The evolution and structure of snake venom phosphodiesterase (svPDE) highlight its importance in venom actions
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Curated by eLife
eLife assessment
This manuscript reports important findings regarding the evolution of snake venom proteins. The conclusions are convincing and are based on appropriate and validated methodology in line with the current state-of-the-art. The findings will be of interest to biologists and biochemists interested in the evolution of venoms as well as those generally interested in the evolution of molecular novelties.
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Abstract
For decades, studies of snake venoms focused on the venom-ome-specific toxins (VSTs). VSTs are dominant soluble proteins believed to contribute to the main venomous effects and emerged into gene clusters for fast adaptation and diversification of snake venoms. However, the conserved minor venom components, such as snake venom phosphodiesterase (svPDE), remain largely unexplored. Here, we focus on svPDE by genomic and transcriptomic analysis across snake clades and demonstrate that soluble svPDE is co-opted from the ancestral membrane-attached ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) gene by replacing the original 5′ exon with the exon encoding a signal peptide. Notably, the exons, promoters, and transcription/translation starts have been replaced multiple times during snake evolution, suggesting the evolutionary necessity of svPDE. The structural and biochemical analyses also show that svPDE shares the similar functions with ENPP family, suggesting its perturbation to the purinergic signaling and insulin transduction in venomous effects.
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Author Response
Reviewer #2 (Public Review):
The work integrated genomic and transcriptomic data to reconstruct the origin of the svPDE gene from the ancestral ENPP3 gene. The authors also analyzed the expression of svPDE along different snake lineages and different tissues in three species of venomous snakes. Finally, they purified an svPDE from the venom of Naja atra and analyzed its crystallographic structure and enzymatic function. The experiments are adequately designed and carefully planned and the conclusions made by the authors are well supported by evidence.
I have the following suggestions:
- I could not find a section where the authors provided information regarding the origin of the analyzed venom and tissues. i.e. muscle tissue from Naja atra and venom for purification of svPDE. It is important to include this …
Author Response
Reviewer #2 (Public Review):
The work integrated genomic and transcriptomic data to reconstruct the origin of the svPDE gene from the ancestral ENPP3 gene. The authors also analyzed the expression of svPDE along different snake lineages and different tissues in three species of venomous snakes. Finally, they purified an svPDE from the venom of Naja atra and analyzed its crystallographic structure and enzymatic function. The experiments are adequately designed and carefully planned and the conclusions made by the authors are well supported by evidence.
I have the following suggestions:
- I could not find a section where the authors provided information regarding the origin of the analyzed venom and tissues. i.e. muscle tissue from Naja atra and venom for purification of svPDE. It is important to include this information.
We thank the reviewer for mentioning this.
The information for the venom purification has been described in Results (LINE 116) as “This svPDE was directly purified from the crude venom of Naja atra captured in Taiwan”. The information for the tissues of sequencing data has been included in Results (LINE 117) as “… with publicly available RNA-Seq data and compared them with the corresponding genomes available in the NCBI Assembly database (SI Appendix, Table S1)”, and Material and Methods (Line 403) as “DNA was extracted from the muscle tissue of a male Naja atra …”.
Also, the SI Appendix Table S1 summarized all samples used for sequence analysis with their tissue origins.
We are still grateful for this comment and have updated the text to make it clearer as follows:
“The target genomes included the draft one of Naja atra sequenced from a muscle tissue (ongoing internal project, see Material and Methods for detail) and the complete one of its sister species, Naja naja, from the public data (Suryamohan et al., 2020).”
We have also updated the text when the first time mentioning the comparative genomics and transcriptomes analysis to indicate where the information is described.
“To test our hypothesis, we comprehensively de novo assembled transcriptomes from the species across 13 clades of Toxicofera (Fig. 1B) with publicly available RNA-Seq data and compared them with the corresponding genomes available in the NCBI Assembly database (see SI Appendix, Table S1 for sample details).”
- The authors mention (Line 156) that "the genomic sequences of svPDE-E1a were present in all species of Serpentes but not in the species of Dactyloidae, Varanidae, and Typhlopidae.". As I understand it, the family Typhlopidae is included in the Suborder Serpentes. The conclusions stand of course, but I believe it is worth revising, for accuracy.
