Zebrafish s100s are distantly related to human S100A8 and S100A9

We started by looking for phylogenetic evidence that fish have a protein orthologous to mammalian S100A8 or S100A9. Orthologous proteins arise by speciation and are thus the same gene in the species being compared. In contrast, paralogous proteins arise by gene duplication and often exhibit gain or loss of function from the ancestral state [35], establishing themselves as new proteins. Fig 1A summarizes the evolutionary history of S100s. This tree was built referencing several published phylogenetic analyses of the family, including two from our group [33,36–38]. The phylogeny at the top shows the current best estimate of the S100 gene tree; the phylogeny on the left shows the evolutionary history of bony vertebrates. Each circle denotes the S100 gene observed in at least one member of the taxonomic group on the left.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Phylogenetic analyses reveal there is no calprotectin ortholog outside of amniotes.

(A) S100 gene tree adapted from Wheeler et al., 2016 shows the evolutionary relationships determined for S100s across vertebrates. The phylogeny on the left shows the relationships between species, with branch point times noted in millions of years ago; the phylogeny on the top shows the estimated S100 gene tree. Circles denote S100 genes from the phylogeny at the top found in at least one member of the taxonomic group from the left. A horizontal line through a circle indicates a single gene that is co-orthologous to multiple S100 genes found in mammals. S100A8, S100A9, and S100A12 form a clade specific to amniotes (orange). Zebrafish genes in the syntenic region shown in panel B are shown in blue. Zebrafish s100a10b, which has been treated as a calprotectin ortholog, is denoted with a star. (B) Syntenic regions of human chromosome 1:151,982,915–154,870,281 (top) and zebrafish chromosome 16:22,682,280–29,387,215 (bottom) identified by ENSEMBL. Arrows denote relative gene length and orientation. Human S100A8, S100A9, and S100A12 are shown in orange; zebrafish s100s and their human orthologs are shown in blue. The non-S100 genes adar/ADAR and KCNN3 are diagnostic for the syntenic region. Not all genes in the region are depicted. (C) A multiple sequence alignment of human S100A8, S100A9 and S100A10 compared to the zebrafish s100s from a similar genomic context. At the top, secondary structure features of human S100A8 are shown. Under this, the bar is colored by amino acid conservation from the alignment (blue = low, red = high). The consensus sequence from all sequences in the alignment is shown above the individual protein sequences. Amino acids found in a particular position in at least 50% of the sequences shown are shaded. The antigen for the “Fish Calprotectin” antibody was raised against the peptide boxed in yellow. (D) A pairwise percent identity matrix of S100 protein sequences from C (darker box indicates higher identity; S1 Table). S100 pair identity values for zebrafish s100s compared to human S100A8 range from 21.4–34.4%, where both s100a10b and s100t score 34.4%. Values comparing zebrafish proteins to S100A9 range from 23.8–39.6% with the highest identity shared with s100a1.

https://doi.org/10.1371/journal.pone.0322649.g001

This evolutionary tree indicates that S100A8 and S100A9 evolved by gene duplication from a single gene in the ancestor of amniotes (shown in orange). Reptiles and birds preserve a single gene (MRP-126), while mammals expanded it into three proteins (S100A8, S100A9 and S100A12). The reptile/bird protein MRP-126 is the earliest diverging protein known to exhibit nutritional immunity and/or Toll-like receptor 4 activation in functional assays [37,39]. These observations indicate that calprotectin evolved in amniotes ~320 million years ago.

The closest evolutionary relatives of S100A8 and S100A9 arose from duplications of a tetrapod-specific S100 gene. In contrast, zebrafish s100a10b (the putative zebrafish calprotectin) is a member of one of the earliest S100 gene subfamilies to evolve, with orthologs present in jawless fishes and the co-orthologous genes S100A10 and S100A11 in tetrapods. S100a10b thus diverged from the lineage that led to mammalian S100A8 and S100A9 at least 563 million years ago, in the last common ancestor of humans and lampreys. Further, after this speciation event, there were at least two more gene duplications on the lineage leading to S100A8 and S100A9. S100a10b is therefore a different gene than S100A8 or S100A9.

Chromosome placement indicates a shared origin but complicated evolution of S100s in humans and zebrafish

To cross-validate the lack of evidence for vertical inheritance from published phylogenies, we used syntenic analysis to identify zebrafish s100 genes in a similar genomic location to human S100A8 and S100A9. We used ENSEMBL to identify the zebrafish genomic region most similar to human chromosome Chr 1:152-155M, which encodes 19 of the 24 human S100s, including S100A8 and S100A9. This region corresponded to zebrafish chromosome 16 (S1 Fig). Specifically, human Chr 1:154.6M-154.7M and zebrafish Chr 16:23.5M-23.7M cover the KCNN3 and ADAR genes adjacent to tandem repeats of S100 genes in both species (Fig 1B).

The existence of this shared cluster indicates that a handful of S100 genes were in this genomic context at least in the bony vertebrate ancestor ~430 million years ago, as established in previous work [33]. The syntenic relationships, however, also give evidence for extensive evolution after the divergence of bony fishes and tetrapods: the orientation and placement of genes are different, and several orthologs to human S100s are missing from this genomic location but present on other zebrafish chromosomes. Further, most of the zebrafish s100s in this region appear to be teleost-specific duplicates [33]. This includes ictacalcin (icn), icn2, s100t, s100s, and s100w. The only clear orthologs to human genes are zebrafish s100a10b, co-orthologous to both human S100A10 and S100A11, s100v1, co-orthologous to both human S100A13 and S100A14, and s100a1, orthologous to human S100A1 (Fig 1A and B).

