IL1R9 Is Evolutionarily Related to IL18BP and May Function As an IL-18 Receptor Chris S

IL1R9 Is Evolutionarily Related to IL18BP and May Function as an IL-18 Receptor Chris S. Booker and David R. Grattan This information is current as J Immunol published online 23 November 2016 of September 28, 2021. http://www.jimmunol.org/content/early/2016/11/23/jimmun ol.1500648 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/11/23/jimmunol.150064 Material 8.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 23, 2016, doi:10.4049/jimmunol.1500648 The Journal of Immunology

IL1R9 Is Evolutionarily Related to IL18BP and May Function as an IL-18 Receptor

Chris S. Booker and David R. Grattan

The IL-1 families of ligands and receptors exhibit similarity of coding sequences, protein structures, and chromosomal positions, suggesting that they have arisen via duplication of ancestral genes. Within these families there is selectivity in ligand–receptor interactions as well as promiscuity. IL-18 and its receptor are members of these families. IL-18 is recognized as binding to the protein products of the IL18R1 and IL18RAP genes, and with high afﬁnity to a separate IL-18 binding protein (IL-18BP). However, IL-18BP is anomalous, as it exhibits little resemblance to IL-18R proteins. Additionally, IL-18 is produced in the brain in medial habenula neurons, which project IL-18–containing axons to the interpeduncular nucleus. However, there is a lack of focal IL-18R expression in their terminal ﬁeld. Given these anomalies, we hypothesized that another receptor for IL-18 may exist,

and that IL18BP is evolutionarily related to this receptor. We examined Ensembl and National Center for Biotechnology Infor- Downloaded from mation databases to identify available IL18BP records (n = 86 species) and show through bioinformatics approaches that across mammalian species with IL18BP genes, IL-18BP is consistently most similar to IL-1R9 (IL-1R accessory protein–like 2), another member of the IL-1R family. IL-1R9 and the related IL-1R8, but not other IL-1R family members, exhibit an amino acid sequence similar to binding site A of human and viral IL-18BPs. Conserved intron/exon boundaries, protein structure, and key binding site amino acids suggest that IL18BP and IL1R9 are evolutionarily related, and that IL-1R9 and IL-1R8 may bind IL-18. The

