Definition of a saxitoxin (STX) binding code enables discovery and characterization of the anuran saxiphilin family

Abstract

American bullfrog ( Rana castesbeiana ) saxiphilin ( Rc Sxph) is a high-affinity “toxin sponge” protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (Na V s). How specific Rc Sxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX “recognition code” enabled engineering of Rc Sxph to improve its ability to rescue Na V s from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

Binding and sequestration of poison frog alkaloids by a plasma globulin

“…Taken together, this supports a model where Os ABG is produced in the liver, binds alkaloids present in the blood or recently absorbed by the intestines, and then may transport alkaloids to the skin for bioaccumulation. The micromolar K D value we identified for Os ABG with PTX is higher than the previously reported value for CBG [34,36], and TBG [54], as well as that reported for the “toxin sponge” protein saxiphilin [55], which are all in the nanomolar range. The lower affinity of Os ABG provides further support to the hypothesis that Os ABG may be acting as a transporter protein to other tissues, and would be in line with the hypothesis that there may be other mechanisms involved in autoresistance to circulating alkaloids not bound by protein [56].…”

Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry

“…Some frogs resist STX poisoning 7 , 8 , 9 , 10 , a property attributed to high-affinity STX binding proteins called saxiphilins (Sxphs). 1 , 9 , 11 Sxph-like activity is found in arthropods, 12 amphibians, 12 , 13 , 14 fish, 12 and reptiles. 12 Except for frog and toad Sxphs 1 and a pufferfish protein, Pufferfish Saxitoxin and Tetrodotoxin Binding Protein, PSTBP, that binds STX and related toxin, tetrodotoxin (TTX), 15 the molecular origins of these STX binding activities are uncharacterized.…”

“… 1 , 9 , 11 Sxph-like activity is found in arthropods, 12 amphibians, 12 , 13 , 14 fish, 12 and reptiles. 12 Except for frog and toad Sxphs 1 and a pufferfish protein, Pufferfish Saxitoxin and Tetrodotoxin Binding Protein, PSTBP, that binds STX and related toxin, tetrodotoxin (TTX), 15 the molecular origins of these STX binding activities are uncharacterized. Our recent structural studies have shown that frog and toad Sxphs have a single, high affinity (Kd∼ 1 nM) STX binding site that uses an STX binding code that shares both structural and energetic binding principles with the Na V STX binding site.…”

“…Our recent structural studies have shown that frog and toad Sxphs have a single, high affinity (Kd∼ 1 nM) STX binding site that uses an STX binding code that shares both structural and energetic binding principles with the Na V STX binding site. 1 , 11 Because Sxphs are soluble proteins that are readily purified and characterized using a host of biophysical methods, this protein family provides an advantageous platform to investigate fundamental principles underlying STX recognition and a scaffold for developing STX binding assays that can be generalized to other toxin sponge proteins.…”

Protein‐Loaded Cellular Nanosponges for Dual‐Biomimicry Neurotoxin Countermeasure

“…[18,19] Interestingly, American bullfrogs (Rana castesbeiana) are resistant to STX poisoning by producing a 91-kDa protein saxiphilin (Sxph), which binds to STX with high affinity. [20,21] Sxph is present in the bullfrogs' plasma and heart, acting as a molecular "sponge" to bind and sequester STX in the event of toxin exposure. Similar toxin resistance mechanisms have been observed in several marine animals, such as pufferfish, cockles, and crabs.…”