De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator

The brown widow spider, Latrodectus geometricus, is a predator of a variety of agricultural insects and is also hazardous for humans. Its venom is a true pharmacopeia representing neurotoxic peptides targeting the ion channels and/or receptors of both vertebrates and invertebrates. The lack of transcriptomic information, however, limits our knowledge of the diversity of components present in its venom. The purpose of this study was two-fold: (1) carry out a transcriptomic analysis of the venom, and (2) investigate the bioactivity of the venom using an electrophysiological bioassay. From 32,505 assembled transcripts, 8 toxin families were classified, and the ankyrin repeats (ANK), agatoxin, centipede toxin, ctenitoxin, lycotoxin, scorpion toxin-like, and SCP families were reported in the L. geometricus venom gland. The diversity of L. geometricus venom was also uncovered by the transcriptomics approach with the presence of defensins, chitinases, translationally controlled tumor proteins (TCTPs), leucine-rich proteins, serine proteases, and other important venom components. The venom was also chromatographically purified, and the activity contained in the fractions was investigated using an electrophysiological bioassay with the use of a voltage clamp on ion channels in order to find if the neurotoxic effects of the spider venom could be linked to a particular molecular target. The findings show that U24-ctenitoxin-Pn1a involves the inhibition of the insect sodium (Nav) channels, BgNav and DmNav. This study provides an overview of the molecular diversity of L. geometricus venom, which can be used as a reference for the venom of other spider species. The venom composition profile also increases our knowledge for the development of novel insecticides targeting voltage-gated sodium channels.

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Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus

eLife ◽

10.7554/elife.04387 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 23

Author(s):

Paul G DeCaen ◽

Yuka Takahashi ◽

Terry A Krulwich ◽

Masahiro Ito ◽

David E Clapham

Keyword(s):

Divalent Cations ◽

Ion Selectivity ◽

Resting Membrane Potential ◽

Na Channel ◽

Ph Homeostasis ◽

Ionic Selectivity ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Nav Channels ◽

Alkaliphilic Bacillus

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na+ to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na+ channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na+, Ca2+ and K+ ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na+ or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

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Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss

GigaScience ◽

10.1093/gigascience/giaa120 ◽

2020 ◽

Vol 9 (11) ◽

Cited By ~ 1

Author(s):

Brooke L Whitelaw ◽

Ira R Cooke ◽

Julian Finn ◽

Rute R da Fonseca ◽

Elena A Ritschard ◽

...

Keyword(s):

Salivary Gland ◽

Serine Proteases ◽

Bacterial Species ◽

Resistance Mutation ◽

Minor Component ◽

Gene Families ◽

Venom Gland ◽

Evolutionary Patterns ◽

Novel Gene ◽

Voltage Gated Sodium Channels

Abstract Background Cephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland. Findings To reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non–tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source. Conclusions We present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

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Venom-gland transcriptomic, venomic, and antivenomic profiles of the spine-bellied sea snake (Hydrophis curtus) from the South China Sea

BMC Genomics ◽

10.1186/s12864-021-07824-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Hong-Yan Zhao ◽

Lin Wen ◽

Yu-Feng Miao ◽

Yu Du ◽

Yan Sun ◽

...

Keyword(s):

South China Sea ◽

South China ◽

Evolutionary History ◽

De Novo ◽

Venom Gland ◽

The South China Sea ◽

Protein Families ◽

The South ◽

Widespread Species ◽

China Sea

Abstract Background A comprehensive evaluation of the -omic profiles of venom is important for understanding the potential function and evolution of snake venom. Here, we conducted an integrated multi-omics-analysis to unveil the venom-transcriptomic and venomic profiles in a same group of spine-bellied sea snakes (Hydrophis curtus) from the South China Sea, where the snake is a widespread species and might generate regionally-specific venom potentially harmful to human activities. The capacity of two heterologous antivenoms to immunocapture the H. curtus venom was determined for an in-depth evaluation of their rationality in treatment of H. curtus envenomation. In addition, a phylogenetic analysis by maximum likelihood was used to detect the adaptive molecular evolution of full-length toxin-coding unigenes. Results A total of 90,909,384 pairs of clean reads were generated via Illumina sequencing from a pooled cDNA library of six specimens, and yielding 148,121 unigenes through de novo assembly. Sequence similarity searching harvested 63,845 valid annotations, including 63,789 non-toxin-coding and 56 toxin-coding unigenes belonging to 22 protein families. Three protein families, three-finger toxins (3-FTx), phospholipase A2 (PLA2), and cysteine-rich secretory protein, were detected in the venom proteome. 3-FTx (27.15% in the transcriptome/41.94% in the proteome) and PLA2 (59.71%/49.36%) were identified as the most abundant families in the venom-gland transcriptome and venom proteome. In addition, 24 unigenes from 11 protein families were shown to have experienced positive selection in their evolutionary history, whereas four were relatively conserved throughout evolution. Commercial Naja atra antivenom exhibited a stronger capacity than Bungarus multicinctus antivenom to immunocapture H. curtus venom components, especially short neurotoxins, with the capacity of both antivenoms to immunocapture short neurotoxins being weaker than that for PLA2s. Conclusions Our study clarified the venom-gland transcriptomic and venomic profiles along with the within-group divergence of a H. curtus population from the South China Sea. Adaptive evolution of most venom components driven by natural selection appeared to occur rapidly during evolutionary history. Notably, the utility of commercial N. atra and B. multicinctus antivenoms against H. curtus toxins was not comprehensive; thus, the development of species-specific antivenom is urgently needed.

