Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels.
García-Fernández R
,
Peigneur S
,
Pons T
,
Alvarez C
,
González L
,
Chávez MA
,
Tytgat J
.
???displayArticle.abstract???
The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the N- and C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the N- and C-terminal ends.
Figure 1. Protease inhibition curves using rShPI-1A. Fixed concentrations of each enzyme were mixed with increasing concentrations of rShPI-1A (displayed in nM). Each enzymatic activity was determined after adding the corresponding substrate (see Materials and Methods for experimental details). The fractional enzymatic activities (vi/v0) were calculated after measuring the initial reaction rates with (vi) and without inhibitor (v0). Each connecting line represents the best fits to the quadratic Morrison equation for tight binding inhibitors described in [21]. Apparent Ki values (Kiapp) were calculated by adjusting the experimental points to that equation and are shown here as the mean ± SE (n = 3).
Figure 2. Activity of rShPI-1A on ion channels expressed in X. laevis oocytes. Traces are representative of at least three independent experiments (n ⥠3). The dotted line indicates the zero current level. The asterisk (*) distinguishes the steady-state current after application of 50 nM peptide.
Figure 3. Characterization of rShPI-1A activity on channel gating. Left panel: current-voltage relationship. Closed symbols are the control condition; open symbols are after application of 50 nM peptide. Right panel: concentration-response curve on Kv1.1 and Kv1.2 channels obtained by plotting the percentage of blocked current as a function of increasing toxin concentrations. All data represent at least three independent experiments (n ⥠3) and are presented as the mean ± standard error.
Figure 4. Sequence alignment of Kv-channel blocking toxins. Residues reported as crucial in the activity toward Kv channels are underlined. Conserved Cys residues are in bold and (*) represent conserved residues. Positions buried to solvent or partially exposed are highlighted in gray. Residues with a negative charge are highlighted in red; positive charge (blue); polar uncharged (magenta); and hydrophobic (green). The numbering system in the corresponding UniProt entry (protein name and UniProt accession code) is used here. Continuous line boxes enclose the sequence regions around CysI and CysV-CysVI, which show an amino acid substitution pattern in most of these proteins. A discontinuous line box encloses key residues around CysII. Additional data about toxins included in this alignment are provided in Table 3 and Table 4. A list of the ShPI-1 interatomic contacts and the accessible surface area (ASA) relative to the residue in a vacuum is provided in the Supplementary File 1.
Figure 5. Structure-based sequence alignment of Kv-channel blocking toxins. Uppercase letters represent the equivalent position in the 3D superimposition. Continuous line boxes enclose those regions that superimpose well among toxins sharing the BPTI-Kunitz fold and those from ShK-type and scorpion-type families, also active against Kv channels. Information about their respective secondary structure elements, helix (h) and β-sheet (e), is shown over each sequence, based on ShPI-1 as representative of all BPTI-Kunitz-type proteins and the toxins ShK, BgK and ChTx. Information about toxin family, including the Pfam database (PF) code, is shown at the right. Conserved Cys residues are in bold, and positions buried to solvent or partially exposed are highlighted in gray. Residues with a negative charge are highlighted in red; positive charge (blue); polar uncharged (magenta); and hydrophobic (green).
Figure 6. Surface electrostatic potential representation of toxins. The surface is colored according to the electrostatic potential: negative regions (in red), positive regions (in blue) and neutral regions (in gray). The orientation of the surface electrostatic potentials is the same as that in the ribbon representation at the bottom right. The list of PDB codes for toxins is as follows: ShPI-1 (3M7Q); LmKTT-1a (2M01); HWTX-XI (2JOT); Conk-S1 (1Y62); α-DTX (1DTX); DTX-K (1DTK); DTX-I (1DEM); BgK (1BGK); ShK (4LFS); and ChTX (1BAH). We also provided a color intensity scale to better represent the electrostatic potential. BPTI-Kunitz-type toxins are displayed in the first two lines. These figures were prepared with the PyMOL Molecular Graphics System, Schrödinger, LLC.
Alvarez,
Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: their interaction with membranes.
2009, Pubmed
Alvarez,
Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: their interaction with membranes.
2009,
Pubmed
Aurell,
New chromogenic peptide substrates for factor X.
1978,
Pubmed
Bayrhuber,
Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.
2005,
Pubmed
Beeton,
Analogs of the sea anemone potassium channel blocker ShK for the treatment of autoimmune diseases.
2011,
Pubmed
Bieth,
Theoretical and practical aspects of proteinase inhibition kinetics.
1995,
Pubmed
Bontems,
Analysis of side-chain organization on a refined model of charybdotoxin: structural and functional implications.
