Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal
Abstract
:1. Introduction
2. Results
2.1. De Novo Tentacle Transcriptome Assembly
2.2. Venom Proteome
2.3. Putative Toxins
2.4. Toxin Tentacle Gene Clusters Detected in Venom
2.5. Exploration of the Dominant E. quadricolor Venom Phenotype
2.5.1. Toxins Assigned to the Unknown Venom Function Category
2.5.2. Haemostatic and Haemorrhagic Venom Category
2.5.3. Protease Inhibitor Venom Category
2.5.4. Mixed Function Enzymes Venom Category
2.5.5. Neurotoxin Venom Category
2.5.6. Pore-Forming Venom Category
3. Discussion
3.1. Putative Toxins
3.2. Venom Proteome
3.3. Where Are the Toxin Genes That Are Expressed Going?
3.4. Dominant Venom Hypothesis
4. Conclusions
5. Materials and Methods
5.1. Study Species and Experimental Set-Up
5.2. Sea Anemone Venom and Tentacle Collection
5.3. Transcriptomics
5.3.1. RNA Isolation and Library Preparation
5.3.2. RNA-Seq Read Quality Control
5.3.3. De Novo Transcriptome Assembly
5.3.4. Contaminant Removal
5.3.5. Clustering and Read Counting
5.3.6. Gene Annotation and Functional Enrichment Analysis
5.3.7. Toxin Annotation Pipeline
5.3.8. Signal P
5.3.9. Normalised Abundance of Transcripts
5.4. Proteomics
5.4.1. Venom Protein Extraction for Mass Spectrometry
5.4.2. Spectral Library Creation via DDA and GPF
5.4.3. Normalised Abundance of Protein Expression Utilising DIA
5.5. Data Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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RNA Gene Clusters | Protein Gene Clusters | |
---|---|---|
ORF sequences | 279,274 | 5375 |
Annotated ORF sequences | 72,218 | 5213 |
ORFs with GO terms | 46,288 | 3901 |
Unique UniRef90 Hits | 18,469 | 3718 |
ORFs with signal sequence | 11,807 | 1224 |
Putative toxin ORFs | 1251 | 230 |
Putative toxin ORFs with signal sequence | 515 | 149 |
Putative toxin unique UniRef90 Hits | 296 | 124 |
Venom Category | RNA Gene Clusters | TF | Protein Gene Clusters | TF | Percentage (%) 1 |
---|---|---|---|---|---|
Allergen and innate immunity | 9 | 3 | 4 | 2 | 44.4 |
Auxiliary | 67 | 1 | 19 | 1 | 28.4 |
Haemostatic and haemorrhagic | 409 | 5 | 55 | 5 | 13.4 |
Mixed function enzymes | 24 | 1 | 1 | 1 | 4.2 |
Neurotoxins | 122 | 9 | 21 | 5 | 17.2 |
Pore forming | 45 | 5 | 16 | 4 | 35.6 |
Protease inhibitors | 38 | 2 | 5 | 1 | 13.2 |
Unknown | 537 | 16 | 109 | 11 | 20.3 |
(a) Venom Category | Toxin Protein Family | RNA Gene Clusters | Rank of GC in Proteome |
---|---|---|---|
Haemostatic and haemorrhagic | Coagulation factor V-like | 315 | 2 |
Unknown | IG-like | 300 | 1 |
Unknown | Uncharacterised toxins | 104 | 3 |
Neurotoxin | ShK-like | 85 | 5 |
Auxiliary | Peptidase M12A | 67 | 4 |
Haemostatic and haemorrhagic | Peptidase S1 | 37 | 3 |
Haemostatic and haemorrhagic | Ficolin lectin family | 30 | 10 |
Unknown | EGF-like | 25 | 11 |
Mixed function enzymes | PLA2 | 24 | 13 |
