Identification of non-venom protein genes highly expressed in the venom gland of Habu (Protobothrops flavoviridis) and their potential contribution to the quality control of venom proteins

Venom proteins act in the immunological interactions between parasitoids and their host insects. The effect of venom proteins on host immunity is not fully understood in pupal parasitoids. We identified the functions of a venom protein, calreticulin (PvCRT), in the pupal ectoparasitoid Pachycrepoideus vindemiae. Here, we report that PvCRT features a signal peptide and two conserved “calreticulin” domains. Multiple sequence alignments show that PvCRT shares 83.54% amino acid identity with CRT from both Pteromalus puparum and Nasonia vitripennis, which infers a close relationship among these three species. Using qPCR analysis, we found a lower expression level of PvCRT (0.27-fold) in the venom apparatus compared to the corresponding carcass. Immunohistochemical localization revealed that PvCRT was ubiquitously expressed in venom gland. The expression of the PvCRT gene in Drosophila transgenic lines via the UAS/Gal4 binary expression system reduced the self-encapsulation phenotype of tu(1)Sz1 mutants. Additionally, studies on humoral immunity indicate that PvCRT does not affect the antimicrobial immune responses of the host. This work on an ectoparasitoid will increase our understanding of venom–mediated host-parasitoid interactions.

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Alternative mRNA Splicing in Three Venom Families Underlying a Possible Production of Divergent Venom Proteins of the Habu Snake, Protobothrops flavoviridis

Toxins ◽

10.3390/toxins11100581 ◽

2019 ◽

Vol 11 (10) ◽

pp. 581 ◽

Cited By ~ 4

Author(s):

Ogawa ◽

Oda-Ueda ◽

Hisata ◽

Nakamura ◽

Chijiwa ◽

...

Keyword(s):

Alternative Splicing ◽

Gene Families ◽

Venom Gland ◽

Potential Contribution ◽

Venom Protein ◽

Divergent Gene ◽

A Genome ◽

Alternative Mrna Splicing ◽

Trans Splicing ◽

Gene Transcripts

Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake.

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Identification of a Novel Family of Snake Venom Proteins Veficolins fromCerberus rynchopsUsing a Venom Gland Transcriptomics and Proteomics Approach

Journal of Proteome Research ◽

10.1021/pr901044x ◽

2010 ◽

Vol 9 (4) ◽

pp. 1882-1893 ◽

Cited By ~ 60

Author(s):

G. OmPraba ◽

Alex Chapeaurouge ◽

Robin Doley ◽

K. Rama Devi ◽

P. Padmanaban ◽

...

Keyword(s):

Snake Venom ◽

Venom Gland ◽

Proteomics Approach ◽

Venom Proteins

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TOXIFY: a deep learning approach to classify animal venom proteins

10.7287/peerj.preprints.27498 ◽

2019 ◽

Author(s):

T Jeffrey Cole ◽

Michael S Brewer

Keyword(s):

Protein Sequence ◽

Software Package ◽

Shotgun Proteomics ◽

Learning Approach ◽

The Novel ◽

Venom Protein ◽

Order Of Magnitude ◽

Gated Recurrent Units ◽

Venom Proteins ◽

Generation Sequencing

In the era of Next-Generation Sequencing and shotgun proteomics, the sequences of animal toxigenic proteins are being generated at rates exceeding the pace of traditional means for empirical toxicity verification. To facilitate the automation of toxin identification from protein sequences, we trained Recurrent Neural Networks with Gated Recurrent Units on publicly available datasets. The resulting models are available via the novel software package TOXIFY, allowing users to infer the probability of a given protein sequence being a venom protein. TOXIFY is more than 20X faster and uses over an order of magnitude less memory than previously published methods. Additionally, TOXIFY is more accurate, precise, and sensitive at classifying venom proteins. Availability: https://www.github.com/tijeco/toxify

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Quality control for unfolded proteins at the plasma membrane

