Linear Peptides-A Combinatorial Innovation in the Venom of Some Modern Spiders.

In the venom of spiders, linear peptides (LPs), also called cytolytical or antimicrobial peptides, represent a largely neglected group of mostly membrane active substances that contribute in some spider species considerably to the killing power of spider venom. By next-generation sequencing venom gland transcriptome analysis, we investigated 48 spider species from 23 spider families and detected LPs in 20 species, belonging to five spider families (Ctenidae, Lycosidae, Oxyopidae, Pisauridae, and Zodariidae). The structural diversity is extraordinary high in some species: the lynx spider Oxyopes heterophthalmus contains 62 and the lycosid Pardosa palustris 60 different LPs. In total, we identified 524 linear peptide structures and some of them are in lycosids identical on amino acid level. LPs are mainly encoded in complex precursor structures in which, after the signal peptide and propeptide, 13 or more LPs (Hogna radiata) are connected by linkers. Besides Cupiennius species, also in Oxyopidae, posttranslational modifications of some precursor structures result in the formation of two-chain peptides. It is obvious that complex precursor structures represent a very suitable and fast method to produce a high number and a high diversity of bioactive LPs as economically as possible. At least in Lycosidae, Oxyopidae, and in the genus Cupiennius, LPs reach very high Transcripts Per Kilobase Million values, indicating functional importance within the envenomation process.

Complex precursor structures of cytolytic cupiennins identified in spider venom gland transcriptomes.

Analysis of spider venom gland transcriptomes focuses on the identification of possible neurotoxins, proteins and enzymes. Here, the first comprehensive transcriptome analysis of cupiennins, small linear cationic peptides, also known as cytolytic or antimicrobial peptides, is reported from the venom gland transcriptome of Cupiennius salei by 454- and Illumina 3000 sequencing. Four transcript families with complex precursor structures are responsible for the expression of 179 linear peptides. Within the transcript families, after an anionic propeptide, cationic linear peptides are separated by anionic linkers, which are transcript family specific. The C-terminus of the transcript families is characterized by a linear peptide or truncated linkers with unknown function. A new identified posttranslational processing mechanism explains the presence of the two-chain CsTx-16 family in the venom. The high diversity of linear peptides in the venom of a spider and this unique synthesis process is at least genus specific as verified with Cupiennius getazi.

Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects.

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupienniussalei venom.

High Specific Efficiency of Venom of Two Prey-Specialized Spiders.

The venom of predators should be under strong selection pressure because it is a costly substance and prey may potentially become resistant. Particularly in prey-specialized predators, venom should be selected for its high efficiency against the focal prey. Very effective venom paralysis has been observed in specialized predators, such as spiders preying on dangerous prey. Here, we compared the toxicity of the venoms of two prey-specialized species, araneophagous Palpimanus sp. and myrmecophagous Zodarion nitidum, and their related generalist species. We injected different venom concentrations into two prey types-the prey preferred by a specialist and an alternative prey-and observed the mortality and the paralysis of the prey within 24 h. We found that the venoms of specialists were far more potent towards the preferred prey than alternative prey. The venoms of generalists were similarly potent towards both prey types. In addition, we tested the efficacy of two venom fractions (smaller and larger than 10 kDa) in araneophagous Palpimanus sp. Compounds larger than 10 kDa paralyzed both prey types, but smaller compounds (<10 kDa) were effective only on preferred prey, suggesting the presence of prey-specific compounds in the latter fraction. Our results confirm that prey-specialized spiders possess highly specific venom that allows them to subdue dangerous prey.

Spider Venom: Components, Modes of Action, and Novel Strategies in Transcriptomic and Proteomic Analyses.

This review gives an overview on the development of research on spider venoms with a focus on structure and function of venom components and techniques of analysis. Major venom component groups are small molecular mass compounds, antimicrobial (also called cytolytic, or cationic) peptides (only in some spider families), cysteine-rich (neurotoxic) peptides, and enzymes and proteins. Cysteine-rich peptides are reviewed with respect to various structural motifs, their targets (ion channels, membrane receptors), nomenclature, and molecular binding. We further describe the latest findings concerning the maturation of antimicrobial, and cysteine-rich peptides that are in most known cases expressed as propeptide-containing precursors. Today, venom research, increasingly employs transcriptomic and mass spectrometric techniques. Pros and cons of venom gland transcriptome analysis with Sanger, 454, and Illumina sequencing are discussed and an overview on so far published transcriptome studies is given. In this respect, we also discuss the only recently described cross contamination arising from multiplexing in Illumina sequencing and its possible impacts on venom studies. High throughput mass spectrometric analysis of venom proteomes (bottom-up, top-down) are reviewed.

