD ca. 0.2 nM) in bullfrog saxiphilin has been in a wide variety of ectothermic vertebrates, including localized to the C-lobe, as determined by assay of a fish and amphibians from the aquatic environment as recombinant form of the protein in which the N-lobe well as reptiles indigenous to semi-arid locales. The $ has been deleted (Morabito et al. 1995). [ H]STX data imply that saxiphilin has an ancient origin in binding to saxiphilin is also inhibited at low pH in a animal evolution, and that it may function in a manner reminiscent of the pH-dependent release of process(es) of broadly-based biological significance. $ Fe + by transferrin (Llewellyn & Moczydlowski 1994), a process which is important in the delivery of iron to 2. METHODS eukaryotic cells by transferrin receptor-mediated endo- cytosis (Thorstensen & Romslo 1990). (a) Materials $ Aside from the recognized role of serum transferrin [ H]STX was purchased from Amersham International, in iron transport, transferrin and lactoferrin are also purified, and standardized according to Moczydlowski et al. $+ $ responsible for maintaining low levels of free Fe in (1988). Several different lots of [ H]STX used in this $+ biological fluids, which inhibits the growth of Fe - study had specific activities in the range of 20200– " requiring microorganisms and protects against the 35100 cpm pmol− . STX was purchased from Calbiochem # $ potential toxicity of Fe +} Fe + in the generation of (La Jolla, CA). The following STX derivatives were hydroxyl free radicals (Crichton 1991). By analogy to generously provided by Dr Sherwood Hall (US Food and this latter chemical defence function, it may be Drug Administration): decarbamoylsaxitoxin (dcSTX), hypothesized that saxiphilin functions as a defence neosaxitoxin (neoSTX), B1 and C1 (see figure 2a for structures). Stock solutions of these toxins were diluted in mechanism against STX intoxication, by sequestering 1 mM citrate buffer, pH 5.0. The common buffers, Mops, any STX that an animal might acquire from microbial Mes, Hepes, and Tris, and the anaesthetics, tricaine sources. Although comparatively little is known about methanesulphonate and sodium brevital, came from Sigma the chemical ecology of STX in the freshwater (St Louis, MO). The cation exchange resin, AG50W-X2, H+ environment, an argument for this hypothesis can be form, 100–200 mesh, was obtained from Bio-Rad (Richmond, drawn from previous observations of tadpole mortality CA). Other chemicals were reagent grade, and came from associated with STX production by the cyanobacterial commercial sources. species, Aphaniomenon flos-aquae (Ikawa et al. 1982), and the occurrence of saxiphilin in Ranid tadpoles and (b) Sources of animals and sample preparation frogs (Mahar et al. 1991). Another question concerns the molecular evolution Some species used in this study were purchased from of saxiphilin. Transferrins have thus far been identified Connecticut Valley Biological (Southampton, MA), Caro- in all classes of vertebrates, several insect species and an lina Biological (Burlington, NC), and Charles Sullivan Co. (Nashville, TN). Various animals were collected in the ascidian (Bartfeld & Law 1990; Jamroz et al. 1993; vicinity of Mount Desert Island Biological Laboratory Kurama et al. 1995; Martin et al. 1984; Welch 1990). (Salisbury Cove, ME) and the Australian Institute of Marine Sequence data imply that the gene duplication Science (Townsville, ueensland, Australia). Numerous underlying the bilobed structure of modern transferrin professional colleagues, listed in the acknowledgements, occurred before the emergence of insects (Bowman et generously donated plasma and tissue samples from animals al. 1988; Bartfeld & Law 1990). Since saxiphilin used in their own studies. Plasma samples from several species contains this same internal duplication, a transferrin of sharks, dolphins and whales were obtained from the New gene may have been the direct ancestral precursor of England Aquarium (Boston, MA) through the assistance of the saxiphilin gene. Evidence has recently emerged Dr Don Anderson at the Woods Hole Oceanographic $ that saxiphilin is not the only example of a non-Fe +- Institute (Woods Hole, MA). Lyophilized plasma from terrestrial mammals, birds, and the Thailand cobra (N. n. binding member of the transferrin family. Fierke and kaouthia), liver extract of the African lungfish (P. aethiopicus) her co-workers have identified a protein inhibitor of and horseshoe crab (L. polphemus) haemolymph were