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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.
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