Human calprotectin and zebrafish s100 protein sequences share low sequence identity

Previous work identified zebrafish s100a10b as calprotectin using human S100A8 as a query in a BLASTP search against the zebrafish proteome [30]. In line with their results, we found that using human S100A8 as a BLASTP query against the NCBI RefSeq database (v. 224) yielded s100a10b as the top zebrafish hit (e-value: 8E-17, percent identity: 36.4%). The zebrafish icn2 and icn proteins were the next most similar hits. We assessed the quality of the hit by reciprocal BLAST, meaning we used the zebrafish s100a10b protein sequence as a query against the human proteome on NCBI. This yielded human S100A1 (9E-34; 55.9%) as the top hit, not S100A8. In fact, S100A8 (5E-16; 36.4%) and S100A9 (4E-18; 42.5%) were far down the hit list, following human proteins S100A10, S100Z, S100P, S100A11, S100B, S100A4, S100A12, S100A5, S100A6, and S100A2. This is consistent with previous phylogenetic analyses that place S100A8 and S100A9 as distant paralogs to zebrafish s100a10b (Fig 1A).

To evaluate sequence similarity and identity, we aligned zebrafish s100 protein sequences from the syntenic region to human S100A8, S100A9, and S100A10 sequences (Fig 1C). As expected, there is high conservation at sites that form the EF-hand and pseudo-EF-hand calcium-binding domains of the S100 proteins. There is low conservation in the region connecting the two EF-hands and at the termini. We next determined the sequence identity shared between zebrafish s100s and human S100A8, S100A9, and S100A10 (Fig 1D; S1 Table). We find similarly low levels of shared identity between human S100A8 and S100A9 and the zebrafish s100s. Overall, there is no obvious candidate zebrafish s100 that is like calprotectin by sequence similarity or identity.

The sequence alignment also allowed us to ask what zebrafish s100 protein(s) might be recognized by the commercially available “Fish Calprotectin” ELISA Kit from MyBioSource. This kit was made with antibodies raised against a 20 amino acid partial peptide of a human calprotectin-like protein (GenBank: AAB33355.1), which forms the N-terminal helix and beginning of the EF-hand 1 domain of human S100A8 (Fig 1C, yellow box). Eight to nine residues in this helix are highly conserved in several zebrafish s100s including s100b, s100a10b, s100a10a, s100a1, s100z, s100s and s100w as well as several other unrelated proteins. This suggests the antibody may have broad, non-specific interactions with multiple zebrafish proteins.

Could a calprotectin paralog have convergently evolved similar immune functions?

We hypothesized that, although there is not a direct calprotectin ortholog in zebrafish, there could be a paralogous s100 that convergently evolved immune functions like calprotectin in zebrafish and other teleost fish. Like the analogous evolution of wings in bats and birds, the emergence of similar immune functions in two different s100 genes would support that divergent species experienced similar environmental pressures and evolved parallel but distinct solutions to this pressure. Zebrafish share six s100 orthologs with mammals as well as eight s100 genes unique to teleost fish [33] (Fig 1A). In the following sections, we will address the possibility that a s100 protein distantly related to calprotectin could have convergently evolved a calprotectin-like role in the zebrafish immune response.

Single cell RNA sequencing dataset analyses identify zebrafish s100s upregulated in immune cells during an immune response

Our bioinformatic analyses revealed zebrafish do not have a calprotectin ortholog; however, it is possible that a different s100 protein convergently evolved calprotectin-like activity. To investigate this possibility, we identified zebrafish s100s that share a similar expression profile to calprotectin. Calprotectin is expressed constitutively in mammalian neutrophils, monocytes, and several epithelial cell types and is upregulated upon infection and injury [40,41]. We queried existing zebrafish single cell RNA sequencing (scRNAseq) datasets for zebrafish s100 genes expressed in immune cells and upregulated in response to injury.

We used the UCSC cell browser to visualize a zebrafish development dataset (NCBI Bioproject: 564810) [42,43] and assessed immune cell expression of s100 genes in whole fish 1-, 2-, and 5-days post-fertilization (Table 1; S2 Fig). Of the genes that share genomic context with S100A8 and S100A9 (Fig 1B), we found that s100a10b, icn, icn2, and s100a1 are expressed in immune cells of developing zebrafish. S100w, s100t, and s100v1, also in this genomic region, show very low expression in immune cell clusters. Four s100 genes from other zebrafish chromosome locations show some expression in immune cells: s100v2, s100u, s100z, and s100a11. Finally, the remaining annotated zebrafish s100 genes—s100s, s100b, and s100a10a—appear in very few immune cells at the 1-, 2-, and 5-day stage of development.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Single-cell RNAseq profiles for zebrafish s100s syntenic to human calprotectin.

https://doi.org/10.1371/journal.pone.0322649.t001

To evaluate whether expression of these genes are upregulated during the innate immune response to injury, we explored a scRNAseq dataset tracking local responses after tail fin amputation (NCBI GEO accession number GSE137971) [44]. Cells were isolated from several 1-year-old (adult) zebrafish caudal fins at the time of amputation (pre-injury) and at 1-, 2-, and 4-days post-amputation (dpa) from the same position.

In this dataset, the macrophage marker mpeg1.1 is expressed above background in a subset of each cell cluster described in the dataset (epithelial, mesenchymal, and hematopoietic) and is highly expressed in the hematopoietic cluster. In response to injury, mpeg1.1 expression slightly decreases at 1dpa, but then surpasses pre-injury levels in days 2 and 4 (S3 Fig). In contrast, neutrophil marker mpx is not detected above background in the hematopoietic cluster; it is only upregulated in a small number of basal epithelial and mesenchymal cells of regenerating fins 4dpa (S3 Fig). Lyz, another neutrophil marker, is found to be highly expressed in a single hematopoietic cell pre-injury but is not detected above baseline post-amputation. These data suggest injury by fin clip does not induce a strong transcriptional response by neutrophils at the site of amputation.

