Binding and sequestration of poison frog alkaloids by a plasma globulin
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eLife assessment
Poison frogs sequester alkaloids to make themselves toxic or unpalatable to predators, but how this sequestration occurs is not well understood. This valuable study identifies an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. The supporting evidence is solid and the study adds to our understanding of how toxic animals resist the effects of their own defenses.
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Abstract
Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of ‘free’ plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.
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Author Response
Reviewer #1 (Public Review):
In this exciting and well-written manuscript, Alvarez-Buylla and colleagues report a fascinating discovery of an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. This work is a major advance in our knowledge of how poison frogs are able to sequester and even resist such a panoply of alkaloids. Their study also adds to our understanding of how toxic animals resist the effects of their own defenses. Although target site insensitivity and other mechanisms acting to prevent the binding of alkaloids to their targets (often ion channels) are well characterized now in poison frogs, less is known regarding how they regulate the movement of toxins throughout the animal and in …
Author Response
Reviewer #1 (Public Review):
In this exciting and well-written manuscript, Alvarez-Buylla and colleagues report a fascinating discovery of an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. This work is a major advance in our knowledge of how poison frogs are able to sequester and even resist such a panoply of alkaloids. Their study also adds to our understanding of how toxic animals resist the effects of their own defenses. Although target site insensitivity and other mechanisms acting to prevent the binding of alkaloids to their targets (often ion channels) are well characterized now in poison frogs, less is known regarding how they regulate the movement of toxins throughout the animal and in blood in particular. In the fugu (pufferfish) a protein binds saxitoxin and tetrodotoxin and in some amphibians possibly the protein saxiphilin has been proposed to be a toxin sponge for saxitoxin. However, little is known about poison frogs in particular and if toxin-binding proteins are involved in their sequestration and auto-resistance mechanisms.
The authors use a clever approach wherein a fluorescently labeled probe of a pumiliotoxin analog (an alkaloid toxin sequestered by some poison frogs) is able to be crosslinked to proteins to which it binds. The authors then use sophisticated mass spectroscopy to identify the proteins and find an outlier 'hit' that is a serpin protein. A competition assay, as well as mutagenesis studies, revealed that this ~50-60 kDa plasma protein is responsible for binding much of the pumiliotoxin and a few other alkaloids known to be sequestered in the in vivo assay, but not nicotine, an alkaloid not sequestered by these frogs.
In general, their results are convincing, their methods and analyses robust and the writing excellent. Their findings represent a major breakthrough in the study of toxin sequestration in poison frogs. Below, a more detailed summary and both major and minor constructive comments are given on the nature of the discoveries and some ways that the manuscript could be improved.
Many thanks for this positive summary of our work! We greatly appreciate your time and thoroughness in giving us feedback.
Detailed Summary
The authors functionally characterize a serine-protease inhibitor protein in Oophaga sylvatica frog plasma, which they name O. sylvatica alkaloid-binding globulin (OsABG), that can bind toxic alkaloids. They show that OsABG is the most highly expressed serpin in O. sylvatica liver and that its expression is higher than that of albumin, a major small molecule carrier in vertebrates. Using a toxin photoprobe combined with competitive protein binding assays, their data suggest that OsABG is able to bind specific poison frog toxins including the two most abundant alkaloids in O. sylvatica skin. Their in vitro isolation of toxin-bound OsABG shows that the protein binds most free pumiliotoxin in solution and suggests that OsABG may play an important role in its sequestration. The authors further show that mutations in the binding pocket of OsABG remove its ability to bind toxins and that the binding pocket is structurally similar to that of other vertebrate serpins.
These results are an exciting advance in understanding how poison frogs, which make and use alkaloids as chemical defenses, prevent self-intoxication. The authors provide convincing evidence that OsABG can function as a toxin sponge in O. sylvatica which sets a compelling precedent for future work needed to test the role of OsABG in vivo.
