Type I and V collagens are the major components of fibrillogenic proteins in fish skin, and their hydrolysis products possess hyaluronidase inhibitory activity. In this study, for the first time, type I and V collagens were isolated from the skin of shortbill spearfish and striped marlin. Type I (2α1[I]α2[I]) and type V (α1[V]α3[V]α2[V]) collagens composed of distinct α-peptide chains with comparable structures were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometric chromatography. After enzymatic digestion, the collagen peptides were purified by using ultrafiltration (30 KDa) and high-performance liquid chromatography (RP-HPLC) to yield CPI-F3 and CPV-F4 fractions with strong hyaluronidase inhibition rates (42.17% and 30.09%, respectively). Based on the results of simulated gastrointestinal fluid, temperature, and pH stability assays, CPI-F3 and CPV-F4 exhibited stability in gastric fluid and showed no significant changes under the temperature range from 50 to 70 °C (p > 0.05). The results of this first research on the bioactivity of type V collagen peptides provide valuable information for the biomedical industry and show the potential for future bioactivity investigations of type V collagen and its peptides.

The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7–6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish.

Tetrodotoxin (TTX)-binding proteins are present in toxic TTX-bearing animals, such as pufferfish and gastropods. These may prevent autotoxicity. However, TTX-binding proteins are also found in the nontoxic marine shore crab, Hemigrapsus sanguineus. Here, we isolated the TTX-binding protein, HSTBP (Hemigrapsus sanguineus TTX-binding protein), from the hemolymph of H. sanguineus and elucidated its primary structure using cDNA cloning. HSTBP, a 400 kDa acidic glycoprotein by gel filtration high-performance liquid chromatography, comprises 3 subunits, 88 kDa (subunit-1), 65 kDa (subunit-2), and 26 kDa (subunit-3) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. The open reading frame of the cDNA comprises 5049 base pairs encoding 1683 amino acid residues, and the mature protein contains 1650 amino acid residues from Arg³⁴ to Ser¹⁶⁸³. The three subunits are arranged in tandem in the following order: subunit-3 (Arg³⁴-Gln²⁶¹), subunit-1 (Asp²⁶²-Phe¹¹³⁸), and subunit-2 (Val¹¹³⁹-Ser¹⁶⁸³). A BLAST homology search showed weak similarity of HSTBP to clotting proteins of crustaceans (29–40%). SMART analysis revealed a von Willebrand factor (vWF)-type (⇒delete hyphen) D domain at Phe¹³⁸⁷-Gly¹⁵⁴⁴. We confirmed that the recombinant protein of HSTBP subunit-2 containing the vWF-type (⇒delete hyphen) D domain bound to TTX at a molecular ratio of 1:1.