Carolina Möller

A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins

APETx3, a novel peptide isolated from the sea anemone Anthopleura elegantissima, is a naturally occurring mutant from APETx1, only differing by a Thr to Pro substitution at position 3. APETx1 is believed to be a selective modulator of human ether‐á‐go‐go related gene (hERG) potassium channels with a Kd of 34 nM. In this study, APETx1, 2, and 3 have been subjected to an electrophysiological screening on a wide range of 24 ion channels expressed in Xenopus laevis oocytes: 10 cloned voltage‐gated sodium channels (NaV 1.2–NaV1.8, the insect channels DmNaV1, BgNaV1–1a, and the arachnid channel VdNaV1) and 14 cloned voltage‐gated potassium channels (KV1.1–KV1.6, KV2.1, KV3.1, KV4.2, KV4.3, KV7.2, KV7.4, hERG, and the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels and provides this toxin the ability to become a potent (EC50 276 nM) modulator of voltage‐gated sodium channels (NaVs) because it slows down the inactivation of mammalian and insect NaV channels. Our study also shows that the homologous toxins APETx1 and APETx2 display promiscuous properties since they are also capable of recognizing NaV channels with IC50 values of 31 nM and 114 nM, respectively, causing an inhibition of the sodium conductance without affecting the inactivation. Our results provide new insights in key residues that allow these sea anemone toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins thus far believed to be highly selective.—Peigneur, S., Béress, L., Möller, C., Marí, F., Forssmann, W.‐G., Tytgat, J. A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins. FASEB J. 26, 5141–5151 (2012). www.fasebj.org

A vasopressin/oxytocin-related conopeptide with γ-carboxyglutamate at position 8

Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin-related peptides that have been found in the venom of cone snails, a genus of marine predatory molluscs that envenom their prey with a complex mixture of neuroactive peptides. In the present paper, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. This novel peptide, designated γ-conopressin-vil, has the sequence CLIQDCPγG* (γ is γ-carboxyglutamate and * is C-terminal amidation). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is γ-carboxyglutamate instead of a neutral or basic residue; therefore it could not be directly classified into either the vasopressin or the oxytocin peptide families. Nano-NMR spectroscopy of the peptide isolated directly from the cone snails revealed that the native γ-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the γ-carboxyglutamate residue. However, the negatively charged residues in the sequence of γ-conopressin-vil may mediate calcium binding by a novel mechanism not observed in other peptides of this family.

9.3 KDa components of the injected venom of Conus purpurascens define a new five-disulfide conotoxin framework.

The 83‐residue conopeptide (p21a) and its corresponding nonhydroxylated analog were isolated from the injected venom of Conus purpurascens. The complete conopeptide sequences were derived from Edman degradation sequencing of fragments from tryptic, chymotryptic and cyanogen bromide digestions. p21a has a unique, 10‐cystine/5‐disulfide 7‐loop framework with extended 10‐residue N‐terminus and a 5‐residue C‐terminus tails, respectively. p21a has a 48% sequence homology with a recently described 13‐cystine conopeptide, con‐ikot‐ikot, isolated from the dissected venom of the fish‐hunting snail Conus striatus. However, unlike con‐ikot‐ikot, p21a does not form a dimer of dimers. MALDI‐TOF mass spectrometry suggests that p21a may form a noncovalent dimer. p21a is an unusually large conotoxin and in so far is the largest isolated from injected venom. p21a provides evidence that the Conus venom arsenal includes larger molecules that are directly injected into the prey. Therefore, cone snails can utilize toxins that are comparable in size to the ones commonly found in other venomous animals.

High molecular weight components of the injected venom of fish-hunting cone snails target the vascular system

UNLABELLED The venom of marine cone snails is a rich source of pharmacotherapeutic compounds with striking target specificity and functional diversity. Small, disulfide-rich peptide toxins are the most well characterized active compounds in cone snail venom. However, reports on the presence of larger polypeptides have recently emerged. The majority of these studies have focused on the content of the dissected venom gland rather than the injected venom itself. Recent breakthroughs in the sensitivity of protein and nucleotide sequencing techniques allow for the exploration of the proteomic diversity of injected venom. Using mass spectrometric analysis of injected venoms of the two fish-hunting cone snails Conus purpurascens and Conus ermineus, we demonstrate the presence of angiotensin-converting enzyme-1 (ACE-1) and endothelin converting enzyme-1 (ECE-1), metalloproteases that activate potent vasoconstrictive peptides. ACE activity was confirmed in the venom of C. purpurascens and was significantly reduced in venom preincubated with the ACE inhibitor captopril. Reverse-transcription PCR demonstrated that these enzymes are expressed in the venom glands of other cone snail species with different prey preferences. These findings strongly suggest that cone snails employ compounds that cause disruption of cardiovascular function as part of their complex envenomation strategy, leading to the enhancement of neurotropic peptide toxin activity. BIOLOGICAL SIGNIFICANCE To our knowledge, this is the first study to show the presence of ACE and ECE in the venom of cone snails. Identification of these vasoactive peptide-releasing proteases in the injected venoms of two fish-hunting cone snails highlights their role in envenomation and enhances our understanding of the complex hunting strategies utilized by these marine predators. Our findings on the expression of these enzymes in other cone snail species suggests an important biological role of ACE and ECE in these animals and points towards recruitment into venom from general physiological processes.