With the exception of s100t and s100v1, zebrafish s100 genes that share genomic context with S100A8 and S100A9 were enriched in hematopoietic cell populations, with s100a1 to a lesser degree. S100a10b and s100a1 exhibit immediate and persistent upregulation after injury, whereas icn, icn2, and s100w initially drop in expression and then return roughly to pre-injury levels by 4dpa, similar to mpeg1.1 (Table 1; S3 Fig). Zebrafish s100 genes from other chromosomal loci show varying levels of expression in hematopoietic cells and in the injury response (S3 Fig). Combining the synteny and scRNAseq analyses, we determined that zebrafish s100a10b, s100a1, s100w, and icn were promising candidates to begin functionally comparing to calprotectin, though this is only a subset of immune-related zebrafish s100 genes.

Recombinant zebrafish s100 proteins fold and interact with calcium

We chose to functionally characterize the zebrafish s100 proteins which seemed most promising to behave like calprotectin based on genomic context and gene expression profiles: s100a10b, s100a1, s100w and icn. We left out icn2 because it is very similar to icn by all metrics including sequence identity (87.37%: they differ by 14 amino acids, 8 of these at the termini; Fig 1). The protein sequences and plasmids used for recombinant expression are shown in S2 Table.

We started by structurally characterizing the four selected zebrafish proteins. We used AlphaFold2 to predict structures for all four proteins [45–47]. Overlaying the predicted structures with the crystal structure of human calprotectin (RCSB ID 5W1F) shows high predicted structural similarity (Fig 2A). High α-helical content is a shared feature of all known S100 proteins, as well as the predicted zebrafish s100 structures. We tested whether this held for our selected zebrafish s100s recombinantly expressed and purified from Escherichia coli (E. coli). We measured their secondary structure content by far-UV circular dichroism (CD). This revealed signal minima at 208 and 222 nm consistent with primarily α helical structures (Fig 2B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Recombinantly expressed zebrafish s100 proteins are folded and respond to calcium.

(A) Overlaid AlphaFold2 structure predictions for all zebrafish s100 homodimers in this dataset, as well as the human S100A8/S100A9 heterodimer (5W1F RCSB ID). Different chains of each homodimer are shown in black or white. (B) Far UV circular dichroism spectra for each protein in the presence of 2 mM Ca⁺⁺ (orange) and then adding 5mM EDTA (blue). Units are in molar ellipticity (deg × cm²/dmol) over wavelength (nm). (C) Fluorescence excitation and emission spectra for each protein in the presence or absence of calcium (orange and blue, respectively). The fluorescence units are arbitrary; the x-axis is wavelength in nanometers. Excitation spectra were collected while observing fluorescence at the maximum emission wavelength; emission spectra were collected while exciting at the maximum excitation wavelength.

https://doi.org/10.1371/journal.pone.0322649.g002

Most S100 proteins bind calcium and undergo a conformational change exposing a hydrophobic binding surface [48,49]. We tested whether this held for the four zebrafish s100 proteins by measuring calcium-induced changes in protein secondary and tertiary structure by far-UV CD and intrinsic fluorescence, respectively. We found that all four recombinantly expressed zebrafish proteins exhibited evidence of calcium-induced conformational change (Fig 2B and C).

Upon addition of saturating calcium (orange), zebrafish s100a10b and s100a1 exhibited an increase in helical content, while s100w and icn, in contrast, show little change in secondary structure (Fig 2B). The intrinsic fluorescence of all four proteins, however, responded to calcium (Fig 2C). Intrinsic fluorescence captures changes in the local chemical environments of tyrosine and tryptophan residues, suggesting that calcium binding induces a change in the tertiary structure of all measured s100 proteins. This is consistent with the canonical calcium-induced rotation of the third helix relative to the other helices of S100s [48].

Taken together, these results show that these four zebrafish s100 proteins are folded, bind to calcium, and undergo the calcium-induced conformational changes expected for members of the family.

Zebrafish s100s do not exhibit nutritional immunity characteristics like human calprotectin

One of the most important biological functions of human calprotectin is antimicrobial activity via nutritional immunity: calprotectin binds to and sequesters transition metals, thus inhibiting bacterial growth. We evaluated the antimicrobial abilities of each of the four zebrafish s100 proteins against human-derived Stapholococcus epidermidis and zebrafish-derived Vibrio ZWU0020 and Aeromonas ZOR001 strains. S. epidermidis was previously shown to be susceptible to human calprotectin [10,50]; the response of the zebrafish-derived strains is unknown, however, both strains elicit immune responses in zebrafish [51]. Fig 3A shows the dose-dependent antimicrobial activity of human calprotectin against each strain over 13 hours in nutrient rich media across three biological replicates. For all three strains, increasing amounts of calprotectin (from blue to green) leads to decreased final OD₆₀₀ values (indicated by black arrows), confirming that the previously uncharacterized zebrafish commensal bacteria are susceptible to calprotectin’s metal sequestration. A positive control for the effects of metal chelation on each strain by EDTA is included in Supplemental S4 Fig.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Zebrafish s100s do not exhibit nutritional immunity activity like human calprotectin.