The Dual Prey-Inactivation Strategy of Spiders-In-Depth Venomic Analysis of Cupiennius salei.

Most knowledge of spider venom concerns neurotoxins acting on ion channels, whereas proteins and their significance for the envenomation process are neglected. The here presented comprehensive analysis of the venom gland transcriptome and proteome of Cupiennius salei focusses on proteins and cysteine-containing peptides and offers new insight into the structure and function of spider venom, here described as the dual prey-inactivation strategy. After venom injection, many enzymes and proteins, dominated by α-amylase, angiotensin-converting enzyme, and cysteine-rich secretory proteins, interact with main metabolic pathways, leading to a major disturbance of the cellular homeostasis. Hyaluronidase and cytolytic peptides destroy tissue and membranes, thus supporting the spread of other venom compounds. We detected 81 transcripts of neurotoxins from 13 peptide families, whereof two families comprise 93.7% of all cysteine-containing peptides. This raises the question of the importance of the other low-expressed peptide families. The identification of a venom gland-specific defensin-like peptide and an aga-toxin-like peptide in the hemocytes offers an important clue on the recruitment and neofunctionalization of body proteins and peptides as the origin of toxins.

Identification of a precursor processing protease from the spider Cupiennius salei essential for venom neurotoxin maturation.

Spider venom neurotoxins and cytolytic peptides are expressed as elongated precursor peptides, which are post-translationally processed by proteases to yield the active mature peptides. The recognition motifs for these processing proteases, first published more than 10 years ago, include the processing quadruplet motif (PQM) and the inverted processing quadruplet motif (iPQM). However, the identification of the relevant proteases was still pending. Here we describe the purification of a neurotoxin precursor processing protease from the venom of the spider Cupiennius salei The chymotrypsin-like serine protease is a 28-kDa heterodimer with optimum activity at venom's pH of 6.0. We designed multiple synthetic peptides mimicking the predicted cleavage sites of neurotoxin precursors. Using these peptides as substrates, we confirm the biochemical activity of the protease in propeptide removal from neurotoxin precursors by cleavage C-terminal of the PQM. Furthermore, the PQM protease also cleaves the iPQM relevant for heterodimerization of a subgroup of neurotoxins. An involvement in the maturing of cytolytic peptides is very likely, due to high similarity of present protease recognition motifs. Finally, bioinformatics analysis, identifying sequences of homolog proteins from 18 spiders of 9 families, demonstrate the wide distribution and importance of the isolated enzyme for spiders. In summary, we establish the first example of a PQM protease, essential for maturing of spider venom neurotoxins. In the future, the here described protease may be established as a powerful tool for production strategies of recombinant toxic peptides, adapted to the maturing of spider venom toxins.

Spider Neurotoxins, Short Linear Cationic Peptides and Venom Protein Classification Improved by an Automated Competition between Exhaustive Profile HMM Classifiers.

Spider venoms are rich cocktails of bioactive peptides, proteins, and enzymes that are being intensively investigated over the years. In order to provide a better comprehension of that richness, we propose a three-level family classification system for spider venom components. This classification is supported by an exhaustive set of 219 new profile hidden Markov models (HMMs) able to attribute a given peptide to its precise peptide type, family, and group. The proposed classification has the advantages of being totally independent from variable spider taxonomic names and can easily evolve. In addition to the new classifiers, we introduce and demonstrate the efficiency of hmmcompete, a new standalone tool that monitors HMM-based family classification and, after post-processing the result, reports the best classifier when multiple models produce significant scores towards given peptide queries. The combined used of hmmcompete and the new spider venom component-specific classifiers demonstrated 96% sensitivity to properly classify all known spider toxins from the UniProtKB database. These tools are timely regarding the important classification needs caused by the increasing number of peptides and proteins generated by transcriptomic projects.

Peptidomic and transcriptomic profiling of four distinct spider venoms.