Pc16a, the first characterized peptide from Conus pictus venom, shows a novel disulfide connectivity

A novel conotoxin, pc16a, was isolated from the venom of Conus pictus. This is the first peptide characterized from this South-African cone snail and it has only 11 amino acid residues, SCSCKRNFLCC*, with the rare cysteine framework XVI and a monoisotopic mass of 1257.6Da. Two peptides were synthesized with two possible conformations: globular (pc16a_1) and ribbon (pc16a_2). pc16a_1 co-eluted with the native peptide, which indicates a disulfide connectivity I-III, II-IV. The structure of pc16a_1 was determined by NMR. Both synthetic peptides were used to elucidate the biological activity. Bioassays were performed on crickets, ghost shrimps, larvae of the mealworm beetle and mice, but no effect was seen. Using two-electrode voltage clamp, a range of voltage-gated ion channels (Na(v) and K(v)) and nicotinic acetylcholine receptors were screened, but again no activity was found. Hence, the specific target of pc16a still remains to be discovered.

Functional Hypervariability and Gene Diversity of Cardioactive Neuropeptides

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

Comparative analysis of proteases in the injected and dissected venom of cone snail species.

The venom of cone snails has been the subject of intense studies because it contains small neuroactive peptides of therapeutic value. However, much less is known about their larger proteins counterparts and their role in prey envenomation. Here, we analyzed the proteolytic enzymes in the injected venom of Conus purpurascens and Conus ermineus (piscivorous), and the dissected venom of C. purpurascens, Conus marmoreus (molluscivorous) and Conus virgo (vermivorous). Zymograms show that all venom samples displayed proteolytic activity on gelatin. However, the electrophoresis patterns and sizes of the proteases varied considerably among these four species. The protease distribution also varied dramatically between the injected and dissected venom of C. purpurascens. Protease inhibitors demonstrated that serine and metalloproteases are responsible for the gelatinolytic activity. We found fibrinogenolytic activity in the injected venom of C. ermineus suggesting that this venom might have effects on the hemostatic system of the prey. Remarkable differences in protein and protease expression were found in different sections of the venom duct, indicating that these components are related to the storage granules and that they participate in venom biosynthesis. Consequently, different conoproteases play major roles in venom processing and prey envenomation.

Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis.

Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components.

Definition of the R-superfamily of conotoxins: Structural convergence of helix-loop-helix peptidic scaffolds

HighlightsUsing the transcriptome and cDNA libraries, we determined the precursor proteins sequences that define the R‐superfamily of conotoxins.The R‐superfamily pre‐pro region is unusually long and with prevalent Pro repeats, which constitutes a proline‐rich motif (PRM).We determined the three dimensional structure of vil14a, which displays a cystine‐stabilized &agr;‐helix‐loop‐helix (Cs &agr;/&agr;) fold.The R‐superfamily conotoxins overlap with Cs &agr;/&agr; scorpion toxins and other native peptides indicating structural convergence for this motif. ABSTRACT The F14 conotoxins define a four‐cysteine, three‐loop conotoxin scaffold that produce tightly folded structures held together by two disulfide bonds with a C‐C‐C‐C arrangement (conotoxin framework 14). Here we describe the precursors of the F14 conotoxins from the venom of Conus anabathrum and Conus villepinii. Using transcriptomic and cDNA cloning analysis, the full‐length of the precursors of flf14a and flf14b from the transcriptome of C. anabathrum revealed a unique signal sequence that defines the new conotoxin R‐superfamily. Using the signal sequence as a primer, we cloned seven additional previously undescribed toxins of the R‐superfamily from C. villepinii. The propeptide regions of the R‐conotoxins are unusually long and with prevalent proline residues in repeating pentads which qualifies them as Pro‐rich motifs (PRMs), which can be critical for protein‐protein interactions or they can be cleaved to release short linear peptides that may be part of the envenomation mélange. Additionally, we determined the three‐dimensional structure of vil14a by solution 1H‐NMR and found that the structure of this conotoxin displays a cysteine‐stabilized &agr;‐helix‐loop‐helix (Cs &agr;/&agr;) fold. The structure is well‐defined over the helical regions (backbone RMSD for residues 2–13 and 17–26 is 0.63 ± 0.14 Å), with conformational flexibility in the triple Gly region of the second loop as well as the N‐ and C‐ termini. Structurally, the F14 conotoxins overlap with the Cs &agr;/&agr; scorpion toxins and other peptidic natural products, and in spite of their different exogenomic origins, there is convergence into this scaffold from several classes of living organisms that express these peptides.

Structural plasticity of mini‐M conotoxins – expression of all mini‐M subtypes by Conus regius

The mini‐M conotoxins are peptidic scaffolds found in the venom of cones snails. These scaffolds are tightly folded structures held together by three disulfide bonds with a CC‐C‐C‐CC arrangement (conotoxin framework III) and belong to the M Superfamily of conotoxins. Here, we describe mini‐M conotoxins from the venom of Conus regius, a Western Atlantic worm‐hunting cone snail species using transcriptomic and peptidomic analyses. These C. regius conotoxins belong to three different subtypes: M1, M2, and M3. The subtypes show little sequence homology, and their loop sizes (intercysteine amino acid chains) vary significantly. The mini‐Ms isolated from dissected venom contains preferentially hydroxylated proline residues, thus augmenting the structural reach of this conotoxin class. Using 2D‐NMR methods, we have determined the 3D structure of reg3b, an M2 subtype conotoxin, which shows a constrained multi‐turn scaffold. The structural diversity found within mini‐M conotoxin scaffolds of C. regius is indicative of structural hypervariability of the conotoxin M superfamily that is not seen in other superfamilies. These stable minimalistic scaffolds may be investigated for the development of engineered peptides for therapeutic applications.