(A) Dose dependence of human calprotectin challenge on human and zebrafish commensal bacteria. Each column specifies which bacteria was used for the set of nutritional immunity assays: human-derived Gram-positive Stapholococcus epidermidis and two zebrafish-derived Gram-negative bacteria, Aeromonas strain: ZOR001 and Vibrio strain: ZWU0020. Bacterial growth was measured by OD₆₀₀ over 13 hours with challenge by increasing doses of human calprotectin noted in the legend on the right. Concentration increases from dark blue to dark green and black arrows indicate how growth is affected as calprotectin increases. Error bars indicate standard error of three biological replicates. (B) Zebrafish s100 dose effects on human and zebrafish commensal bacterial growth compared to human calprotectin. Each datapoint shows the change in the area under the curve from the absence of s100 protein to the indicated s100 concentration, measured from growth curves like those shown in panel A (S4 Fig). The dotted line at zero represents no effect on bacterial growth.

https://doi.org/10.1371/journal.pone.0322649.g003

Each bacterial strain exhibited unique growth phases and phase-dependent responses to calprotectin (Fig 3A). We wanted to evaluate the overall nutritional immunity effect of calprotectin across all phases of growth for each strain, rather than comparing OD₆₀₀ at snapshots in time. To do this, we first calculated the area under the curve (AUC) for all OD₆₀₀ curves from 0–13 hrs. A stronger antimicrobial effect results in a smaller AUC. We quantified the consequence of increasing the calprotectin challenge on overall bacterial growth by calculating the difference in the AUC value for growths at each concentration of calprotectin against the unchallenged control growth (ΔAUC). We then generated dose-response curves for each strain by plotting ΔAUC as a function of protein concentration. A negative ΔAUC value indicates growth inhibition, while a zero or positive value indicates no antimicrobial activity. The results showed that increasing the calprotectin dose increases overall growth inhibition of all three bacterial strains (Fig 3B, yellow curves). This reproduces what the literature already knows about the S. epidermidis response to calprotectin and reveals that zebrafish-derived Aeromonas ZOR001 and Vibrio ZWU0020 are susceptible to nutritional immunity by calprotectin’s metal chelation properties.

To assess the nutritional immunity capacity of the zebrafish s100s, we performed identical experiments and analyses. The OD₆₀₀ dose response curves for zebrafish s100s are show in S4 Fig. We calculated ΔAUC curves for each bacterial strain under increasing concentrations of each s100 (Fig 3B). Unlike the effect of human calprotectin, none of the four zebrafish s100 proteins exhibited nutritional immunity (Fig 3B; S4 Fig). Human calprotectin was the only protein to exhibit nutritional immunity under these conditions (Fig 3B, yellow curve). Bacteria treated with zebrafish s100a1 showed improved growth relative to bacteria in the absence of s100 (Fig 3B, brown curve). Zebrafish s100a10b increased growth of S. epidermidis and had no effect on growth of zebrafish-derived bacterial strains (Fig 3B, orange). Zebrafish s100w (purple) improved S. epidermidis growth, showed a possible slight inhibitory effect on Aeromonas ZOR001 for concentrations at or above 50 μM, and did not affect Vibrio ZWU0020 growth at any concentration. Similarly, zebrafish icn (blue) improved S. epidermidis growth. We performed an ANOVA analysis on the antimicrobial activity at 50 μM protein for every condition and found that human calprotectin had a statistically different effect than any of the zebrafish s100 proteins (p < 0.0039), but that the zebrafish proteins could not be distinguished from one another (S3 Table).

Zebrafish s100s do not exhibit pro-inflammatory activity like S100A9

Antimicrobial activity is not the only function of mammalian calprotectin. Extracellular calprotectin is thought to act as a cytokine, activating immune receptors like the Receptor for Advanced Glycation Endproducts (RAGE) and Toll-like receptor 4 (TLR4) [20]. However, RAGE evolved in mammals from a family of cell adhesion molecules [52], so is not biologically relevant to zebrafish s100 function. TLR4 is an ancient receptor that arose in the ancestor of all bony vertebrates [53,54]. The S100A9 component of calprotectin has been demonstrated to drive TLR4 activation, inducing nuclear localization of NF-κB and transcription of a wide variety of pro-inflammatory proteins [55,56]. This activity can be reproduced in an in vitro functional assay by transfecting human embryonic kidney-293T (HEK293T) cells with plasmids encoding the proteins of the TLR4 complex (TLR4, MD-2, and CD14), as well as a plasmid placing luciferase behind an NF-κB promoter. We treat the cells with exogenous S100A9 and measure the expression and activity of luciferase produced in response [37].

We wanted to see if our purified zebrafish s100 proteins could play a similar pro-inflammatory role; therefore, we tested the ability of these proteins to activate TLR4 in this assay (Fig 4). Zebrafish have three ohnologs of tetrapod TLR4: tlr4ba, tlr4bb, and tlr4al [53,54]. Zebrafish Tlr4ba has been shown to induce inflammation in response to endotoxin, the small molecule lipopolysaccharide (LPS) derived from Gram-negative bacterial outer membranes, but neither Tlr4bb nor Tlr4al showed activity [53].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Zebrafish s100s do not exhibit the pro-inflammatory characteristics of S100A9.

The bar graph shows relative activation of human and zebrafish TLR4 complexes in the presence of 1 μM zebrafish s100 proteins. The positive controls for this experiment included human TLR4 and zebrafish Tlr4ba treated with endotoxin (blue, outlined), and human TLR4 treated with 1 μM human S100A9 (light blue, outlined). There is no known agonist for zebrafish Tlr4bb and Tlr4al complexes. The negative control was vehicle treatment with 200 ng/uL Polymyxin B (PB) in PBS. All data was background subtracted and normalized to the signal from human TLR4 treated with endotoxin. Bars show the average signal across three biological replicates, with error bars indicating standard error. Statistics shown are p-values <0.05 from unpaired Welch t-tests.

https://doi.org/10.1371/journal.pone.0322649.g004

We validated our assay by testing the ability of each complex to activate in response to endotoxin, the canonical agonist for the receptor (blue, outlined). As expected, the human TLR4 and zebrafish Tlr4ba complexes responded strongly to endotoxin. As observed previously [53], Tlr4bb and Tlr4al did not show signal above vehicle treatment (light blue). We next challenged all four complexes with human S100A9 (light blue, outlined) and calprotectin (yellow). We show that although pure calprotectin is not pro-inflammatory in our assay, 1 μM human S100A9 potently activates human TLR4, as expected, but none of the zebrafish Tlr4 complexes. This suggests that sterile inflammation via a mechanism similar to S100A9 activation of TLR4 is not a conserved function in zebrafish.

