A serine protease inhibitor from the anemone Radianthus macrodactylus: Isolation and physicochemical characteristics

Abstract

A serine protease inhibitor with a molecular mass of 6106 +/- 2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed alpha/beta or alpha + beta polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 x 10(-9) M) and alpha-chymotrypsin (Ki 2.17 x 10(-8) M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.

Atypical Reactive Center Kunitz-Type Inhibitor from the Sea Anemone Heteractis crispa

“…This scheme was described previously [19]. In brief, it includes a hydrophobic chromatography on Polychrome-1, gel filtration chromatography on Bio-gel P-4 and ion-exchange chromatography on SP-Sephadex G-25.…”

“…To date about 40 low molecular mass protease inhibitors (6000 Da) from sea anemones have been isolated and partially characterized [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23]. However, the complete amino acid sequences have only been determined for a few of them.…”

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor

“…Analgesic peptides, APHC1, APHC2, APHC3 [28,29], Jn-IV [14], InhVJ [16], and representatives of HCGS peptide family with P1Thr from the combinatorial library, obtained from H. crispa cDNA [11]: HCGS1.10, HCGS1.36, HCGS1.34, HCGS1.26, and HCGS2.3; and BPTI from bovine pancreas [55]. P1—amino acid residue of the inhibitor reactive center.…”

“…To date, a number of highly homologous native serine protease inhibitors has been isolated from different species: Anemonia sulcata [7,13], Heteractis crispa [14,15,16,17,18], Anthopleura aff. xanthogrammica [19,20], Anthopleura fuscoviridis [21], Stichodactyla helianthus [22,23,24], and Stichodactyla haddoni [8].…”

“…HCRG1 and HCRG2 are the first two representatives of a new Kunitz-type HCRG peptide subfamily [18]. These peptides are more potent inhibitors of trypsin and α-chymotrypsin than some known representatives of the HCGS subfamily [14,16,17] and they have exhibited an anti-inflammatory activity through inhibition of inflammatory mediators [18]. …”