The study could be improved by shifting the focus to O. sylvatica specifically rather than the convergent evolution of sequestration among different dendrobatid species. The reason for this is that most of the results (aside from some of the photoprobe binding results presented in Fig. 1 and Fig. 4) and the proteomics identification of OsABG itself are based on O. sylvatica. It's unclear whether ABG proteins are major toxin sponges in D. tinctorius or E. tricolor since these frogs may contain different toxin cocktails. The competitive binding results suggest that putative ABG proteins in D. tinctorius and E. tricolor have reduced binding affinity at higher toxin concentrations than ABG proteins in O. sylvatica. Although molecular convergence in toxin sponges may be at play in the dendrobatid poison frogs, more work is needed in non-O. sylvatica species to determine the extent of convergence.
We understand and appreciate you raising this concern. As is partially described in the “essential revisions” section above, we have been more cautious throughout the results and discussion to not describe the plasma binding in E. tricolor and D. tinctorius as definitively due to ABG proteins, and to shift the overall focus to O. sylvatica.
Major constructive comments:
Although the protein gels in Fig.1-2 show clearly the role of ABG, a ~50 kDa protein, it's unclear whether transferrin-like proteins, which are ~80 kDa, may also play a role because the gels show proteins between 39-64 kDa (Fig.1). The gel in Fig.2A is specific to one O. sylvatica and extends this range, but the gel does not appear to be labeled accordingly, making it unclear whether other larger proteins could have been detected in addition to ABG. Clarifying this issue would facilitate the interpretation of the results.
Thank you for this suggestion, please see our response above in the “essential revisions” section.
There is what seems to be a significant size difference between the O. sylvatica bands and bands from the other toxic frog species, namely D. tinctorius and E. tricolor. Could the photoprobe be binding to other non-ABG proteins of different sizes in different frog species? Given that O. sylvatica bands are bright and this species was the only one subject to proteomics quantification, a possible conclusion may be that the ABG toxin sponge is a lineage-specific adaptation of O. sylvatica rather than a common mechanism of toxin sequestration among multiple independent lineages of poison frogs. It would be helpful if the authors could address this observation of their binding data and the hypothesis flowing from that in the manuscript.
Thank you for this suggestion, please see our response above in the “essential revisions” section.
Figure 1B: The species names should be labeled alongside the images in the phylogeny. In addition, please include symbols indicating the number of times toxicity has evolved (for example, once in the ancestors of O. sylvatica and D. tinctorius frogs and once in the ancestors of E. tricolor frogs).
These suggested changes have been added to Figure 1B. We were not able to fit the full species names into the figure, instead we added an abbreviated version that is spelled out completely in the figure caption.
Figure 4B-C: Photoprobe binding results in the presence of epi and nicotine appear to be missing for D. tinctorius and those in the presence of PTX and nicotine are missing for D. tricolor. Adding these results would make for a more complete picture of alkaloid binding by ABG in non-O. sylvatica species.
Thank you for this suggestion, please see our response above in the “essential revisions” section.
Using recombinant proteins with mutations at residues forming the binding pocket of O. sylvatica ABG (as inferred from docking simulations), the authors found that all binding pocket mutations disrupted photoprobe binding completely in vitro (L221-222, Fig. 4E). However, there is no information presented on non-binding pocket mutations. Mutations outside of the binding pocket would presumably maintain photoprobe binding - barring any indirect structural changes that might disrupt binding pocket interactions with the photoprobe. This result is important for the conclusion that the binding pocket itself is the sole mediator of toxin interactions. The authors do show that one binding pocket mutation (D383A) results in some degree of photoprobe binding (Fig. 4E) but more detail on the mutations in the binding pocket per se being causal would be helpful.
Thank you for this suggestion, please see our response above in the “essential revisions” section.
Please include concentrations in the descriptions of gel lanes in the main figures. The relative concentrations of the photoprobe and other toxins (eg., PTX, DHQ, epi, and nic) are essential for interpreting the competitive binding images. For example, this was done in Fig. S1 (e.g., PB + 10x PTX).
The photoprobe and competitor concentrations have been added beneath the gels in Figures 1, 4, and 6 as suggested. Additionally, in the crosslinking experiments involving purified protein the amount of protein per well has been added to the top of the TAMRA gel.
For clarity, the section "OsABG sequesters free PTX in solution with high affinity" could be presented directly after the section titled "Proteomic analysis identifies an alkaloid-binding globulin". The former highlights in vitro experiments confirming the binding affinity of the ABG protein identified in the latter.