We thank the reviewer for noticing this issue.
We have updated the text as follows to prevent misleading:
From “the genomic sequences of svPDE-E1a were present in all species of Serpentes but not in the species of Dactyloidae, Varanidae, and Typhlopidae. This suggests an early emergence of svPDE-E1a in the common ancestor of Serpentes and became …”
To
“the genomic sequences of svPDE-E1a were present in all species of Serpentes except for the earliest diverged Typhlopidae. This suggest an early emergence of svPDE-E1a in the Serpentes evolution and became …”
- During the discussion (Line 315), it is stated that the expression of svPDE in Lamprophiidae is probably associated with the adaptation of prey selection as a dietary generalist compared to Viperidae and Elapidae. Provided that both of these clades have several species considered dietary generalists, I believe this statement is not strongly supported.
We agreed with the reviewer’s comment that we overstated it without solid support. However, here we believe it is worth mentioning and providing a hint for future studies that Lamprophiidae, a less-known clade, has svPDE expression and is not lower than several species of Elapidae. Therefore, we have revised this paragraph to include the finding without further speculations.
“Comparative transcriptomics is a powerful tool to reveal species-specific or tissue-specific novel transcripts, providing new insights for further studies. For example, the svPDE expression of Lamprophiidae, even higher than several species of Elapidae, indicates the worth of further study for this less-known clade to fill the knowledge gap.”
- Also in the discussion (Line 320), the authors mention that Colubridae is traditionally regarded as a non-venomous clade. This statement is far from accurate given that Colubridae is a very diverse clade and several species within it have been shown to be at least moderately venomous. Various species have been shown to produce secretions comparable to those of front-fanged snakes. Furthermore, despite their difference in morphology, I believe there is little to no evidence that suggests Duvernoy's glands in colubrids have any functions differing from the venom glands of front-fanged snakes.
We thank reviewer’s comment for revising the interpretation. This paragraph has been rewritten to as follows:
“Interestingly, the svPDE expression in Duvernoy’s glands of Colubridae, although low, several species within the diverse Colubridae clade have been shown to be moderately venomous. The expression of svPDE in the Duvernoy’s glands also highlights its potential function despite that Duvernoy’s glands exhibit morphological difference from the venom glands of front-fanged snakes”
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eLife assessment
This manuscript reports important findings regarding the evolution of snake venom proteins. The conclusions are convincing and are based on appropriate and validated methodology in line with the current state-of-the-art. The findings will be of interest to biologists and biochemists interested in the evolution of venoms as well as those generally interested in the evolution of molecular novelties.
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Reviewer #1 (Public Review):
In this work, Pan et al. investigate the properties of the underexplored snake venom phosphodiesterase (svPDE) from a genomic, transcriptomic, and structural perspective. These analyses are complemented by comparisons with similar ENPP proteins to better understand the elements that may underline the specific role of svPDE in envenomation. The data support a role for svPDE that may be related to its interactions with partner proteins or due to its phosphodiesterase activity to enhance the cytotoxic effects of other venoms present in the environment.
Overall, the authors have done a good job of investigating the origins and function of svPDE. The evolutionary analyses are adequate and informative, which are expanded by further experiments to determine the structure and interactions of svPDE. The …
Reviewer #1 (Public Review):
In this work, Pan et al. investigate the properties of the underexplored snake venom phosphodiesterase (svPDE) from a genomic, transcriptomic, and structural perspective. These analyses are complemented by comparisons with similar ENPP proteins to better understand the elements that may underline the specific role of svPDE in envenomation. The data support a role for svPDE that may be related to its interactions with partner proteins or due to its phosphodiesterase activity to enhance the cytotoxic effects of other venoms present in the environment.
Overall, the authors have done a good job of investigating the origins and function of svPDE. The evolutionary analyses are adequate and informative, which are expanded by further experiments to determine the structure and interactions of svPDE. The protein-protein interaction experiments and the svPDE activity experiments with different substrate types shed light on the possible role of the protein in the context of its cellular environment and point to the potential role of glycosylation as part of the mode of action of svPDE. These results will pose a good prelude for further research into the mechanism and interactions of svPDE from other species. Further, the mechanistic insights from this work may also help the development of antivenom compounds that target svPDE.