Venom based research is exploited to find novel candidates for the development of innovative pharmacological tools, drug candidates and new ingredients for cosmetic and agrochemical industries. Moreover, venomics, as a well-established approach in systems biology, helps to elucidate the genetic mechanisms of the production of such a great molecular biodiversity. Today the advances made in the proteomics, transcriptomics and bioinformatics fields, favor venomics, allowing the in depth study of complex matrices and the elucidation even of minor compounds present in minute biological samples. The present study illustrates a rapid and efficient method developed for the elucidation of venom composition based on NextGen mRNA sequencing of venom glands and LC-MS/MS venom proteome profiling. The analysis of the comprehensive data obtained was focused on cysteine rich peptide toxins from four spider species originating from phylogenetically distant families for comparison purposes. The studied species were Heteropoda davidbowie (Sparassidae), Poecilotheria formosa (Theraphosidae), Viridasius fasciatus (Viridasiidae) and Latrodectus mactans (Theridiidae). This led to a high resolution profiling of 284 characterized cysteine rich peptides, 111 of which belong to the Inhibitor Cysteine Knot (ICK) structural motif. The analysis of H. davidbowie venom revealed a high richness in term of venom diversity: 95 peptide sequences were identified; out of these, 32 peptides presented the ICK structural motif and could be classified in six distinct families. The profiling of P. formosa venom highlighted the presence of 126 peptide sequences, with 52 ICK toxins belonging to three structural distinct families. V. fasciatus venom was shown to contain 49 peptide sequences, out of which 22 presented the ICK structural motif and were attributed to five families. The venom of L. mactans, until now studied for its large neurotoxins (Latrotoxins), revealed the presence of 14 cysteine rich peptides, out of which five were ICK toxins belonging to the CSTX superfamily. This in depth profiling of distinct ICK peptide families identified across the four spider species highlighted the high conservation of these neurotoxins among spider families.

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami.

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE: Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES: Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-ß-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging.

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70-80%) and granulocytes (20-30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionisation-mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide.

Cupiennins are small cationic α-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

Multicomponent venom of the spider Cupiennius salei: a bioanalytical investigation applying different strategies.

The multicomponent venom of the spider Cupiennius salei was separated by three different chromatographic strategies to facilitate subsequent analysis of peptidic venom components by tandem mass spectrometry (MALDI-TOF-MS and ESI-MS), Edman degradation and amino acid analysis: (a) desalting of the crude venom by RP-HPLC only, (b) chromatographic separation of the crude venom into 42 fractions by RP-HPLC, and (c) multidimensional purification of the crude venom by size exclusion and cation exchange chromatography and RP-HPLC. A total of 286 components were identified in the venom of C. salei by mass spectrometry and the sequence of 49 new peptides was determined de novo by Edman degradation and tandem mass spectrometry; 30 were C-terminally amidated. The novel peptides were assigned to two main groups: (a) short cationic peptides and (b) Cys-containing peptides with the inhibitor cystine knot motif. Bioinformatics revealed a limited number of substantial similarities, namely with the peptides CpTx1 from the spider Cheiracantium punctorium and U3-ctenitoxin-Asp1a from the South American fishing spider (Ancylometes sp.) and with sequences from a Lycosa singoriensis venom gland transcriptome analysis. The results clearly indicate that the quality of the data is strongly dependent on the chosen separation strategy. The combination of orthogonal analytical methods efficiently excludes alkali ion and matrix adducts, provides indispensable information for an unambiguous identification of isomasses, and results in the most comprehensive repertoire of peptides identified in the venom of C. salei so far.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Structural and biochemical characterization of native and recombinant single insulin-like growth factor-binding domain protein (SIBD-1) from the Central American hunting spider Cupiennius salei (Ctenidae).

Cupiennius salei single insulin-like growth factor binding domain protein (SIBD-1) is an 8.6 kDa Cys-, Pro-, and Gly-rich protein, discovered in the hemocytes of the Central American hunting spider Cupiennius salei. SIBD-1 exhibits high sequence similarity to the N-terminal domain of the insulin-like growth factor-binding protein superfamily and has been reported to play an important role in the spider's immune system. Here, the recombinant expression and the elucidation of the three-dimensional structure of recombinant SIBD-1 and the characterization of the sugar moiety at Thr2 of native SIBD-1 is described in detail.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei.

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells.

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-D: - and all-L: -cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of L: -cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Expression of defensins in non-infected araneomorph spiders.

Defensins are a major family of antimicrobial peptides found throughout the phylogenetic tree. From the spider species: Cupiennius salei, Phoneutria reidyi, Polybetes pythagoricus, Tegenaria atrica, and Meta menardi, defensins belonging to the 'ancestral' class of invertebrate defensins were cloned and sequenced. The deduced amino acid sequences contain the characteristic six cysteines of this class of defensins and reveal precursors of 60 or 61 amino acid residues. The mature peptides consist of 37 amino acid residues, showing up to 70% identities with tick and scorpion defensins. In C. salei, defensin mRNA was found to be constitutively expressed in hemocytes, ovaries, subesophageal nerve mass, hepatopancreas, and muscle tissue. This is the first report presenting and comparing antimicrobial peptides belonging to the family of defensins from spiders.

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei.

Three novel glycine-rich peptides, named ctenidin 1-3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mass.