We next tested the ability of zebrafish s100s to activate each TLR4 complex at 1 μM. We observed no statistically significant agonist activity for any protein against any zebrafish Tlr4. We detected a small amount of signal for treatment with zebrafish s100a10b (orange) and zebrafish s100w (purple) against human TLR4; however, this was narrowly above background and much lower than the response for either endotoxin or human S100A9. We repeated the same experiment with 10 μM zebrafish s100s and observed no convincing agonist activity (S5 Fig).

These zebrafish s100 proteins show no evidence of activity against human TLR4 nor the three zebrafish Tlr4 ohnologs. Given the potent response of human TLR4 and zebrafish Tlr4ba to positive controls (human S100A9 and/or endotoxin), and that false positives are common in this assay due to endotoxin contamination from recombinant protein expression in E. coli, the lack of signal thus gives strong evidence that these zebrafish s100s cannot activate TLR4 in the same fashion as human S100A9. This does not rule out the possibility that they might activate a yet uncharacterized zebrafish immune receptor, but if such a receptor cannot be activated by calprotectin, S100A8, or S100A9 then this activity would also not be characterized as convergent evolution.

Structure predictions do not support that convergent evolution of a calprotectin-like metal chelation site occurred in zebrafish s100s

We next wanted to extend our analysis beyond the four zebrafish s100 homodimers we experimentally characterized. There are ten additional zebrafish s100 genes in the genome. These may form homodimers, heterodimers, or even high-order complexes that play functional roles in fish physiology and innate immunity. Experimentally measuring the functional properties of all 196 possible zebrafish s100 homodimers and heterodimers would, however, be a massive undertaking. We therefore took a computational approach.

We chose to structurally assess all zebrafish s100 homo- and hetero-dimers for possible convergently evolved antimicrobial activity by looking for calprotectin-like hexa-histidine sites. Because the structural basis by which S100A9 activates TLR4 remains unknown, we only assessed predicted antimicrobial function.

The crystal structure of human calprotectin bound to Zn²⁺ (PDB: 8SJC) illustrates that the metal-chelating hexa-histidine site is constructed from two residues from human S100A8 (H17 and H27) and four residues from human S100A9 (H87, H91, H99 and H101) (Fig 5A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Predicted structures of zebrafish s100 homodimers and heterodimers.

(A) A close-up of calprotectin’s (gray) hexa-histidine site coordinating a zinc ion (purple sphere) via polar contacts (dashed yellow lines). (B) A view of the calprotectin heterodimer from crystal structure PDB: 8JSC. Calcium ions are represented as green spheres. (C and D) are overlay views of AlphaFold3 model structure predictions of homodimers and heterodimers, respectively, aligned to the calprotectin heterodimer. The first chain of each model dimer is in blue, and the second chain is in green. This excludes the s100t homodimer (S7 Fig). (E and F) Histograms showing the number of predicted homodimer (E) and heterodimer (F) models that have a certain number of histidine alpha carbons within 9 Å of the calprotectin Zn²⁺ ion. Arrows indicate that human calprotectin has seven histidines within 9 Å of the Zn²⁺ ion.

https://doi.org/10.1371/journal.pone.0322649.g005

For our computational analysis, we used AlphaFold3 [57] to make five model predictions for each zebrafish homo- and hetero-dimer in the presence of four calcium ions and one zinc ion. We used the full-length sequence for all zebrafish proteins, except for s100u, for which we used only residues 1–89. (Positions 90–240 of s100u extend beyond the calprotectin dimer alignment, are predicted to be disordered, and have no histidine residues that could chelate metals as part of a hexa-histidine site.) After generating our structural models for all 196 possible zebrafish s100 homo- and heterodimers, we aligned them to the crystal structure of Zn²⁺ bound calprotectin dimer (PDB: 8SJC; chains A and C). All predicted dimers, except for the s100t homodimer, aligned well with calprotectin. (In the presence of zinc, s100t is predicted to form a unique S100 dimer fold which may be interesting to explore in the future (S7 Fig)).

We next quantified the number of histidine residues per predicted model within 9 Å of the calprotectin-bound Zn²⁺ ion (Fig 5). This is a generous cutoff that would allow for conformational rearrangements to bring histidine residues into proximity with the ion binding site. For reference, the human calprotectin structure has seven histidine residues within 9 Å of its bound Zn²⁺ ion.

Across our five models for each of the 196 homo- and heterodimers, we observed no more than three histidine residues within 9 Å of the calprotectin Zn²⁺ ion (Fig 5E-F). 87% of the structural models had zero or one histidine within this cutoff; the remaining 13% had two or three histidine residues. All the proteins we functionally characterized in the nutritional immunity assay—s100a1, s100a10b, s100w, and icn—are predicted to have a single histidine near the calprotectin hexa-histidine site. This is consistent with their lack of observed antimicrobial activity.

Our computational analysis suggests that none of the zebrafish homo- or hetero-dimer s100 complexes convergently evolved calprotectin’s hallmark high-affinity metal chelating hexa-histidine site. While this finding does not exclude antimicrobial activity via some other mechanism, this would be entirely convergent, and thus, a separate evolutionary function than what is seen in mammalian calprotectin.

Protein sequence analysis

In our BLAST and reciprocal BLAST study, we used the NCBI web interface and BLASTP with default parameters to query the NCBI RefSeq database (v. 244). We aligned and calculated S100 sequence identity using Clustal Omega [62]. We viewed and generated figures displaying the alignment and consensus sequence in SnapGene Viewer 5.2.4.

