Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

This article focuses on the enrichment and glyco-proteomic characterization of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes). Using ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography, the study efficiently isolated TTX-binding proteins, primarily identifying a 125-kDa isoform X1 protein—characterized as a novel lipocalin family member with three tandemly repeated domains—and 88- and 79-kDa isoform X2 variants with two domains. Five N-glycosylation sites were empirically determined in the X2 isoform, and the complexity of PSTBP (pufferfish saxitoxin and tetrodotoxin-binding protein) proteoforms arising from a single gene via alternative splicing and posttranslational modifications was elucidated. The findings provide a foundation for further research into the functional diversity of these proteins in toxin binding, accumulation, and resistance in pufferfish.