Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation

To demonstrate the application of the SeqAPASS tool and highlight new features, two case studies are described representing instances in which protein conservation predicts that there are differences in chemical susceptibility across species (human transthyretin) and that there are no differences (µ opioid receptor [MOR]). The first of these examples addresses protein sequence/structural comparisons to predict the domain of applicability for adverse outcome pathways (AOPs, see Table 2 for definition), while the second is focused on developing research hypotheses relevant to cross-species susceptibility to opioids present in wastewater. The basic approaches described in these case studies can be applied to any chemical and demonstrate the broad utility of this tool for decision-making and research.

Thyroid hormones are essential for normal growth and development. They are synthesized in the thyroid gland and secreted into the bloodstream where they bind to distribution proteins and are circulated throughout the body^{14,15,16,17,18}. Recent studies have shown that environmental contaminants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and per- and polyfluoroalkyl substances (PFAS), can competitively bind to the distribution protein transthyretin (TTR) and disrupt normal thyroid processes^{19,20,21,22,23,24,25}. An AOP has been developed describing competitive binding to TTR leading to human neurodevelopmental toxicity (https://aopwiki.org/aops/152). While there is evidence that this AOP is also applicable to rodents, the applicability to other taxonomic groups has yet to be defined. As TTR-binding chemicals are present in the environment, it is important to understand the taxonomic relevance of this AOP, a challenge that can be addressed in part through SeqAPASS analysis. Using the tool’s problem formulation strategy, the objective of the analysis can be stated as follows: With the knowledge that TTR-binding compounds lead to adverse outcomes in humans, what taxonomic groups would be predicted to share similar susceptibility? 

The human transthyretin protein is well characterized, and there are several well-studied ligands known to bind at the human TTR (hTTR) binding site, making it an optimal target for SeqAPASS analysis^8,9,13. Using the NCBI accession for human transthyretin, P02766.1, a Level 1 analysis was conducted with default settings. The results of the Level 1 analysis set the percent similarity cutoff at 49%, with mammals (Mammalia), birds (Aves), reptiles (Testudines, Lepidosauria, Crocodylia), amphibians (Amphibia), and most fish species (Actinopteri, Coelacanthimorpha, Cladista, Chondrichthyes) falling above this cutoff (Figure 3). Thus, all species from these taxonomic groups resulted in a susceptibility prediction of "Y" (i.e., yes) and are likely susceptible to chemicals known to interact with hTTR (Figure 3 and Supplemental File 1).

For the Level 2 assessment of functional domain(s), the NCBI Conserved Domain Database was used to identify TR_THY (accession smart00095) as a conserved domain comprising the mature chain of the TTR subunit protein from residues 27 to 147. As the protein sequence of TTR reported in NCBI includes a 20 amino acid pre-segment not relevant to the current analysis, focusing the comparison on the mature chain provides an additional, more specific, line of evidence toward the conservation of this protein across species. From the Level 2 evaluation, a percent similarity cutoff of 58% was reported, with mammals, birds, reptiles, amphibians, and most fish species again falling above this cutoff (Figure 4). Consequently, SeqAPASS concluded a susceptibility prediction of "Y" (i.e., yes) for species from these taxonomic groups, indicating they are likely susceptible to chemicals that interact with the hTTR protein (Figure 4 and Supplemental File 1). Overall, results from Level 1 and Level 2 analyses suggest that most vertebrate species share conservation of the hTTR and are likely to be susceptible to chemicals known to interact with this protein.

Figure 3: SeqAPASS Level 1 analysis of transthyretin conservation across taxonomic groups with available sequence information relative to the human protein. Percent similarity of the protein amino acid sequence is displayed on the Y-axis; taxonomic group is displayed on the X-axis. Open circles (○) indicate the query sequence, and closed circles (●) indicate the species within the taxonomic group with the highest percent similarity. Within the plot, the top and bottom of each box represent the 75^th and 25^th percentiles, the whiskers extend to 1.5 times the interquartile range, and the mean and median values are represented by horizontal black lines on the box. The dashed line indicates the cutoff for susceptibility predictions. Abbreviations: TTR = transthyretin; SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility. Please click here to view a larger version of this figure.