Single-cell RNA sequencing datasets

We used interactive data visualization platforms for existing zebrafish single cell RNA sequencing (scRNAseq) datasets to look for zebrafish s100 genes expressed in immune cells and upregulated in response to injury (Table 1). We used the UCSC cell browser to visualize a zebrafish development dataset (NCBI Bioproject: 564810) [42,43] which sampled whole fish 1-, 2-, and 5-days post-fertilization (S2 Fig). We then used a web platform built by the authors, Hou et al., (https://k326xh.shinyapps.io/FinRegenerationSCRNA/) to access their fin amputation and regeneration dataset (NCBI GEO accession number GSE137971) [44] (S3 Fig). Cells were isolated from the same position in the caudal fin of several 1-year old (adult) zebrafish at the time of amputation (pre-injury) and at 1-, 2-, and 4-days post-amputation (dpa). Differential gene expression for both datasets was determined by comparing the gene expression profile of each cell with those of the rest of the cells in the dataset using a Wilcoxon rank sum test in Seurat.

Protein purification

We purchased all zebrafish s100 genes from GenScript in the pET-28a(+)-TEV vector with an N-terminal 6x-Histidine tag and TEV protease cleavage site (S2 Table). All genes were codon-optimized for expression in E. coli. We expressed human calprotectin (with S100A8 containing the C42S mutation and S100A9 containing the C3S mutation) and human S100A9/C3S in a pET-Duet vector without purification tags. We transformed Rosetta2(DE3)pLysS E. coli cells with plasmids. We used transformant glycerol stocks to inoculate cultures in 15 mL Luria broth (LB) with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol. We incubated cultures overnight at 37 °C, shaking at 250 rpm. The following day, we diluted 15 mL saturated cultures into 1.5 L of LB with antibiotics. When the OD₆₀₀ reached 0.6–1.0, we induced recombinant protein expression with 1 mM IPTG and 0.2% glucose and then grew overnight at 16 °C, shaking at 250 rpm. We pelleted cells at 3,000 rpm for at least 15 minutes in an F6B rotor in a Beckman Coulter preparative centrifuge. We stored pellets at -20 °C for up to one month.

We prepared protein lysates for purification with the following method: We vortexed pellets (6–9 g) in 45 mL buffer from the first chromatography step (see below) until cells were resuspended, added 15 µ L each of DNase I and Lysozyme (ThermoFisher Scientific), and incubated at room temperature with gentle shaking for at least 10 minutes. We lysed the cells by sonication at 55% amplitude with 0.3 second pulse on, 0.7 second pulse off, for 3–5 minutes. We pelleted cell debris by centrifugation at 15,000 rpm at 4 °C for at least 20 minutes in a JA-20 rotor in a Beckman Coulter preparative centrifuge and collected the supernatant. To remove remaining large debris, we filtered lysate supernatant through a 0.2 µm pore syringe filter immediately prior to purification chromatography.

We purified all proteins using an Äkta PrimePlus Fast Protein Liquid Chromatography system using two stacked 5 mL HiTrap columns at each step. We used HisTrap FF columns for Ni-affinity and Q HP columns for anion exchange (GE Health Science). All chromatography was performed at 4 °C. At the end of purification, we confirmed protein purity was > 95% by SDS-PAGE (S6 Fig). Then, we dialyzed each protein overnight into 4 L of 25 mM Tris, 100 mM NaCl, pH 7.4 at 4 °C. We placed 2 g/L Chelex 100 resin (Bio-Rad) in the dialysis buffer to chelate divalent metal ions. We concentrated each protein to roughly 2 mg/mL and syringe-filtered through a 0.22 µm filter directly into liquid nitrogen to sterilize and flash freeze before storing at -80 °C.

We purified TEV-cleavable 6xHis-tagged zebrafish s100 proteins with the following scheme. We used 25 mM Tris, 100 mM NaCl, pH 7.4 buffer as the base for all chromatography buffers. We ran our protein lysate over a Ni-affinity column with a 50 mL wash and eluted over a 75 mL gradient from 25–1000 mM imidazole to collect proteins with strong Ni binding capacity. We determined which fractions contained our desired protein by SDS-PAGE and pooled these fractions. To separate our recombinant proteins from their Ni-binding His-tag, we added 5 mM DTT and 6xHis-tagged TEV protease to the pooled fractions and incubated the reaction at room temperature with gentle shaking for at least 5 hours. We then dialyzed the protein solution overnight into 4 L buffer with 25 mM imidazole and 5 mM DTT to allow cleavage to come to completion and to remove excess imidazole from the sample. We performed a second round of Ni-affinity chromatography. Without the His-tag, the zebrafish s100s have low affinity for Ni. Therefore, we isolated pure, non-tagged zebrafish s100 proteins at this step during a 50 mL wash in 25 mM imidazole and then used a step gradient to 1 M imidazole to elute His-tagged and other contaminant proteins that had higher affinity for the Ni column. Purified zebrafish s100s were prepared for storage as described above.

We purified human calprotectin using Ni-affinity chromatography at pH 7.4 and anion exchange at pH 8. When expressing calprotectin, S100A8 and S100A9 homodimers are also expressed and must be removed during chromatography. In the presence of calcium, S100A9 and calprotectin bind divalent metal ions like Ni, but S100A8 and most other lysate proteins do not. We loaded our Ni-affinity column with calprotectin lysate, washed away most A8 and contaminants in a 50 mL wash, and then eluted calprotectin and S100A9 over a 75 mL gradient from 0–1000 mM imidazole and 1–0 mM CaCl₂ in 25 mM Tris, 100 mM NaCl, pH 7.4 buffer. We pooled elution peak fractions containing calprotectin and contaminant S100A9 homodimers, as determined by SDS-PAGE, and dialyzed overnight in 4 L of 25 mM Tris, 100 mM NaCl at pH 8. We loaded our sample onto an anion exchange chromatography column in 25 mM Tris, 100 mM NaCl at pH 8 with 100 mM NaCl. Because S100A9 has a lower pI than calprotectin, it binds the anion column more strongly at pH 8. We used a 50 mL wash in 100 mM NaCl to isolate calprotectin and then used a step gradient increasing the salt to 1 M NaCl to remove S100A9 and other contaminants from the column. At this point, calprotectin was pure and prepared for storage as described, and fractions with S100A9 and other contaminants were discarded.