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Reviewer #2 (Public Review):
The work integrated genomic and transcriptomic data to reconstruct the origin of the svPDE gene from the ancestral ENPP3 gene. The authors also analyzed the expression of svPDE along different snake lineages and different tissues in three species of venomous snakes. Finally, they purified an svPDE from the venom of Naja atra and analyzed its crystallographic structure and enzymatic function. The experiments are adequately designed and carefully planned and the conclusions made by the authors are well supported by evidence.
I have the following suggestions:
I could not find a section where the authors provided information regarding the origin of the analyzed venom and tissues. i.e. muscle tissue from Naja atra and venom for purification of svPDE. It is important to include this information.
The authors …
Reviewer #2 (Public Review):
The work integrated genomic and transcriptomic data to reconstruct the origin of the svPDE gene from the ancestral ENPP3 gene. The authors also analyzed the expression of svPDE along different snake lineages and different tissues in three species of venomous snakes. Finally, they purified an svPDE from the venom of Naja atra and analyzed its crystallographic structure and enzymatic function. The experiments are adequately designed and carefully planned and the conclusions made by the authors are well supported by evidence.
I have the following suggestions:
I could not find a section where the authors provided information regarding the origin of the analyzed venom and tissues. i.e. muscle tissue from Naja atra and venom for purification of svPDE. It is important to include this information.
The authors mention (Line 156) that "the genomic sequences of svPDE-E1a were present in all species of Serpentes but not in the species of Dactyloidae, Varanidae, and Typhlopidae.". As I understand it, the family Typhlopidae is included in the Suborder Serpentes. The conclusions stand of course, but I believe it is worth revising, for accuracy.
During the discussion (Line 315), it is stated that the expression of svPDE in Lamprophiidae is probably associated with the adaptation of prey selection as a dietary generalist compared to Viperidae and Elapidae. Provided that both of these clades have several species considered dietary generalists, I believe this statement is not strongly supported.
Also in the discussion (Line 320), the authors mention that Colubridae is traditionally regarded as a non-venomous clade. This statement is far from accurate given that Colubridae is a very diverse clade and several species within it have been shown to be at least moderately venomous. Various species have been shown to produce secretions comparable to those of front-fanged snakes.
Furthermore, despite their difference in morphology, I believe there is little to no evidence that suggests Duvernoy's glands in colubrids have any functions differing from the venom glands of front-fanged snakes. -
Reviewer #3 (Public Review):
The biochemical identity and the crystal structure of the snake venom phosphodiesterase (svPDE) were determined using protein purified from the crude venom of a snake (Naja atra) captured in Taiwan. The crystal structure was determined with and without AMP bound. The quality of the structure is excellent and the coordination of the bound AMP makes sense based on the coordination by side-chain residues and the known coordination of bound AMP to structural homologues (ENPP3). Naturally, it's interesting that snake venom produces a soluble variant of the membrane-anchored PDE found in humans.
Although the structure and the catalytic site seem overall similar, it is unclear what the role of the snake enzyme is in the host infection. Furthermore, there are a number of human ENPP enzymes and they have different …
Reviewer #3 (Public Review):
The biochemical identity and the crystal structure of the snake venom phosphodiesterase (svPDE) were determined using protein purified from the crude venom of a snake (Naja atra) captured in Taiwan. The crystal structure was determined with and without AMP bound. The quality of the structure is excellent and the coordination of the bound AMP makes sense based on the coordination by side-chain residues and the known coordination of bound AMP to structural homologues (ENPP3). Naturally, it's interesting that snake venom produces a soluble variant of the membrane-anchored PDE found in humans.
Although the structure and the catalytic site seem overall similar, it is unclear what the role of the snake enzyme is in the host infection. Furthermore, there are a number of human ENPP enzymes and they have different substrate preferences and physiological roles. More detailed biochemistry would help to put the role of the svPDE into a physiological context.
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