Pore Forming | Actinoporins | 23 | 7 |
(b) Venom Category | Toxin Protein Family | Protein Gene Clusters | Rank of GC in Transcriptome |
Unknown | IG-like | 56 | 2 |
Haemostatic and haemorrhagic | Coagulation factor V-like | 22 | 1 |
Haemostatic and haemorrhagic | Peptidase S1 | 21 | 6 |
Unknown | Uncharacterised toxins | 21 | 3 |
Auxiliary | Peptidase M12A | 19 | 5 |
Neurotoxin | ShK-like | 14 | 4 |
Unknown | U15 | 11 | 11 |
Pore forming | Actinoporins | 7 | 10 |
Haemostatic and haemorrhagic | Peptidase M12B | 6 | 12 |
Unknown | Z3 | 6 | 13 |
Unknown | U12 | 6 | 17 |
Pore forming | DELTA-alicitoxin-Pse2b-like | 5 | 18 |
Protease inhibitor | Venom Kunitz-type family | 5 | 15 |
Haemostatic and haemorrhagic | Ficolin lectin family | 4 | 7 |
(a) Venom Category | Toxin Protein Family | RNA Normalised Abundance 1 | Rank of TF in Proteome |
---|---|---|---|
Unknown | Z3 | 56,348 | 1 |
Unknown | Sea Anemone 8 | 54,210 | Not present |
Neurotoxin | ShK-like | 48,215 | 18 |
Unknown | IG-like | 34,585 | 7 |
Unknown | Uncharacterised toxins | 24,521 | 16 |
Unknown | EGF-like | 19,030 | 5 |
Mixed function enzyme | PLA2 | 11,305 | 4 |
Auxiliary | Peptidase M12A | 8987 | 13 |
Haemostatic and haemorrhagic | Factor V-like | 8767 | 19 |
Unknown | CREC | 6308 | 15 |
(b) Venom Category | Toxin Protein Family | Protein Normalised Abundance 2 | Rank of TF in Transcriptome |
Unknown | Z3 | 16,933,968 | 1 |
Haemostatic and haemorrhagic | Peptidase S1 | 9,294,230 | 11 |
Protease inhibitor | Venom Kunitz | 6,409,895 | 13 |
Mixed function enzyme | PLA2 | 4,873,099 | 7 |
Unknown | EGF-like | 4,773,700 | 6 |
Unknown | U15 | 4,298,257 | 27 |
Unknown | IG-like | 3,884,603 | 4 |
Unknown | U12 | 1,416,547 | 17 |
Allergen and innate immunity | CAP | 1,367,399 | 14 |
Neurotoxin | NEP 3 family | 911,820 | 16 |
Time | Solvent B |
---|---|
0 min | 2% |
5 min | 2% |
10 min | 8% |
60 min | 31.2% |
66 min | 50% |
69 min | 100% |
72 min | 100% |
75 min | 2% |
Method | m/z |
---|---|
1 | 350–1200 |
2 | 350–500 |
3 | 490–550 |
4 | 540–610 |
5 | 600–710 |
6 | 700–810 |
7 | 800–1200 |
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Hoepner, C.M.; Stewart, Z.K.; Qiao, R.; Fobert, E.K.; Prentis, P.J.; Colella, A.; Chataway, T.; Burke da Silva, K.; Abbott, C.A. Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal. Toxins 2024, 16, 85. https://doi.org/10.3390/toxins16020085
Hoepner CM, Stewart ZK, Qiao R, Fobert EK, Prentis PJ, Colella A, Chataway T, Burke da Silva K, Abbott CA. Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal. Toxins. 2024; 16(2):85. https://doi.org/10.3390/toxins16020085
Chicago/Turabian StyleHoepner, Cassie M., Zachary K. Stewart, Robert Qiao, Emily K. Fobert, Peter J. Prentis, Alex Colella, Tim Chataway, Karen Burke da Silva, and Catherine A. Abbott. 2024. "Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal" Toxins 16, no. 2: 85. https://doi.org/10.3390/toxins16020085