Figure 4: SeqAPASS Level 2 analysis of the transthyretin receptor ligand-binding domain conservation across taxonomic groups with available sequence information relative to the human protein LBD. Percent similarity of the ligand binding domain amino acid sequence is displayed on the Y-axis; taxonomic group is displayed on the X-axis. Open circles (○) indicate the query sequence, and closed circles (●) indicate the species within the taxonomic group with the highest percent similarity. Within the plot, the top and bottom of each box represent the 75^th and 25^th percentiles, the whiskers extend to 1.5 times the interquartile range, and the mean and median values are represented by horizontal black lines on the box. The dashed line indicates the cutoff for susceptibility predictions. Abbreviations: TTR = transthyretin; SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility. Please click here to view a larger version of this figure.

Through the analysis of molecular modeling and protein crystallography studies, amino acids were identified in the binding region of TTR that are predicted to interact with the endogenous ligand 3,3′,5,5′-tetraiodo-L-thyronine (T4, PDB 2ROX), as well as three environmental chemicals: perfluorooctane sulfonate (PFOS, PDB 5JIM), tetrabromobisphenol A (TBBPA, PDB 5HJG), and diethylstilbestrol (DES, PDB 1TZ8)^19,21,22,26. The amino acid residues Lys35, Ser137, Leu130, Ala128, Ala129, and Thr139 were all identified as playing a key role in protein-ligand interactions, either through direct hydrogen bond interactions or van der Waals interactions. These six amino acid residues were evaluated in a Level 3 analysis across species using hTTR as the template sequence and excluding non-homologous, hypothetical, partial, and low-quality sequences (Supplemental File 1). As it was previously determined that TTR is conserved only across vertebrate species, invertebrate species were excluded from this analysis (Figure 3 and Figure 4). Additionally, it is important to note that amino acid positions reported in the literature exclude a 20 amino acid pre-segment that is absent in the mature hTTR protein and, for this reason, positions submitted in Level 3 were adjusted from those reported in the literature to ensure accurate alignment to the selected template protein¹⁵ (Supplemental File 1).

In the Level 3 analysis of TTR, 294 vertebrate species were selected for alignment (mammals, birds, amphibians, reptiles, and fish). Of the species evaluated, 18 displayed differences in key amino acids resulting in a susceptibility prediction of "N" (i.e., no). Interestingly, five species of marine mammals presented with an amino acid substitution at position 2 (128A), while four species of fish demonstrated substitutions at either position 2 (128A) or position 6 (139T) (Figure 5). As these amino acids play important roles in protein-ligand interactions in the binding channel of TTR, these data suggest that TTR ligands may interact differently in these species and would result in different chemical susceptibility relative to humans.

Figure 5: SeqAPASS Level 3 analysis of the conservation of amino acid residues important for TTR-chemical binding. (A) Level 3 summary table displaying the number of species with available sequence data across all taxonomic groups, the number of species predicted to be similarly susceptible (Y), and the number of species predicted to not be similarly susceptible (N). (B) Level 3 heatmap displaying select species predicted to not be similarly susceptible relative to the human transthyretin TTR protein, demonstrating full, partial, and non-matching amino acids. Abbreviations: TTR = transthyretin; SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility. Please click here to view a larger version of this figure.

In response to stakeholder and user feedback, new features have been designed and incorporated into the SeqAPASS tool, including the ability to connect to empirical data for different applications. The interoperability of this tool with the ECOTOX knowledgebase (Table 1) was achieved by both embedding external links in the Level 1 and Level 2 data tables for accessions present in ECOTOX and creating an ECOTOX widget within the tool to directly filter to the most relevant toxicity data in ECOTOX. Through the links and the widget, users can rapidly query ECOTOX and gather corresponding toxicity data for species with SeqAPASS susceptibility predictions. Currently, SeqAPASS predictions are connected to ECOTOX through a chemical stressor and species; however, toxicity data are not yet linked to specific genes/proteins, which would allow for direct connections to the specific endpoints/molecular targets of interest in SeqAPASS. While linking predictions to toxicity data based on a chemical stressor is not ideal, as data may not be specific to a given pathway, establishing a connection to bring results together is the first step. As the first iteration of a SeqAPASS-ECOTOX integration, the current approach provides users with all available toxicity data for the chemical stressor(s) and species at a broad level. These data, when combined with SeqAPASS predictions, can provide context at broad levels (vertebrate vs. invertebrate) and can be considered within the context of the AOP framework.