While we see how this rearrangement might work, we think that the current order of figures creates a more compelling story and provides the evidence in a more intuitive manner. For instance, it is necessary to show that recombinant protein recapitulates the plasma photoprobe results and that binding pocket mutants disrupt photoprobe binding (Figure 4), prior to showing the direct binding assays with the recombinant wild type and mutant proteins. For this reason, we believe that this rearrangement might cause confusion, and are leaving it as is.
Fig. 6E-F should be included as part of Fig. 1 or 2. Although complementary to the RNA sequencing data, these protein results are more closely related to the results in the first two figures which show the degree of competitive binding affinity of PB in the presence of different toxins. The expanded competitive binding results for total skin alkaloids and the two most abundant skin alkaloids from wild samples are most appropriate here.
We understand the reasoning behind this, however we feel that including these results in Figure 6 is more appropriate and that moving it would disrupt the flow of the story. The identification of ABG and its binding activity happened before we fully understood the alkaloid profiles of wild-collected O. sylvatica, therefore we did not think to test additional alkaloids like histrionicotoxin and indolizidines till we saw that these were very abundant on the skin of field collected poison frogs. Furthermore, we would like to leave this section at the end because we feel it contributes important ecological relevance that we want to leave readers with.
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Sure! So I wish I had started out with E. coli, but I started with HEK cell expression because that was what I had access to most easily. That did give some expression, but not nearly enough protein to do any meaningful biochemistry. One thought I had at the time was that it might be an amphibian-specific PTM, so I also tried Xenopus egg extract expression - which didn't work for the same reasons (low protein production). Then I started doing E. coli expression, and was definitely getting close but troubleshooting some inclusion body issues when we got the insect cell expression/purification back from the company we had asked to do it, and that just worked beautifully so we continued with that. I think with more time and troubleshooting, E. coli (or yeast) probably would have worked and been higher throughput, but the way things worked …
Sure! So I wish I had started out with E. coli, but I started with HEK cell expression because that was what I had access to most easily. That did give some expression, but not nearly enough protein to do any meaningful biochemistry. One thought I had at the time was that it might be an amphibian-specific PTM, so I also tried Xenopus egg extract expression - which didn't work for the same reasons (low protein production). Then I started doing E. coli expression, and was definitely getting close but troubleshooting some inclusion body issues when we got the insect cell expression/purification back from the company we had asked to do it, and that just worked beautifully so we continued with that. I think with more time and troubleshooting, E. coli (or yeast) probably would have worked and been higher throughput, but the way things worked out continuing with the company made more sense. Also, we knew that another frog protein, saxiphilin, had been expressed using insect cells which seemed like a good precedent. Hope that helps clarify a bit!
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This is a great point - the affinities we have been seeing are very low, which we think adds weight to the idea of it primarily being a transport system rather than an autoresistance mechanism. That being said, the fact that there is promiscuity for multiple different alkaloids may require that the affinity for each individual alkaloid is low, and is then compensated by a very high expression (which we do see) to shift the reaction kinetics towards binding. Regardless, most tested alkaloids have potencies in the nM-uM range, and ABG is solidly in the mM range, so it is definitely a weaker interaction.
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Thank you so much Adair! I'm a big fan of your work with bacteriophage work too :)
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Alkaloid-binding globulin (ABG) represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points towards serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.
This is such a cool paper, and exciting conclusion!
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The lower affinity of OsABG provides further support to the hypothesis that OsABG may be acting as a transporter protein to other tissues, and would be in line with the hypothesis that there may be other mechanisms involved in autoresistance to circulating alkaloids not bound by protei
I'm curious about how the affinity for OsABG for alkaloids compares to the affinity of these alkaloids for their molecular targets. I would imagine that a sponge/autoresistance protein would need a higher affinity for the alkaloid than the target in order to compete effectively for the molecule, whereas a transport protein may not have such strict requirements.
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Recombinant ABG proteins were expressed by Kemp Proteins (Maryland, USA) through their custom insect cell protein expression and purification services.