1992,
Pubmed
Castañeda,
Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus.
1995,
Pubmed
Chase,
p-Nitrophenyl-p'-guanidinobenzoate HCl: a new active site titrant for trypsin.
1967,
Pubmed
Chen,
Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family.
2012,
Pubmed
Chen,
Computational Studies of Venom Peptides Targeting Potassium Channels.
2015,
Pubmed
Claeson,
Designing of peptide substrates. Different approaches exemplified by new chromogenic substrates for kallikreins and urokinase.
1978,
Pubmed
Czapinska,
Structural and energetic determinants of the S1-site specificity in serine proteases.
1999,
Pubmed
Dauplais,
On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures.
1997,
Pubmed
de Almeida Nogueira,
Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease.
2013,
Pubmed
Delfín,
Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla helianthus.
1996,
Pubmed
DelMar,
A sensitive new substrate for chymotrypsin.
1979,
Pubmed
Dy,
Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail.
2006,
Pubmed
ERLANGER,
The preparation and properties of two new chromogenic substrates of trypsin.
1961,
Pubmed
Frazão,
Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview.
2012,
Pubmed
García-Fernández,
Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme.
2015,
Pubmed
García-Fernández,
Structure of the recombinant BPTI/Kunitz-type inhibitor rShPI-1A from the marine invertebrate Stichodactyla helianthus.
2012,
Pubmed
García-Fernández,
Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor.
2012,
Pubmed
Gasparini,
Delineation of the functional site of alpha-dendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins.
1998,
Pubmed
Gil,
Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris.
2011,
Pubmed
Gladkikh,
Atypical reactive center Kunitz-type inhibitor from the sea anemone Heteractis crispa.
2012,
Pubmed
Gladkikh,
New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.
2015,
Pubmed
Harvey,
Twenty years of dendrotoxins.
2001,
Pubmed
Harvey,
Recent studies on dendrotoxins and potassium ion channels.
1997,
Pubmed
Hernández González,
Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L.
2015,
Pubmed
Honma,
Novel peptide toxins from the sea anemone Stichodactyla haddoni.
2008,
Pubmed
Kem,
Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus.
1989,
Pubmed
Kisiel,
Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin.
1979,
Pubmed
Krissinel,
Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.
2004,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Lanio,
Purification and characterization of two hemolysins from Stichodactyla helianthus.
2001,
Pubmed
MacKinnon,
Structural conservation in prokaryotic and eukaryotic potassium channels.
1998,
Pubmed
Mattler,
Serine protease specificity for peptide chromogenic substrates.
1977,
Pubmed
Nakajima,
Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site.
1979,
Pubmed
Owen,
The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells.
1997,
Pubmed
Peigneur,
A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties.
2011,
Pubmed
,
Xenbase
Pennington,
Development of highly selective Kv1.3-blocking peptides based on the sea anemone peptide ShK.
2015,
Pubmed
Robertson,
Novel effects of dendrotoxin homologues on subtypes of mammalian Kv1 potassium channels expressed in Xenopus oocytes.
1996,
Pubmed
,
Xenbase
Rodriguez,
Homology modeling, model and software evaluation: three related resources.
1998,
Pubmed
Rodríguez,
A novel sea anemone peptide that inhibits acid-sensing ion channels.
2014,
Pubmed
Rodríguez,
Peptide fingerprinting of the neurotoxic fractions isolated from the secretions of sea anemones Stichodactyla helianthus and Bunodosoma granulifera. New members of the APETx-like family identified by a 454 pyrosequencing approach.
2012,
Pubmed
Schechter,
On the size of the active site in proteases. I. Papain.
1967,
Pubmed
Scheidig,
Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.
1997,
Pubmed
Schweitz,
Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels.
1995,
Pubmed
,
Xenbase
Scott,
Kinetics of inhibition of human plasma kallikrein by a site-specific modified inhibitor Arg15-aprotinin: evaluation using a microplate system and comparison with other proteases.
1987,
Pubmed
Scott,
Antibodies specific for distinct Kv subunits unveil a heterooligomeric basis for subtypes of alpha-dendrotoxin-sensitive K+ channels in bovine brain.
1994,
Pubmed
Shon,
kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel.
1998,
Pubmed
,
Xenbase
Sievers,
Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.
2011,
Pubmed
Silva-Lopez,
Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes.
2007,
Pubmed
Tabakmakher,
Analgesic effect of novel Kunitz-type polypeptides of the sea anemone Heteractis crispa.
2015,
Pubmed
Yuan,
Discovery of a distinct superfamily of Kunitz-type toxin (KTT) from tarantulas.
2008,
Pubmed