TTR presents a good case example for examining this connection as the existing AOP (AOP 152) provides context for interpreting potentially relevant ECOTOX toxicity data. Starting with the ligands examined in SeqAPASS Level 3, environmental toxicity data were collected across species for four chemicals known to interact with the TTR ligand binding domain (diethylstilbestrol [DES], perfluorohexanoic acid [PFHxA], perfluorooctane sulfonate [PFOS], and tetrabromobisphenol A [TBBPA])^19,21,23,24. For each chemical, ECOTOX was queried for Aquatic and Terrestrial data by Chemical Abstracts Service (CAS) number using custom search parameters (Supplemental File 1). Data were filtered to species groups of interest (amphibians, birds, fish, invertebrates, mammals, reptiles). Within the filtered query results, an average of the study's minimum and maximum effect concentrations was calculated and implemented as an approximation for the mean for any hits that did not report a mean effect concentration value (Figure 6A and Supplemental File 1). Within the context of a single chemical, Kruskal-Wallis tests were conducted to compare the mean effect concentrations of different taxonomic groups as the data did not meet ANOVA test assumptions. Post-hoc pairwise comparison tests were then conducted using the Dunn's Test for all chemicals, as the taxonomic groups consisted of unequal sample sizes. Aquatic and Terrestrial results were analyzed separately, as data between the two types of exposures are not directly comparable. Within ECOTOX, aquatic toxicity data for the selected chemicals were available for amphibians, birds, invertebrates, and fish species (Figure 6A). Terrestrial toxicity data for the selected chemicals were only available for mammals and DES (Supplemental File 1).

Figure 6: Linking SeqAPASS results with empirical data. (A) Mean effect concentrations across taxonomic groups with data available in the ECOTOXicology knowledgebase for select chemicals known to bind to the human TTR protein. (B) Overlap in the number of species included in each SeqAPASS analysis with the species for which ECOTOX data was available. In panel A, parentheses along the x-axis indicate the number of query hits for which data was aggregated. Asterisks indicate pairs of significantly different effect concentrations between species groups within the context of a single chemical (Dunn's test, p < 0.05), where higher numbers of asterisks indicate stronger levels of significance (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). The center lines within each box represent the median, with box edges demonstrating the interquartile range. The whiskers extend up to 1.5 times the interquartile range. Outliers falling outside of that range are shown as individual points. Abbreviations: TTR = transthyretin; SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility; ECOTOX = ECOTOXicology knowledgebase. Please click here to view a larger version of this figure.

Overall, these data suggest that bioactivity for the chemicals assessed occurs in vertebrate species but does not occur in invertebrates. While the lack of biological target and pathway information in ECOTOX makes it impossible to directly link these empirical data to TTR, these results do support the SeqAPASS predictions that invertebrate species do not share susceptibility. All vertebrate species with available data demonstrated susceptibility to both PFOS and TBBPA, although the mean effect concentrations were significantly higher in fish and birds than amphibians. These data suggest potential differences in sensitivity between taxonomic groups that may be attributed to biological pathway differences (including TTR). It is noteworthy that other variables such as metabolism and excretion may also play a role in differences in sensitivity. For PFHxA and PFOS, fish were found to be significantly more sensitive than both invertebrates and birds, and, for DES, amphibians presented with significantly higher mean effect concentrations than invertebrates. Again, these data support our SeqAPASS prediction that invertebrates do not share susceptibility with vertebrate species. Of all species assessed using this tool for which there were available TTR sequence data, only a small number had corresponding ECOTOX data for the four chemicals of interest (Figure 6B, Supplemental Table S1, and Supplemental Table S2). For those species lacking apical data, SeqAPASS predictions of susceptibility add additional lines of evidence that related species may behave similarly to those with apical data. Data for all SeqAPASS and ECOTOX analyses are available in Supplemental File 1.