We used a similar protocol to purify human S100A9. We performed Ni-affinity chromatography as described for calprotectin. We then performed anion exchange chromatography using a 50 mL wash and collected fractions over a 70 mL gradient elution from 100–1000 mM NaCl in 25 mM Tris, pH 8 buffer to isolate S100A9 from contaminant proteins that also bind the anion column. We used SDS-PAGE to confirm fractions with S100A9, pooled and dialyzed these fractions overnight into 4 L of 25 mM Tris, 100 mM NaCl at pH 6. As a final step, we loaded the S100A9 sample onto an anion exchange column in 25 mM Tris, 100 mM NaCl buffer at pH 6. S100A9 binds weakly to the anion column at pH 6. Therefore, we collected S100A9 in a 50 mL wash in 100 mM NaCl, and then removed contaminants from the column with a step elution at 1 M NaCl. Pure S100A9 was prepared for storage as described.

Far-UV circular dichroism and fluorescence spectroscopy

Prior to biophysical measurements, we thawed and equilibrated all proteins into 25 mM Tris, 100 mM NaCl, pH 7.4 via overnight dialysis in 4 L buffer at 4 °C. We determined protein concentrations by Bradford Assay using bovine serum albumin (BSA) standards and the molecular weight of each dimeric structure, then diluted to ~ 10 µ M in dialysis buffer.

For all spectroscopic measurements, we assessed metal-induced changes to the spectra by measuring the spectrum in the presence of 2 mM CaCl₂ and then adding excess EDTA at 5 mM and re-measuring the spectrum. We collected far-UV circular dichroism data between 200–250 nm using a J-815 CD spectrometer (Jasco) with a 1 mm quartz spectrophotometer cell (Starna Cells, Inc. Catalog No. 1-Q-1). We collected 3 scans for each condition, and then averaged the spectra and subtracted a blank buffer spectrum using the Jasco spectra analysis software suite. We converted raw ellipticity into mean molar ellipticity using the concentration and number of residues in each protein.

We collected intrinsic tyrosine and/or tryptophan fluorescence using the J-815 CD spectrometer (Jasco) with an attached model FDT-455 fluorescence detector (Jasco) using a 1 cm quartz cuvette (Starna Cells, Inc.). We collected a single excitation and emission scan at 10 nm/min with a 10 nm bandwidth, 1 nm data pitch, and 1 sec D.I.T. for each condition and then subtracted a blank buffer spectrum using the Jasco spectra analysis software suite. Depending on the sample signal, we set the detector sensitivity to either 630 or 800 Volts. We conducted excitation scans by measuring 305 nm light emitted at for all zebrafish proteins and 345 nm emitted light for human S100A9 for each excitation wavelength from 200–295 nm. For emission scans we used 280 nm light to excite zebrafish proteins and 288 nm for human S100A9, and measured light emitted at all wavelengths from 285–425 nm.

Nutritional immunity assay

We measured the antimicrobial activity of zebrafish s100s and human calprotectin against human- and zebrafish-derived bacterial strains using a modified version of a well-established assay that will be described here [5,50,63,64]. Bacterial strains used in this assay include 1) Staphylococcus epidermidis, a human commensal strain previously shown to respond to calprotectin [50,64]; 2) Aeromonas ZOR001, isolated from zebrafish and not previously characterized for response to calprotectin; and 3) Vibrio ZWU0020, isolated from zebrafish and not previously characterized for response to calprotectin but related to human-derived Vibrio cholerae shown to respond to calprotectin [29]. We obtained both zebrafish-derived strains from the Guillemin lab at the University of Oregon.

Each week, we plated bacterial strains from glycerol stocks onto antibiotic-free LB agar and grew at 30 °C overnight before storing plates at 4 °C. The day before an experiment, we inoculated a 5 mL culture in liquid LB media with a single colony from each strain and grew overnight at 30 °C with shaking. The following day, we diluted cultures 1:100 in 5 mL LB and grew to an OD₆₀₀ around 0.8 by the time of the experiment. Aeromonas ZOR001 and Vibrio ZWU0020 were diluted 2 hours before the experiment. S. epidermidis grew more slowly so required dilution 4 hours prior to the experiment.

The day before each experiment, we thawed a single S100 protein from -80 °C, concentrated to at least 200 μM using a Nanosep 3K Omega spin concentrator (Pall Corporation), and dialyzed overnight at 4 °C into 4 L of Experimental Buffer (25 mM Tris, 100 mM NaCl, pH 7.4) with 2 g/L Chelex 100 resin (Bio-Rad) to chelate residual transition metal ions. After dialysis, we filter-sterilized the protein through a Ultrafree-MC-VV centrifugal filter with Durapore PVDF 0.1 µm and kept at 4 °C until time of experiment.

To start the experiment, we made a protein dilution series by mixing a desired amount of protein in sterile Experimental Buffer with the appropriate amount of LB to achieve a ratio of 62:38, respectively. We then brought the volume of these protein solutions up to 1.7 mL in Experimental Media (EM). We made EM by mixing Experimental Buffer in LB at a ratio of 62:38, respectively, and filter-sterilized. We distributed each sample in aliquots of 160 µ L across ten wells of a clear Falcon 96-Well, Cell Culture-Treated, Flat-Bottom Microplate. At this time, we diluted each bacterial strain to an estimated OD₆₀₀ of.008 in 5 mL Experimental Media with calcium (EMC). We made EMC by adding 10.2 μM CaCl₂ to EM, and sterile-filtered. Then, we added 40 µ L of dilute bacteria or EMC without bacteria (contamination control) to each well, bringing the final volume to 200 µ L/well, and making technical triplicate conditions for bacterial strains. To counteract sample evaporation, the outermost wells of the plate contained 160 μL EM and 40 μL EMC, and we wrapped the plate in a single layer of parafilm.

