Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis

Abstract

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 ± 0.97 nmol·mg−1 protein and a Kd of 14.6 ± 0.33 nm for [3H]saxitoxin in equilibrium binding assays. [3H]Saxitoxin (10 nm) binding to PSTBPs was half‐inhibited by the presence of tetrodotoxin and saxitoxin at 12 µm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino‐acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino‐acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N‐terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.

Transfer profile of intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes

“…TTX/PST-binding proteins have been isolated from the blood plasma of marine pufferfish (Matsui et al, 2000;Yotsu-Yamashita et al, 2001), and may be involved in the transportation mechanism.…”

RT-PCR- and MALDI-TOF Mass Spectrometry-Based Identification and Discrimination of Isoforms Homologous to Pufferfish Saxitoxin- and Tetrodotoxin-Binding Protein in the Plasma of Non-Toxic Cultured Pufferfish (Takifugu rubripes)

“…4) Two cDNAs encoding distinct PSTBP isoforms (PSTBP1 and PSTBP2, with 93% amino acid sequence identity) were cloned and sequenced. 4) Although it has been suggested that PSTBPs appear as non-covalently linked dimers of a single subunit, 4) it is unknown whether PSTBPs form homodimers or heterodimers with both isoforms or either one (PSTBP1 or PSTBP2). Western blot analysis using IgG antibody against recombinant PSTBP1 suggested that a PSTBP homolog of 124 kDa (as estimated by SDS-PAGE) is present in the plasma of wild T. rubripes.…”

Developmental Biology of Neoplastic Growth

“…The pufferfish Fugu pardalis possesses yet another STX binding protein but unlike saxiphilin it can also bind TTX and its sequence was unrelated to the Na channel or bullfrog saxiphilin (Yotsu-Yamashita et al 2001;Yotsu-Yamashita et al 2002). Similar TTX binding proteins have been observed in horseshoe crabs (Ho et al 1994) and malacostracan crustacea (Nagashima et al 2002).…”

“…Temperature may affect the potency of TTX and STX circulating in the blood of certain ectothermic animals which come into contact with these guanidinium toxins. Besides prey, predators which ingest toxic prey may be more sensitive at lower temperatures (Yotsu-Yamashita et al 2001). …”