According to the Centers for Disease Control and Prevention (CDC), in 2017, opioids contributed to about 47,600 deaths by overdose in the United States, a number that continues to rise²⁷. Wastewater plants in the US are regulated nationally by the US Environmental Protection Agency's National Pollutant Discharge Elimination System, which does not require testing for opioids or other pharmaceuticals in their discharge²⁸. In recent years, an effort has been made to use wastewater-based epidemiology as a tool to map community opioid use. Opioid monitoring efforts have detected concentrations as high as 1.27 µg/L in wastewater effluent and 0.7 µg/L in surface waters^29,30. Recent toxicity studies assessing the effect of opioid exposure on fish have reported the development of addictive behaviors and adverse immunological effects (e.g., higher infection rates, downregulation of immune genes)^31,32,33. Overall, these studies suggest there is potential for adverse environmental opioid exposures and highlight the importance of understanding the risk these chemicals pose to aquatic species. Given the range of species that may encounter these compounds in the environment, identifying potentially susceptible species using SeqAPASS may be important for the prioritization of testing or monitoring efforts.

The MOR constitutes the main opioid target for the management of pain and is responsible for the powerful analgesic and addictive properties of opiate alkaloids in humans^34,35. Due to the importance of this receptor to human health, MOR ligands are well known, and high-quality X-ray crystallography studies are available, making this target ideal for SeqAPASS analysis^8,9,13. Using the NCBI accession for human µ opioid receptor, ACM90349.1, a Level 1 analysis was conducted using default settings. The susceptibility cutoff was established at 55% for Level 1, with percent similarities for mammals (Mammalia), birds (Aves), reptiles (Testudines, Lepidosauria, Crocodylia), amphibians (Amphibia), and most fish species (Actinopteri, Coelacanthimorpha, Cladista, Chondrichthyes) falling above this cutoff; therefore, species from these taxonomic groups resulted in a susceptibility prediction of "Y" (i.e. yes), indicating they would likely be susceptible to chemicals known to interact with human MOR (Figure 7 and Supplemental File 1). Using the NCBI Conserved Domain Database, 7tmA_Mu_opioid_R was identified (accession cd15090) as a functional domain comprising all seven helices of the MOR protein from 133 to 411, including a putative ligand binding site. Compared to Level 1, the Level 2 results identified a higher susceptibility cutoff of 88% similarity, with mammals, birds, reptiles, amphibians, and most fish species found above this cutoff and resulting in a susceptibility prediction of "Y" for yes (Figure 8). Overall, results from Level 1 and Level 2 analyses suggest that most vertebrate species share conservation of the MOR and are likely to be susceptible to chemicals known to interact with human MOR.

Figure 7: SeqAPASS Level 1 analysis of µ opioid receptor conservation across taxonomic groups with available sequence information relative to the human protein. Percent similarity of the protein amino acid sequence is displayed on the Y-axis; taxonomic group is displayed on the X-axis. Open circles (○) indicate the query sequence, and closed circles (●) indicate the species within the taxonomic group with the highest percent similarity. Within the plot, the top and bottom of each box represent the 75th and 25th percentiles, the whiskers extend to 1.5 times the interquartile range, and the mean and median values are represented by horizontal black lines on the box. The dashed line indicates the cutoff for susceptibility predictions. Abbreviations: MOR = mu-opioid receptor; SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility. Please click here to view a larger version of this figure.

Figure 8: SeqAPASS Level 2 analysis of the µ opioid receptor ligand-binding domain conservation relative to the domain in the human protein. Percent similarity of the ligand-binding domain amino acid sequence is displayed on the Y-axis; taxonomic group is displayed on the X-axis. Open circles (○) indicate the query sequence, and closed circles (●) indicate the species within the taxonomic group with the highest percent similarity. Within the plot, the top and bottom of each box represent the 75th and 25th percentiles, the whiskers extend to 1.5 times the interquartile range, and the mean and median values are represented by horizontal black lines on the box. The dashed line indicates the cutoff for susceptibility predictions. Abbreviations: MOR = mu-opioid receptor; SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility. Please click here to view a larger version of this figure.

