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Biomedicines
2020 Nov 04;811:. doi: 10.3390/biomedicines8110473.
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Kunitz-Type Peptides from the Sea Anemone Heteractis crispa Demonstrate Potassium Channel Blocking and Anti-Inflammatory Activities.
Gladkikh I
,
Peigneur S
,
Sintsova O
,
Lopes Pinheiro-Junior E
,
Klimovich A
,
Menshov A
,
Kalinovsky A
,
Isaeva M
,
Monastyrnaya M
,
Kozlovskaya E
,
Tytgat J
,
Leychenko E
.
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The Kunitz/BPTI peptide family includes unique representatives demonstrating various biological activities. Electrophysiological screening of peptides HCRG1 and HCRG2 from the sea anemone Heteractis crispa on six Kv1.x channel isoforms and insect Shaker IR channel expressed in Xenopus laevis oocytes revealed their potassium channels blocking activity. HCRG1 and HCRG2 appear to be the first Kunitz-type peptides from sea anemones blocking Kv1.3 with IC50 of 40.7 and 29.7 nM, respectively. In addition, peptides mainly vary in binding affinity to the Kv1.2 channels. It was established that the single substitution, Ser5Leu, in the TRPV1 channel antagonist, HCRG21, induces weak blocking activity of Kv1.1, Kv1.2, and Kv1.3. Apparently, for the affinity and selectivity of Kunitz-fold toxins to Kv1.x isoforms, the number and distribution along their molecules of charged, hydrophobic, and polar uncharged residues, as well as the nature of the channel residue at position 379 (Tyr, Val or His) are important. Testing the compounds in a model of acute local inflammation induced by the introduction of carrageenan administration into mice paws revealed that HCRG1 at doses of 0.1-1 mg/kg reduced the volume of developing edema during 24 h, similar to the effect of the nonsteroidal anti-inflammatory drug, indomethacin, at a dose of 5 mg/kg. ELISA analysis of the animals blood showed that the peptide reduced the synthesis of TNF-α, a pro-inflammatory mediator playing a leading role in the development of edema in this model.
19-74-20088 RSF, 13.1902.21.0012 Grant of the Ministry of Science and Education, Russian Federation, GOC2319 N, GOA4919 N and G0E7120N F.W.O.-Vlaanderen, CELSA/17/047 BOF, KU Leuven, PDM/19/164 KU Leuven funding, n. 2016/04761-4 São Paulo Research Foundation, n. 88881.186830/2018-01 Coordination for the Improvement of Higher Education Personnel
Figure 1. RP-HPLC elution profile of mutant peptide HCRG21 S5L, obtained as the result of hydrolysis of the fusion protein TRX-HCRG21 S5L by BrCN. The fraction containing the mature peptide is underlined. The measured molecular mass of HCRG21 S5L after RP-HPLC is indicated.
Figure 2. 1H NMR spectrum of HCRG21 S5L.
Figure 3. Determination of HCRG21 S5L Ki for trypsin by Dixon method. Substrate concentrations were 0.1 (◊), 0.16 (●), and 0.256 (○) mM. The constants were calculated based on the results of three independent experiments (n ≥ 3).
Figure 4. Multiple alignment of Kunitz peptides, blockers of Kv channels and BPTI. APEKTx1 (P61541) from the sea anemone A. elegantissima, AsKC1–AsKC3 (Q9TWG0, Q9TWF9, Q9TWF8) from A. sulcata, ShPI-1 (P31713) from S. helianthus, SHTXIII (B1B5I8) from S. haddoni, HCRG1, HCRG2, HCRG21, HCRG21 S5L, and InhVJ from H. crispa, HW11c4 (A0A023WBH6) from spider Ornithoctonus huwena; LmKTT-1a (P0DJ46) from scorpion Lychas mucronatus, Hg1 (P0C8W3) from Hadrurus gertschi; DTX1 (P00979), DTX-K (P00981) from snake Dendroaspis polylepis, DTX-α (P00980), DTX-δ (P00982) from Dendroaspis angusticeps; Conk-S1 (P0C1X2) from cone snail Conus striatus; BPTI (P00974) from bovine Bos taurus. Identical amino acids are shownondark-grey and conservative on light-gray background. Hydrophobic amino acids are indicated by green, positively charged by blue, negatively charged by red, and polar non-charged by pink letters. Frames highlight regions responsible for interaction with Kv channels [19]. Asterisks indicate a reactive site with P1 residue and site of weak interaction with serine proteases.
Figure 5. Electrophysiological analysis of HCRG1 and HCRG2 activities on several cloned voltage-gated potassium channel isoforms expressed in X. laevis oocytes. Representative whole-cell current traces in control and toxin conditions are shown. The blue line marks steady-state current traces after the application of 1 μM of peptides. Traces shown are representative traces of at least three independent experiments (n ≥ 3).
Figure 6. Dose–response curves of HCRG1 and HCRG2 on Kv1.1, Kv1.2, Kv1.3, Kv1.6, and Shaker IR channels obtained by plotting the percentage of blocked current as a function of increasing toxin concentrations. Traces shown are representative traces of at least three independent experiments (n ≥ 3).
Figure 7. (a) Electrophysiological analysis of HCRG21 S5L activity on Kv1.1–Kv1.3 isoforms expressed in X. laevis oocytes. Representative whole-cell current traces in control and toxin conditions are shown. The blue line marks steady-state current traces after the application of 10 μM of peptide. (b) Dose–response curve for HCRG21 S5L on Kv1.1 channels. Traces shown are representative traces of at least three independent experiments (n ≥ 3).
Figure 8. Effect of peptide HCRG1 on (a) paw swelling and (b) TNF-α production in mice with acute local inflammation induced by carrageenan administration. Control animals received a similar volume of saline (negative control) or indomethacin solution at a dose of 5 mg/kg (positive control). Intact animals on (b) were not subjected to any manipulation. The reliability of differences is calculated by the Student’s t-criterion. The value * p < 0.05, ** p < 0.01 is considered reliable in comparison with the saline group.
Figure 9. Spatial structures of HCRG1, HCRG2, HCRG21, and HCRG21S5L. 3D-Models of peptides are represented as a ribbon diagram with translucent surfaces accessible to the solvent, painted in accordance with the electrostatic potential: blue indicates the region of positive values, red—negative and gray—neutral. The side chains of positive (Arg and Lys) and hydrophobic (Leu5) amino acid residues are shown as sticks. The distance between the α-carbons of the above amino acid residues is indicated. The ShPI-1 (PDB ID 1SHP) from the sea anemone S. helianthus was used as a template. 3D models were made using Discovery Studio and UCSF Chimera.
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