Cloning and Functional Analysis of Saxiphilin, A Saxitoxin-Binding Protein fromthe Bullfrog

摘要

Saxitoxin (STX), a potent neurotoxin, is the causative agent of paralyticshellfish poisoning in humans. The North American bullfrog, Rana catesbeiana, contains a plasma protein called saxiphilin that binds STX with high affinity and specificity. Since saxiphilin may be useful as a detection reagent or an antidote for STX, we have investigated the biochemical properties of saxiphilin and cloned cDNA encoding this protein. Native saxiphilin is a polypeptide of 825 amino acid residues (Mr = 90,901) that contains one binding site for (3H)STX per molecule with an equilibrium dissociation constant of KD = 0.2 nM. The amino acid sequence of saxiphilin, deduced from cDNA isolated from bullfrog liver, exhibits substantial homology to members of the transferrin family of Fe+-binding proteins. However, biochemical and immunochemical analyses confirm that saxiphilin is a unique protein that is distinct from bullfrog serum transferrin. The mechanism of (3H)STX binding to saxiphilin, including the pH-dependence and temperature-dependence, was characterized in detail. Recombinant saxiphilin was expressed in insect cells using a baculovirus vector and the STX-binding site was localized to the C-lobe domain of the protein. A phylogenetic survey found saxiphilin-like activity in arthropods, fish, amphibians and reptiles.