Localization of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) in the tissues of the pufferfish, Takifugu pardalis, analyzed by immunohistochemical staining.

Pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) was previously isolated from the plasma of the marine pufferfish, Takifugu pardalis. In this study, we investigated distribution pattern of PSTBP in intestine, liver, ovary, skin, and skeletal muscle of T. pardalis by immunohistochemical staining for the study of functions of this protein. In the skin, dermis around the tetrodotoxin secreting gland was positive, while this secreting gland itself was negative. In the ovary containing vitellogenic oocytes, ovarian wall and vitelline envelope were positive, while yolk and nucleus were negative. In the liver, hepatocytes with large fat droplets and capillaries were positive. In the intestine, the lamina propria mucosae were positive, while the mucosal epithelium was negative. In the skeletal muscle, only capillaries were positive. Furthermore, liver specific expression of PSTBP was confirmed by Northern blot analysis. Based on these results together with reported tetrodotoxin localization pattern in pufferfish, PSTBP was assumed to be a carrier protein to transfer tetrodotoxin among the tissues, especially liver, ovary, and skin.

Distribution of homologous proteins to puffer fish saxitoxin and tetrodotoxin binding protein in the plasma of puffer fish and among the tissues of Fugu pardalis examined by Western blot analysis.

Puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP) is a glycoprotein (200 kDa as a dimer) that we previously isolated from the plasma of Fugu pardalis (Yotsu-Yamashita et al., 2001). For the study on functions of PSTBP, here we examined distribution of homologous proteins to PSTBP in the plasma of seven species of puffer fish, and among the tissues of F. pardalis by Western blot analysis probed with a polyclonal IgG against unglycosylated PSTBP1 expressed in Echelichia coli. One or two major positive broad bands were detected at 105-140 kDa molecular weight range in the plasma (0.5 microg protein) of all species of puffer fish tested, while no band was detected in the plasma (5 microg protein) of fish other than puffer fish. Glycopeptidase F treated plasma of all species of puffer fish tested commonly showed the bands at approximately 42 kDa that was consistent to the molecular weight of unglycosylated PSTBP. These data suggest that puffer fish commonly possess glycoproteins homologous to PSTBP, but the sizes of N-glycan are specific to the species. Among soluble protein extracts (5 microg protein) from the tissues of F. pardalis, PSTBP was detected in all tissues examined, most prominently in heart, skin, and gall.