I'm curious if you tried to express these in e. coli or yeast first before escalating to insect cells? Would you mind sharing some of your decision making around expression platforms for these molecules?
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eLife assessment
Poison frogs sequester alkaloids to make themselves toxic or unpalatable to predators, but how this sequestration occurs is not well understood. This valuable study identifies an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. The supporting evidence is solid and the study adds to our understanding of how toxic animals resist the effects of their own defenses.
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Reviewer #1 (Public Review):
In this exciting and well-written manuscript, Alvarez-Buylla and colleagues report a fascinating discovery of an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. This work is a major advance in our knowledge of how poison frogs are able to sequester and even resist such a panoply of alkaloids. Their study also adds to our understanding of how toxic animals resist the effects of their own defenses. Although target site insensitivity and other mechanisms acting to prevent the binding of alkaloids to their targets (often ion channels) are well characterized now in poison frogs, less is known regarding how they regulate the movement of toxins throughout the animal and in blood in …
Reviewer #1 (Public Review):
In this exciting and well-written manuscript, Alvarez-Buylla and colleagues report a fascinating discovery of an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. This work is a major advance in our knowledge of how poison frogs are able to sequester and even resist such a panoply of alkaloids. Their study also adds to our understanding of how toxic animals resist the effects of their own defenses. Although target site insensitivity and other mechanisms acting to prevent the binding of alkaloids to their targets (often ion channels) are well characterized now in poison frogs, less is known regarding how they regulate the movement of toxins throughout the animal and in blood in particular. In the fugu (pufferfish) a protein binds saxitoxin and tetrodotoxin and in some amphibians possibly the protein saxiphilin has been proposed to be a toxin sponge for saxitoxin. However, little is known about poison frogs in particular and if toxin-binding proteins are involved in their sequestration and auto-resistance mechanisms.
The authors use a clever approach wherein a fluorescently labeled probe of a pumiliotoxin analog (an alkaloid toxin sequestered by some poison frogs) is able to be crosslinked to proteins to which it binds. The authors then use sophisticated mass spectroscopy to identify the proteins and find an outlier 'hit' that is a serpin protein. A competition assay, as well as mutagenesis studies, revealed that this ~50-60 kDa plasma protein is responsible for binding much of the pumiliotoxin and a few other alkaloids known to be sequestered in the in vivo assay, but not nicotine, an alkaloid not sequestered by these frogs.
In general, their results are convincing, their methods and analyses robust and the writing excellent. Their findings represent a major breakthrough in the study of toxin sequestration in poison frogs. Below, a more detailed summary and both major and minor constructive comments are given on the nature of the discoveries and some ways that the manuscript could be improved.
Detailed Summary
The authors functionally characterize a serine-protease inhibitor protein in Oophaga sylvatica frog plasma, which they name O. sylvatica alkaloid-binding globulin (OsABG), that can bind toxic alkaloids. They show that OsABG is the most highly expressed serpin in O. sylvatica liver and that its expression is higher than that of albumin, a major small molecule carrier in vertebrates. Using a toxin photoprobe combined with competitive protein binding assays, their data suggest that OsABG is able to bind specific poison frog toxins including the two most abundant alkaloids in O. sylvatica skin. Their in vitro isolation of toxin-bound OsABG shows that the protein binds most free pumiliotoxin in solution and suggests that OsABG may play an important role in its sequestration. The authors further show that mutations in the binding pocket of OsABG remove its ability to bind toxins and that the binding pocket is structurally similar to that of other vertebrate serpins.
These results are an exciting advance in understanding how poison frogs, which make and use alkaloids as chemical defenses, prevent self-intoxication. The authors provide convincing evidence that OsABG can function as a toxin sponge in O. sylvatica which sets a compelling precedent for future work needed to test the role of OsABG in vivo.
