New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

Abstract

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

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

“…Nevertheless, a slightly higher value of K i for rHCRG21 compared to those of APHC1–APHC3, can be explained by the presence of a N-terminal Arg, which noticeably increases the energy of Kunitz-type H. crispa peptides binding with proteases. This energy benefit has been shown previously by computational methods for HCRG1 and HCRG2 [18] (Table 1). …”

“…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].…”

“…Moreover, this peptide, as well as rHCGS1.19 and rHCGS1.36, possess antihistamine activity by inhibiting the increase of the concentration of calcium ions in mouse bone marrow derived macrophages [26,27]. 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].…”

“…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]. …”

Marine natural products

“…856 Absolute congurations were assigned by stereoselective synthesis of the corresponding enantiomers, with the magnitudes of optical rotation observed indicating the natural products had been isolated as scalemic (partially racemic) mixtures. Further studies of toxins from anemones has revealed two new examples of HCRG polypeptides (>6 kDa) from Heteractis crispa, 857 the toxicity of the a-pore-forming toxin equinatoxin II depends upon its ability to assemble into oligomers on the cell surface, 858 while N-terminus modied analogues of the 35-residue disulderich toxin ShK from Stichodactyla helianthus showed enhanced selectivity towards voltage-gated potassium channel Kv1.3 versus other subtypes, making them of clinical interest for the treatment of autoimmune diseases. alignment study of cnidarian toxins suggests a common origin of sodium channel and a subtype of potassium channel toxins in sea anemones and that pore-forming toxins have evolved under strong evolutionary constraints.…”