The Journal of Cell Biology ◽

10.1083/jcb.201006012 ◽

2010 ◽

Vol 191 (3) ◽

pp. 553-570 ◽

Cited By ~ 39

Author(s):

Pirjo M. Apaja ◽

Haijin Xu ◽

Gergely L. Lukacs

Keyword(s):

Quality Control ◽

Plasma Membrane ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cellular Protein ◽

Temperature Sensitive ◽

Potential Contribution ◽

Unfolded Proteins ◽

Endosomal Sorting ◽

V2 Receptor

Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2–E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera–accelerated internalization, impaired recycling, and endosomal sorting complex required for transport–dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.

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Identification and characterization of novel proteins from Arizona bark scorpion venom that inhibit Nav1.8, a voltage-gated sodium channel regulator of pain signaling

10.1101/2021.06.24.449822 ◽

2021 ◽

Author(s):

Tarek Mohamed Abd El-Aziz ◽

Yucheng Xiao ◽

Jake Kline ◽

Harold Gridley ◽

Alyse Heaston ◽

...

Keyword(s):

Sodium Channel ◽

Venom Gland ◽

Reversed Phase ◽

Scorpion Venom ◽

Voltage Gated Sodium Channel ◽

Voltage Gated ◽

Novel Proteins ◽

Centruroides Sculpturatus ◽

Current Mass ◽

Venom Proteins

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, high-lighting the potential to serve as a drug target. However, the biophysical mechanisms that regu-late Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibi-tory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcrip-tome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes 8 cysteines and a CXCE motif where X = an aro-matic residue (tryptophan, tyrosine or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

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Rapid and differential evolution of the venom composition of a parasitoid wasp depending on the host strain

10.1101/796649 ◽

2019 ◽

Author(s):

Fanny Cavigliasso ◽

Hugo Mathé-Hubert ◽

Laurent Kremmer ◽

Christian Rebuf ◽

Jean-Luc Gatti ◽

...

Keyword(s):

Differential Evolution ◽

Rapid Evolution ◽

Intraspecific Variability ◽

Protein Composition ◽

Host Strain ◽

Parasitoid Wasps ◽

Evolutionary Potential ◽

Venom Protein ◽

Venom Composition ◽

Venom Proteins

AbstractParasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with crosses of two lines of Leptopilina boulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance / susceptibility to the parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results evidenced a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened.Key ContributionThe venom protein composition of parasitoid wasps can evolve rapidly and differently depending on the host strain. Studying this evolution can help identify new venom proteins possibly involved in parasitism success on a given host.

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Proteo-Transcriptomic Characterization of the Venom from the Endoparasitoid Wasp Pimpla turionellae with Aspects on Its Biology and Evolution

Toxins ◽

10.3390/toxins11120721 ◽

2019 ◽

Vol 11 (12) ◽

pp. 721 ◽

Cited By ~ 4

Author(s):

Rabia Özbek ◽

Natalie Wielsch ◽

Heiko Vogel ◽

Günter Lochnit ◽

Frank Foerster ◽

...

Keyword(s):

