Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes)
Yafei ZhangRyoma MinamiRyohei TatsunoWei GaoM. UenoAkinori YamadaAsami YoshidaMary Grace SedanzaKazunari ArimaTomohiro TakataniKenichi YamaguchiYuji OshimaOsamu Arakawa
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Abstract:
Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.Keywords:
Takifugu rubripes
Saxitoxin
Grass carp
Tetrodotoxin
Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.
Takifugu rubripes
Saxitoxin
Grass carp
Tetrodotoxin
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Four genes of Takifugu rubripes, tentatively designated Tr1-Tr4, encoding homologs of pufferfish saxitoxin- and tetrodotoxin-binding protein, were identified by BLAST search and 3'-RACE. RT-PCR and MALDI-TOF mass spectrometry allowed the identification and discrimination of Tr isoforms from the non-toxically cultured specimens. The expression of Tr1 and Tr3 mRNAs exclusively in the liver and the presence of their products as 120-kDa plasma proteins were confirmed.
Takifugu rubripes
Saxitoxin
Tetrodotoxin
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Sepharose
Soybean agglutinin
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The receptor proteins for lectins of bovine milk fat globule membrane were probed by affinity chromatography on Concanavalin A (ConA)- and wheat germ agglutinin (WGA)-Sepharose in the presence of sodium dodecyl sulfate. Affinity chromatography on lectin column distinguished minor differences of specificity for lectin in a glycoprotein having the same mobility on electrophoresis. Of seven major glycoproteins (PAS-1 to 7), PAS-1 and -2 were bound to both Con A and WGA. Most of PAS-3 was retained on Con A. PAS-4 was retained on both WGA and Con A. A striking difference was observed between PAS-6 and -7 glycoproteins in the affinity to Con A, that is, PAS-6 was bound to Con A while PAS-7 not retained on Con A. However, most of PAS-6 and -7 was not retained on WGA, Part of PAS-5 was bound firmly while other parts failed to bind to both Con A and WGA. Our results suggest that the structure of saccharide chains of the glycoproteins of MFGM is very complicated.
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Takifugu rubripes
Tetrodotoxin
Saxitoxin
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Takifugu rubripes
Saxitoxin
Tetrodotoxin
Fugu
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Binding and Pharmacokinetics of the Sodium Channel Blocking Toxins (Saxitoxin and the Tetrodotoxins)
Tetrodotoxin (TTX) found in diverse variety of animals including puffer fishes, some newts, frogs and limited number of non-vertebrate species (6 different phyla). The saxitoxin (STX) and the TTX are small molecules composed of 7,8,9 guanidinium and 1,2,3 guanidinium groups, respectively in their structures. These groups provide positive charge to the molecules and are believed to interact with negatively charged Glu755 and Asp400 residues in domain II and I of the sodium channel strongly. The pharmacokinetic studies (absorption, distribution and accumulation) reported on Takifugu rubripes, Takifugu pardalis, Takifugu niphobles, Takifugu vermicularis, Takifugu snyderi, etc. revealed that higher concentration of TTX is accumulated in liver than in the skin or other tissues. Although TTX is also accumulated in the skin of various marine species (secretory glands) and the excess of TTX are emitted through skin which acts as a defence agent for those species. STX showed high toxicity on crab and other animals, due to its accumulation in the tissues and resistance to the sodium channel proteins. It concluded that TTX and STX based toxicities are developed on the species by the absorption, distribution and accumulation of toxins in tissues. Also the ingestion of these species (marine species) as food may allow transferring toxin to the human being.
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Saxitoxin
Tetrodotoxin
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Agglutination (biology)
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