Inhibitor Cystine Knot Peptides Research Articles

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

Immunosuppressive, antimicrobial and insecticidal activities of inhibitor cystine knot peptides produced by teratocytes of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae).

Teratocytes are specialized cells released by parasitoid wasps into their hosts. They are known for producing regulatory molecules that aid the development of immature parasitoids. We have recently reported the primary structures of cystine-rich peptides, including some containing inhibitor cystine knot (ICK) motifs, produced by teratocytes of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae). ICKs are known for their stability and diverse biological functions. In this study, we produced four putative ICK peptides from the teratocytes of C. flavipes using solid-phase peptide synthesis or recombinant expression in E. coli, and investigated their functions on host immune modulation as well their potential to impair the development of two lepidopterans after ingestion of the peptides. In addition, the peptides were assayed against pathogens and human cells. The peptides did not influence total hemocyte count but suppressed cellular immunity, detectable as a reduction of hemocyte encapsulation (CftICK-I, CftICK-II, CftICK-III) and spread indexes (CftICK-IV) in the host. None of the peptides influenced the activities of prophenoloxidase and phenoloxidase in the hemolymph of larval Diatraea saccharalis (Lepidoptera: Crambidae). CftICK-I and CftICK-II with previously unknown function showed antifungal activity against Candida albicans but were non-toxic to human cells. CftICK-I, CftICK-II, and CftICK-III increased larval mortality and reduced leaf consumption of D.saccharalis, a permissive host for C. flavipes. The CftICK-III also increased larval mortality and reduced leaf consumption of Spodoptera frugiperda (Lepidoptera: Noctuidae), a non-permissive host for C. flavipes. This study highlights biological functions and biotechnological potential of ICK peptides from the teratocytes of C. flavipes.

Stability and Safety of Inhibitor Cystine Knot Peptide, GTx1-15, from the Tarantula Spider Grammostola rosea.

Inhibitor cystine knot (ICK) peptides are knotted peptides with three intramolecular disulfide bonds that affect several types of ion channels. Some are proteolytically stable and are promising scaffolds for drug development. GTx1-15 is an ICK peptide that inhibits the voltage-dependent calcium channel Cav3.1 and the voltage-dependent sodium channels Nav1.3 and Nav1.7. As a model molecule to develop an ICK peptide drug, we investigated several important pharmaceutical characteristics of GTx1-15. The stability of GTx1-15 in rat and human blood plasma was examined, and no GTx1-15 degradation was observed in either rat or human blood plasma for 24 h in vitro. GTx1-15 in blood circulation was detected for several hours after intravenous and intramuscular administration, indicating high stability in plasma. The thermal stability of GTx1-15 as examined by high thermal incubation and protein thermal shift assays indicated that GTx1-15 possesses high heat stability. The cytotoxicity and immunogenicity of GTx1-15 were examined using the human monocytic leukemia cell line THP-1. GTx1-15 showed no cytotoxicity or immunogenicity even at high concentrations. These results indicate that GTx1-15 itself is suitable for peptide drug development and as a peptide library scaffold.

Structural Basis of Nav1.7 Inhibition by the Tarantula Toxin Protoxin-II

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Neurotoxins have provided important insights into Nav channels, but the structural basis for toxin modulation of channel gating is not well-defined. Protoxin-II (ProTx2) is an inhibitor cystine-knot peptide and a selective antagonist of the human Nav1.7 channel. Here, using X-ray crystallography and cryo-electron microscopy, we have captured ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct structural features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule of VSD2 to antagonize S4 gating charge movement through an electrostatic mechanism. ProTx2 engages a small interaction surface and few direct contacts on VSD2, allowing the S4 helix to be visualized in both activated and deactivated states. Our results illuminate pharmacological concepts employed by membrane-partitioning peptide toxins, clarify the structural basis of electromechanical coupling in voltage-gated ion channels, and provide new templates to design selective Nav channel antagonists.

Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

Molecular diversity of fungal inhibitor cystine knot peptides evolved by domain repeat and fusion.

Peptides with the inhibitor cystine knot (ICK) motif are extensively present in animals and plants where they exert a diversity of biological functions. However, few studies have been undertaken on this class of peptides in fungi. In this work, we identify a total of 386 fungal ICK peptides and proteins containing this motif by computational data mining of fungal genome databases, which exhibit 14 different exon-intron structures. According to their domain architectures, these proteins are classified into three distinct structural types, including single domains, tandem repeat domains and fusion domains, in which six families belonging to single or tandem repeat domains show remarkable sequence similarity to those from animals and plants, suggesting their orthologous relationship. Extremely high molecular diversity in fungal ICKs might be attributable to different genetic mechanisms, such as gene/domain duplication and fusion. This work not only enlarges the number of ICK peptides in multicellular organisms, but also uncovers their complex evolutionary history in a specific lineage.

Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.

Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.

The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a.

The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.

Cloning and Genomic Characterization of a Natural Insecticidal Peptide LaIT1 with Unique DDH Structural Fold

Two native peptides with disulfide-directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full-length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the "simple" DDH peptide originates from the "complex" ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH-fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.

Novel Inhibitor Cystine Knot Peptides from Momordica charantia

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

A novel sea anemone peptide that inhibits acid-sensing ion channels

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100nM), and also voltage-gated K+ currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na+ voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.

A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: Cloning, heterologous expression and immunological cross-reactivity approaches

The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3–45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3–10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.

Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon–intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.

A new family of cystine knot peptides from the seeds of Momordica cochinchinensis

Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor cystine knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3–MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active.

A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle

The inhibitor cystine knot (ICK) structural motif has been found in several small proteins and peptides from plants, insects, marine molluscs, and also in human. It is defined by a triple β-sheet that is held together by three intramolecular disulfide bonds built by six conserved cysteine residues that generate a highly rigid and stable fold. We describe a procedure for the production of ICK peptides with correct disulfide bond connectivities via expression in Escherichia coli as fusion proteins with an enzymatically inactive variant of the Bacillus amyloliquefaciens RNAse barnase. Barnase directs the fused peptide to the culture medium and the fusion protein can be isolated by combined cation exchange/reverse-phase chromatography. The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides.

Evolutionary origin of inhibitor cystine knot peptides.

The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel have been found to adopt an ICK fold. We have isolated and identified both cDNA and genomic clones for this family of ICK peptides from the scorpion Opistophthalmus carinatus. The gene characterized by three well-delineated exons respectively coding for three structural and functional domains in the toxin precursors illustrates the correlation between exon and module as suggested by the "exon theory of genes." Based on the analysis of precursor organization and gene structure combined with the 3-D fold and functional data, our results highlight a common evolutionary origin for ICK peptides from animals. In contrast, ICK peptides from plant and fungus might be independently evolved from another ancestor.