A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 22A c c e p t e d M a n u s c r i p t Abstract Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K V 's), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus leavis oocytes: 13 cloned voltage-gated potassium channels (K V 1.1-K V 1.6, K V 1.1 triple mutant, K V 2.1, K V 3.1, K V 4.2, K V 4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotidesensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na V 1.2-Na V 1.8 and the insect channel DmNa V 1). Our data shows that APEKTx1 selectively blocks K V 1.1 channels in a very potent manner with an IC 50 value of 0.9 nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124 nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K V 1.1 channels with that of a competitive inhibitor of trypsin.Keywords Anthopleura elegantissima · K V channel inhibitor · sea anemone toxin · protease inhibitor ·

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

“…1). Hg1 and BmKTT-3 belong to the first group, which adopted classical disulfide pairings similar to HWTX-XI toxin from spider (20), dendrotoxin K from snake (32), and APEKTx1 from sea anemone (23). LmKTT-1a, LmKTT-1b, LmKTT-1c, and BmKTT-1 belong to the second group, which adopted a unique cystine framework that we described in our previous work (26).…”

“…For example, Kunitz-type toxin bungaruskunin, isolated from snake venom, is a serine protease inhibitor (22), but ␣-dentrotoxin, ␦-dentrotoxin, dentrotoxin K, and dentrotoxin I, also from snake venom, are potent Kv1.1 channel inhibitors (21). Kunitz-type toxins HWTX-XI from spider and APEKTx1, AKC1, AKC2, and AKC3 from sea anemone are bifunctional toxin peptides with both protease and potassium channel-inhibiting properties (20,23,24). Conkunitzin-S1, a 60-residue cone snail Kunitz-type toxin cross-linked by two disulfide bridges, interacts with the shaker potassium channel (19,25).…”

Mechanisms of Immune Responses in Cnidarians

“…For example, the kazal-type protease inhibitor isolated in Hydra shows strong in vitro bactericidal activity against Staphylococcus aureus . Other protease inhibitors, the kunitz-type protease inhibitor and the alpha-2-macroglobulin, have been identified in anemones and are thought to play a role in immunity by inactivating virulence factors from bacteria (Fujito et al, 2010;Kimura et al, 2009;Peigneur et al, 2011). AMPs have been identified in Hydra (Bosch, 2013) and in the coral P. damicornis (Vidal-Dupiol et al, 2011).…”

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker

“…Kalicludines peptides (AsKC 1-3) from Anemonia viridis and APEKTx1 from Anthopleura elegantissima belong to type 2, with 58-65 amino acid residues and three disulfide bridges. These toxins share a structural homology with the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins (from mamba snakes) which are highly potent blockers of K V channels [10,11]. The third type is composed of toxins containing 41 or 42 amino acid residues and three disulfide bridges: BDS I and II toxins (A. viridis) inhibit K V 3.1, K V 3.2 and K V 3.4 currents by acting as channel-gating modulators and APETx1 (A. elegantissima) is a gating modifier toxin of K V 10.1 and K V 11.1 [12,13].…”