Primary Structure and Conformation of a Tetrodotoxin-Binding Protein in the Hemolymph of Non-Toxic Shore Crab Hemigrapsus sanguineus

Tetrodotoxin (TTX)-binding proteins are present in toxic TTX-bearing animals, such as pufferfish and gastropods. These may prevent autotoxicity. However, TTX-binding also found the nontoxic marine shore crab, Hemigrapsus sanguineus. Here, we isolated protein, HSTBP (Hemigrapsus sanguineus protein), from hemolymph of H. elucidated its primary structure using cDNA cloning. HSTBP, a 400 kDa acidic glycoprotein by gel filtration high-performance liquid chromatography, comprises 3 subunits, 88 (subunit-1), 65 (subunit-2), 26 (subunit-3) via sodium dodecyl sulfate-polyacrylamide electrophoresis under reduced conditions. The open reading frame 5049 base pairs encoding 1683 amino acid residues, mature protein contains 1650 residues Arg34 to Ser1683. three subunits arranged tandem following order: subunit-3 (Arg34-Gln261), subunit-1 (Asp262-Phe1138), subunit-2 (Val1139-Ser1683). A BLAST homology search showed weak similarity clotting crustaceans (29–40%). SMART analysis revealed von Willebrand factor (vWF)-type (⇒delete hyphen) D domain at Phe1387-Gly1544. We confirmed that recombinant containing vWF-type bound TTX molecular ratio 1:1.