Through analysis of molecular modeling and protein crystallography studies, amino acids were identified in the binding region of MOR that are predicted to interact with known ligands. Although the diverse set of ligands that bind well to opioid receptors results in complex pharmacology, some consistent ligand-protein interactions are observed^36,37. Based on molecular docking to various MOR crystal structures, both morphine and fentanyl, high-affinity MOR agonists, interact with D147, Y148, M151, W293, I296, H297, V300, I322, and Y326^36,38. Residues D147, Y148, M151, and H297 are also implicated in crystal structures of MOR bound to the morphine agonist BU72, while D147, M151, H297, and Y326 are also critical in binding to the irreversible morphine antagonist β-funaltrexamine³⁷. Considering these lines of evidence, nine residues were selected (D147, Y148, M151, W293, I296, H297, V300, I322, Y326) for Level 3 evaluation using human MOR as a template sequence and excluding partial, predicted, hypothetical, and low-quality sequences. Importantly, amino acid positions reported in the literature exclude a 64-amino-acid segment relative to the NCBI protein accession and, for this reason, positions in Level 3 were selected to represent those that aligned with the template sequence for correct alignment sequences.

In the Level 3 analysis of the human MOR, 284 species were assessed across vertebrate species (mammals, birds, amphibians, reptiles, and fish). Across all species evaluated, the nine amino acids were either a total match or a partial match based on side-chain classification and molecular weight; consequently, all species assessed resulted in a susceptibility prediction of "Y" for yes (Table 3 and Supplemental File 1). As these amino acids are important in the binding of both strong MOR agonists and strong antagonists, these data suggest that opioid compounds targeting human µ opioid receptors may interact similarly with receptors across vertebrate species. Although there is little empirical data available to date within the ECOTOX knowledgebase for opioid compounds, several studies suggest that fish are likely susceptible^31,32,33. Overall, results from SeqAPASS point to the potential for broader environmental impacts of MOR-modulating chemicals across species, indicating that more research and perhaps monitoring may be valuable. Data for all analyses are available in Supplemental File 1.

Table 3: SeqAPASS Level 3 analysis of the conservation of amino acid residues important for chemical binding to the µ opioid receptor. Summary table displaying the number of species with available sequence data across all taxonomic groups, the number of species predicted to be similarly susceptible (Y), and the number of species predicted to not be similarly susceptible, as well as full, partial, and non-matching amino acids. Abbreviation: SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility. Please click here to download this Table.

Supplemental Table S1: Species with available ECOTOX data for the four chemicals of interest known to bind to the human transthyretin protein. Data available for each chemical is aligned with SeqAPASS predictions of similar Susceptibility across levels 1, 2, and 3. All SeqAPASS predictions relative to the human transthyretin sequence. Abbreviations: SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility; DES = diethylstilbestrol; PFHxA = perfluorohexanoic acid; PFOS = perfluorooctane sulfonic acid; TBBPA = tetrabromobisphenol A. Please click here to download this Table.

Supplemental Table S2: Total number of species with available data across SeqAPASS and ECOTOX Evaluations for select chemicals known to bind to the human transthyretin protein. SeqAPASS provides means for predicting species susceptibility across many species for which empirical toxicity data is unavailable. Please click here to download this Table.

Supplemental File 1: SeqAPASS and ECOTOX data for all representative results. File contains ToC followed by the following data sheets: Tab 1-hTTR SeqAPASS Results Level 1, Tab 2-hTTR SeqAPASS Results Level 2, Tab 3-hTTR SeqAPASS Results Level 3, Tab 4-EcoTox data for DES, Tab 5-EcoTox data for PFOS, Tab 6-EcoTox data for PFHxA, Tab 7-EcoTox data for TBBPA, Tab 8-EcoTox Group Mean Calculations, Tab 9-SeqAPASS EcoTox Data Comparisons, Tab 10-hMOR SeqAPASS Results Level 1, Tab 11-hMOR SeqAPASS Results Level 2, and Tab 12-µ-hMOR SeqAPASS Results Level 3. Abbreviations: SeqAPASS = Sequence Alignment to Predict Across Species Susceptibility; ToC = Table of Contents; hTTR = human transthyretin; ECOTOX = Ecotoxicology knowledgebase; DES = diethylstilbestrol; PFOS = perfluorooctane sulfonic acid; PFHxA = perfluorohexanoic acid; TBBPA = tetrabromobisphenol A; hMOR = human mu-opioid receptor. Please click here to download this File.