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Transcriptomic and Proteomic Analyses Reveal the Diversity of Venom Components from the Vaejovid Scorpion Serradigitus gertschi

Toxins ◽

10.3390/toxins10090359 ◽

2018 ◽

Vol 10 (9) ◽

pp. 359 ◽

Cited By ~ 14

Author(s):

Maria Romero-Gutiérrez ◽

Carlos Santibáñez-López ◽

Juana Jiménez-Vargas ◽

Cesar Batista ◽

Ernesto Ortiz ◽

...

Keyword(s):

Serine Proteases ◽

De Novo ◽

Sequence Similarity ◽

Scorpion Venom ◽

Secretory Proteins ◽

Host Defense Peptides ◽

The Family ◽

Pathogenesis Related ◽

Molecular Masses

To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as “other venom components”. A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.

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The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na+channel (ENaC): IUPHAR Review 19

British Journal of Pharmacology ◽

10.1111/bph.13533 ◽

2016 ◽

Vol 173 (18) ◽

pp. 2671-2701 ◽

Cited By ~ 62

Author(s):

Emilie Boscardin ◽

Omar Alijevic ◽

Edith Hummler ◽

Simona Frateschi ◽

Stephan Kellenberger

Keyword(s):

Ion Channels ◽

Na Channel ◽

Acid Sensing Ion Channels ◽

Epithelial Na Channel

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Partial proteomic analysis of brown widow spider (Latrodectus geometricus) venom to determine the biological activities

Toxicon X ◽

10.1016/j.toxcx.2020.100062 ◽

2020 ◽

Vol 8 ◽

pp. 100062

Author(s):

Pornsawan Khamtorn ◽

Prapenpuksiri Rungsa ◽

Nisachon Jangpromma ◽

Sompong Klaynongsruang ◽

Jureerut Daduang ◽

...

Keyword(s):

Proteomic Analysis ◽

Biological Activities ◽

Latrodectus Geometricus ◽

Brown Widow Spider ◽

Widow Spider

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Spider Knottin Pharmacology at Voltage-Gated Sodium Channels and Their Potential to Modulate Pain Pathways

Toxins ◽

10.3390/toxins11110626 ◽

2019 ◽

Vol 11 (11) ◽

pp. 626 ◽

Cited By ~ 7

Author(s):

Yashad Dongol ◽

Fernanda Caldas Cardoso ◽

Richard J Lewis

Keyword(s):

Chronic Pain ◽

Sodium Channels ◽

Structural Features ◽

Voltage Gated ◽

Subtype Selectivity ◽

Voltage Gated Sodium Channels ◽

Neuronal Signalling ◽

Pain Pathways ◽

Nav Channels ◽

Chronic Pain Conditions

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.

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Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.4.f613 ◽

2000 ◽

Vol 278 (4) ◽

pp. F613-F619 ◽

Cited By ~ 95

Author(s):

Alexander Shigaev ◽

Carol Asher ◽

Hedva Latter ◽

Haim Garty ◽

Eitan Reuveny

Keyword(s):

De Novo ◽

Rat Kidney ◽

Kidney Cortex ◽

Na Channel ◽

Xenopus Laevis Oocytes ◽

Fourfold Increase ◽

Tight Epithelia ◽

High Level

Aldosterone is the major corticosteroid regulating Na+ absorption in tight epithelia and acts primarily by activating the epithelial Na+ channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514–2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgkmRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+ channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

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Engineering prokaryotic channels for control of mammalian tissue excitability

Nature Communications ◽

10.1038/ncomms13132 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 12

Author(s):

Hung X. Nguyen ◽

Robert D. Kirkton ◽

Nenad Bursac

Keyword(s):

Action Potential ◽

Sodium Channels ◽

Connexin 43 ◽

De Novo ◽

Interstitial Fibrosis ◽

Human Fibroblasts ◽

Mammalian Tissue ◽

Voltage Gated Sodium Channels ◽

Inward Rectifying Potassium Channel

Abstract The ability to directly enhance electrical excitability of human cells is hampered by the lack of methods to efficiently overexpress large mammalian voltage-gated sodium channels (VGSC). Here we describe the use of small prokaryotic sodium channels (BacNav) to create de novo excitable human tissues and augment impaired action potential conduction in vitro. Lentiviral co-expression of specific BacNav orthologues, an inward-rectifying potassium channel, and connexin-43 in primary human fibroblasts from the heart, skin or brain yields actively conducting cells with customizable electrophysiological phenotypes. Engineered fibroblasts (‘E-Fibs’) retain stable functional properties following extensive subculture or differentiation into myofibroblasts and rescue conduction slowing in an in vitro model of cardiac interstitial fibrosis. Co-expression of engineered BacNav with endogenous mammalian VGSCs enhances action potential conduction and prevents conduction failure during depolarization by elevated extracellular K+, decoupling or ischaemia. These studies establish the utility of engineered BacNav channels for induction, control and recovery of mammalian tissue excitability.

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