We measured bacterial growth by OD₆₀₀ every 15 minutes over 13 hours in a Molecular Devices SpectraMax i3. The plate was shaken for 5 seconds before the first read, then for 10 minutes between each subsequent read. We set the plate reader temperature to 25 °C, however, over the course of the overnight growth, the actual temperature reached 37 °C. The final concentration of metals in the media without bacteria was measured using ICP-MS at the USR Elemental Analysis Core. The measured concentrations were Ni: 45.4 μM, Ca: 107.3 μM, Cu: 157.4 μM, Mg: 160.5 μM, Mn: 216.6 μM, Fe: 1.1 mM, and Zn: 5.9 mM.

For the analysis, we background subtracted each experimental condition using OD₆₀₀ values for the matching concentration of S100 protein concentration in buffer without bacteria added. We used Prism to average the replicates by condition, determine the standard error of the mean, and graph the results. We performed an ANOVA analysis on the antimicrobial activity at 50 μM protein for every condition and reported the results in Supplemental S3 Table.

Pro-inflammatory activity assay

We tested the S100A9-like pro-inflammatory activity of zebrafish s100s using a well-established assay [37,50,53]. This assay measures relative activation of the TLR4-mediated immune response through NF-κB. For each experiment, we thawed all zebrafish s100 proteins and human S100A9 from -80 °C, buffer exchanged into endotoxin-free PBS, then treated with endotoxin removal spin columns (ThermoFisher Scientific) to remove residual LPS from the purification process.

We performed each experiment in technical triplicate and followed the Dual-Glo Luciferase Assay System protocol (Promega). We transiently transfected adherent HEK293T cells (ATCC; CRL-11268) in a Falcon 96-Well, Cell Culture-Treated, Flat-Bottom Microplate with pcDNA vector plasmids using PLUS and Lipofectamine Reagents (ThermoFisher Scientific). Plasmids contained genes for human or zebrafish TLR4 complex components and Renilla luciferase enzyme under constitutively active promoters, and the firefly luciferase gene controlled by an NF-κB promoter. For human TLR4 complex transfections, we transfected 10 ng human TLR4, 0.5 ng human LY96 (MD-2), and 1 ng human CD14 plasmids per well. For zebrafish Tlr4 complex transfections, we used 10 ng zebrafish tlr4ba, bb, or al, 20 ng zebrafish ly96, and 1 ng mouse Cd14 plasmids per well, as this ratio gives us the best signal to noise ratio. Zebrafish do not have an annotated CD14. Previous studies have shown zebrafish Tlr4ba can be activated in the presence of mouse and human CD14, but more strongly with mouse. We also transfected all wells with 1 ng Renilla plasmid, 20 ng elam-Luc (firefly), and brought the total DNA mass per well to 100 ng with empty pcDNA vector in a total media volume of 200 μL per well.

After 20–24 hours incubation at 37 °C in 5% CO₂, we removed all 200 µ L of transfection mix from each well. We then treated transfected HEK293T cells with 100 µ L of one of the following treatment mixes: 1) 1 µ M S100 protein and 200 ng/μL Polymyxin B to bind up LPS in media, 2) 0.1 ng/ µ L LPS-R (tlrl-eklps; Invivogen) as a positive control for human TLR4, 3) 1 ng/ µ L lipid IVa as a positive control for zebrafish Tlr4ba activation, or 4) PBS and 200 ng/μL Polymyxin B as a negative vehicle control treatment. Because there is no known activator of zebrafish Tlr4bb and Tlr4al complexes [53], we treated these transfected cells with 1 ng/ µ L lipid IVa for consistency. After incubating again at 37 °C in 5% CO₂ for 3–4 hours, we removed and discarded 60 µ L of treatment mix from each well. We chemically lysed the cells by adding 30 µ L Dual Glo lysis reagent containing firefly luciferin and incubated in the dark for 7 minutes. We then mechanically lysed the cells by scraping the bottom of each well with a pipet tip and transferring 60 µ L of cell solution to an opaque 96-well plate. After a 7-minute incubation in the dark at room temperature, we measured luminescence per well produced by firefly luciferase activity using a Molecular Devices SpectraMax i3. Then we added 30 μL of Dual-Glo Stop & Glo buffer containing firefly luciferase quencher and Renilla luciferase reagent, incubated for 7 more minutes, and measured luminescence.

For the analysis, we took the firefly signal for each experimental condition and background subtracted the averaged firefly signal of wells transfected with the corresponding complex but treated with buffer without agonist. We did the same for the Renilla signal, with background signal considered as the averaged signal from wells with same treatment condition but transfected only with vector. We divided the background-subtracted firefly signal for each well by the background-subtracted Renilla signal for that same well. To simplify comparisons across experiments, we normalized the firefly/Renilla value for each well to the triplicate average of the firefly/Renilla values for human TLR4 complex treated with 0.2 ng/μL LPS-R. Statistical analyses were done using the multiple unpaired Welch t-tests in Prism v10.3.1 and p-values were reported on the graph.

Protein structure prediction and analysis

We collected zebrafish s100 polypeptide sequences identified in The Zebrafish Information Network (ZFIN). We fed these sequences into the AlphaFold3 web server [57] to build all possible homodimer and heterodimer complexes in the presence of four calcium ions and one zinc ion. We then opened the five output models from each AlphaFold3 prediction in Open-Source PyMol and aligned them to the crystal structure of calprotectin bound to zinc (PDB: 8JSC, chains A and C). We used PyMol to determine which histidine residues were within 9 Å of the calprotectin-bound zinc ion for each model. This information was used to make histogram plots for homodimer and heterodimer datasets. We also qualitatively noted when plausible alternate zinc-binding sites were predicted.