Transcriptome Assembly ◽

Parasitoid Wasp ◽

Venom Gland ◽

Hymenopteran Species ◽

Robber Flies ◽

And Function ◽

Wasp Venoms ◽

Venom Proteins ◽

Proteins And Peptides

Within mega-diverse Hymenoptera, non-aculeate parasitic wasps represent 75% of all hymenopteran species. Their ovipositor dual-functionally injects venom and employs eggs into (endoparasitoids) or onto (ectoparasitoids) diverse host species. Few endoparasitoid wasps such as Pimpla turionellae paralyze the host and suppress its immune responses, such as encapsulation and melanization, to guarantee their offspring’s survival. Here, the venom and its possible biology and function of P. turionellae are characterized in comparison to the few existing proteo-transcriptomic analyses on parasitoid wasp venoms. Multiple transcriptome assembly and custom-tailored search and annotation strategies were applied to identify parasitoid venom proteins. To avoid false-positive hits, only transcripts were finally discussed that survived strict filter settings, including the presence in the proteome and higher expression in the venom gland. P. turionella features a venom that is mostly composed of known, typical parasitoid enzymes, cysteine-rich peptides, and other proteins and peptides. Several venom proteins were identified and named, such as pimplin2, 3, and 4. However, the specification of many novel candidates remains difficult, and annotations ambiguous. Interestingly, we do not find pimplin, a paralytic factor in Pimpla hypochondriaca, but instead a new cysteine inhibitor knot (ICK) family (pimplin2), which is highly similar to known, neurotoxic asilid1 sequences from robber flies.

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Toxins from scratch? Diverse, multimodal gene origins in the predatory robber fly Dasypogon diadema indicate a dynamic venom evolution in dipteran insects

GigaScience ◽

10.1093/gigascience/giz081 ◽

2019 ◽

Vol 8 (7) ◽

Cited By ~ 8

Author(s):

Stephan Holger Drukewitz ◽

Lukas Bokelmann ◽

Eivind A B Undheim ◽

Björn M von Reumont

Keyword(s):

Single Gene ◽

Draft Genome ◽

Evolutionary Analysis ◽

Orphan Genes ◽

High Quality ◽

Venom Protein ◽

Robber Flies ◽

Venom Evolution ◽

Toxin Evolution ◽

Venom Proteins

Abstract Background Venoms and the toxins they contain represent molecular adaptations that have evolved on numerous occasions throughout the animal kingdom. However, the processes that shape venom protein evolution are poorly understood because of the scarcity of whole-genome data available for comparative analyses of venomous species. Results We performed a broad comparative toxicogenomic analysis to gain insight into the genomic mechanisms of venom evolution in robber flies (Asilidae). We first sequenced a high-quality draft genome of the hymenopteran hunting robber fly Dasypogon diadema, analysed its venom by a combined proteotranscriptomic approach, and compared our results with recently described robber fly venoms to assess the general composition and major components of asilid venom. We then applied a comparative genomics approach, based on 1 additional asilid genome, 10 high-quality dipteran genomes, and 2 lepidopteran outgroup genomes, to reveal the evolutionary mechanisms and origins of identified venom proteins in robber flies. Conclusions While homologues were identified for 15 of 30 predominant venom protein in the non-asilid genomes, the remaining 15 highly expressed venom proteins appear to be unique to robber flies. Our results reveal that the venom of D. diadema likely evolves in a multimodal fashion comprising (i) neofunctionalization after gene duplication, (ii) expression-dependent co-option of proteins, and (iii) asilid lineage-specific orphan genes with enigmatic origin. The role of such orphan genes is currently being disputed in evolutionary genomics but has not been discussed in the context of toxin evolution. Our results display an unexpected dynamic venom evolution in asilid insects, which contrasts the findings of the only other insect toxicogenomic evolutionary analysis, in parasitoid wasps (Hymenoptera), where toxin evolution is dominated by single gene co-option. These findings underpin the significance of further genomic studies to cover more neglected lineages of venomous taxa and to understand the importance of orphan genes as possible drivers for venom evolution.

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Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis

Toxins ◽

10.3390/toxins13020140 ◽

2021 ◽

Vol 13 (2) ◽

pp. 140

Author(s):

Chien-Chun Liu ◽

Ya-Han Yang ◽

Yung-Chin Hsiao ◽

Po-Jung Wang ◽

Jo-Chuan Liu ◽

...

Keyword(s):

Liquid Chromatography ◽

Cation Exchange ◽

Human Plasma ◽

Rapid Test ◽

Major Protein ◽

Snake Venoms ◽

Simple Method ◽

Venom Protein ◽

Strong Cation Exchange ◽

Venom Proteins

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims’ plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange–high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10–15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

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