The study could be improved by shifting the focus to O. sylvatica specifically rather than the convergent evolution of sequestration among different dendrobatid species. The reason for this is that most of the results (aside from some of the photoprobe binding results presented in Fig. 1 and Fig. 4) and the proteomics identification of OsABG itself are based on O. sylvatica. It's unclear whether ABG proteins are major toxin sponges in D. tinctorius or E. tricolor since these frogs may contain different toxin cocktails. The competitive binding results suggest that putative ABG proteins in D. tinctorius and E. tricolor have reduced binding affinity at higher toxin concentrations than ABG proteins in O. sylvatica. Although molecular convergence in toxin sponges may be at play in the dendrobatid poison frogs, more work is needed in non-O. sylvatica species to determine the extent of convergence.
Major constructive comments:
Although the protein gels in Fig.1-2 show clearly the role of ABG, a ~50 kDa protein, it's unclear whether transferrin-like proteins, which are ~80 kDa, may also play a role because the gels show proteins between 39-64 kDa (Fig.1). The gel in Fig.2A is specific to one O. sylvatica and extends this range, but the gel does not appear to be labeled accordingly, making it unclear whether other larger proteins could have been detected in addition to ABG. Clarifying this issue would facilitate the interpretation of the results.
There is what seems to be a significant size difference between the O. sylvatica bands and bands from the other toxic frog species, namely D. tinctorius and E. tricolor. Could the photoprobe be binding to other non-ABG proteins of different sizes in different frog species? Given that O. sylvatica bands are bright and this species was the only one subject to proteomics quantification, a possible conclusion may be that the ABG toxin sponge is a lineage-specific adaptation of O. sylvatica rather than a common mechanism of toxin sequestration among multiple independent lineages of poison frogs. It would be helpful if the authors could address this observation of their binding data and the hypothesis flowing from that in the manuscript.
Figure 1B: The species names should be labeled alongside the images in the phylogeny. In addition, please include symbols indicating the number of times toxicity has evolved (for example, once in the ancestors of O. sylvatica and D. tinctorius frogs and once in the ancestors of E. tricolor frogs).
Figure 4B-C: Photoprobe binding results in the presence of epi and nicotine appear to be missing for D. tinctorius and those in the presence of PTX and nicotine are missing for D. tricolor. Adding these results would make for a more complete picture of alkaloid binding by ABG in non-O. sylvatica species.
Using recombinant proteins with mutations at residues forming the binding pocket of O. sylvatica ABG (as inferred from docking simulations), the authors found that all binding pocket mutations disrupted photoprobe binding completely in vitro (L221-222, Fig. 4E). However, there is no information presented on non-binding pocket mutations. Mutations outside of the binding pocket would presumably maintain photoprobe binding - barring any indirect structural changes that might disrupt binding pocket interactions with the photoprobe. This result is important for the conclusion that the binding pocket itself is the sole mediator of toxin interactions. The authors do show that one binding pocket mutation (D383A) results in some degree of photoprobe binding (Fig. 4E) but more detail on the mutations in the binding pocket per se being causal would be helpful.
Please include concentrations in the descriptions of gel lanes in the main figures. The relative concentrations of the photoprobe and other toxins (eg., PTX, DHQ, epi, and nic) are essential for interpreting the competitive binding images. For example, this was done in Fig. S1 (e.g., PB + 10x PTX).
For clarity, the section "OsABG sequesters free PTX in solution with high affinity" could be presented directly after the section titled "Proteomic analysis identifies an alkaloid-binding globulin". The former highlights in vitro experiments confirming the binding affinity of the ABG protein identified in the latter.
Fig. 6E-F should be included as part of Fig. 1 or 2. Although complementary to the RNA sequencing data, these protein results are more closely related to the results in the first two figures which show the degree of competitive binding affinity of PB in the presence of different toxins. The expanded competitive binding results for total skin alkaloids and the two most abundant skin alkaloids from wild samples are most appropriate here.
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Reviewer #2 (Public Review):
Poison frogs are able to sequester alkaloids to make themselves toxic or unpalatable to predators. Despite much research, the proteins that accomplish this sequestering role are not well known. Here, biochemical and proteomic analysis identifies a liver-derived alkaloid binding globulin (ABG) as the main alkaloid binding molecule in the blood of poison frogs. The results are solid and address a major void in our understanding of plasma alkaloid transport in frogs. While some additional analysis of ABG mutants would further enhance the interpretations, the study represents an important starting point that suggests specific new roles for serpins in animal ecophysiology.
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