Journal of Immunology, 2017, 198: 000–000. http://www.jimmunol.org/

nterleukin-18 is a proinflammatory cytokine that activates A further anomaly regarding the IL-18 ligand/binding protein/ NF-kB, JNK, and p38-MAPK signaling pathways down- receptor system concerns the role of brain-produced IL-18, I stream of the IL-18R complex (1). IL-18 is constitutively which is constitutively present in select medial habenula (MHb) present in the circulation in the absence of an inflammatory neurons, principally located in the superior subnucleus (5, 6), with stimulus, and its activity is regulated by a natural antagonist, IL-18 strong projections to the interpeduncular nucleus (IPN). Currently binding protein (IL-18BP). A common evolutionary feature of there is no known role for MHb-produced IL-18, and studies ex- many cytokines is the presence of circulating soluble receptors, amining IL-18 receptor subunit distribution in the brain have not generated from transmembrane receptor mRNA through alternate shown a focus of receptor expression in the IPN (7–10); this by guest on September 28, 2021 exon usage, or by shedding extracellular portions of transmem- anomaly suggests the existence of a different receptor for IL-18. brane receptors upon ligand binding (2). Unlike other cytokine The idea that IL-18BP evolved from a transmembrane receptor binding proteins, IL-18BP is not derived from the transmembrane has been previously raised. In one of the papers describing the IL-18R subunits, and it shares little resemblance with either subunit; discovery of IL-18BP in 1999, Novick et al. (11) documented that both IL-18R subunits possess three Ig-like domains, and IL-18BP no reading frame existed that could generate a transmembrane possesses only one, and yet it shows higher affinity to IL-18 than receptor, noting, “It is possible that IL-18BP evolved from a pri- does the ligand-binding IL-18Ra-chain (3, 4). As such, IL-18BP is mordial cell-surface protein that lost its membrane-anchoring an oddity in the cytokine field, being a cytokine-specific inhibitory domain.” Similarities between IL-18BP and the inhibitory recep- binding protein unrelated to that cytokine’s transmembrane tor for IL-1 (IL-1R2, also known as IL-1RII) were noted by receptor. Novick et al. (11) and later explored by Watanabe et al. (12) who concluded “that IL-18BP and IL-1R2 had a common ancestral gene.” The IL-1 ligand and receptor families, to which IL18 and Centre for Neuroendocrinology, Department of Anatomy, University of Otago, IL1R2, respectively, belong, appear to have evolved via duplica- Dunedin 9054, New Zealand tion of both ligand and receptor genes, given the high degree of ORCID: 0000-0001-8095-7547 (C.S.B.). similarity among members of each group and their clustering Received for publication March 19, 2015. Accepted for publication November 2, 2016. along specific chromosomal loci (e.g., the IL-1 ligand family cluster at q13 and the IL-1R family cluster at q11.2 of chromo- This work was supported by Health Research Council of New Zealand Grants 08/076 and 11/1076. some 2 in humans). Thus, the hypothesis that IL18BP and IL1R2 Address correspondence and reprint requests to Dr. Chris S. Booker, Centre for had a common ancestral gene that underwent duplication fits well Neuroendocrinology, Department of Anatomy, University of Otago, P.O. Box 913, with the apparent proliferation of IL-1 ligand and receptor family Dunedin 9054, New Zealand. E-mail address: [email protected] members by gene duplication. However, because there is no evi- The online version of this article contains supplemental material. dence that IL-1R2 is capable of binding IL-18, if IL18BP and Abbreviations used in this article: BLAST, basic local alignment search tool; IL1R2 derived from a common ancestral gene, then IL18BP and IL-18BP, IL-18 binding protein; IL-18BPa, IL-18 binding protein isoform a; IL-1RAP, IL-1R accessory protein; IL-1RAPL, IL-1R accessory protein–like; IPN, interpeduncu- the regions of IL1R2 encoding extracellular portions of IL-1R2 lar nucleus; MHb, medial habenula; NUMA1, nuclear mitotic apparatus 1; PHI, pattern would need to have diverged sufficiently for the protein product of hit–initiated; PTPRD, protein tyrosine phosphatase receptor d; RNF121, ring finger IL18BP to consist of one Ig-like domain and bind IL-18 with high protein 121. affinity, and the protein product of IL1R2 to consist of three Ig-like Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 domains and not bind IL-18.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500648 2 IL1R9 AND IL18BP BINDING MOTIFS

We hypothesized that the above anomalies could be overcome if alignment to make use of the crystal structures for ectromelia virus IL18BP were in fact related to a gene encoding a transmembrane IL-18BP (PDB ID 3F62) and Yaba-like disease virus IL-18BP (4EEC) receptor for IL-18 that has not been discovered or recognized to to guide alignment. Alignments were then passed through the MView visualization tool from the European Bioinformatics Institute to generate date. This would bring IL-18BP into line with other known cy- consensus sequences and residues involved in IL-18 binding or inhibition tokine inhibitors, which are typically related to, or arise from, a and were mapped as shown in Fig. 1 and Supplemental Fig. 1. transmembrane receptor for that cytokine. It would also open Three pattern hit–initiated (PHI) patterns were derived from the multiple new avenues of investigation by suggesting a ligand–receptor re- sequence alignment and consensus sequences: PHI pattern 1 included a key motif present at the 70% consensus level spanning many of the residues forming lationship between IL-18 and a previously unrecognized receptor. site A of poxvirus IL-18BP as defined by Krumm et al. (14, 16): Y-W-X(5,12)- Given the number of organisms in which entire genomes have F-X-E-X-L-X(5,7)-E (depicted in Fig. 1). PHI pattern 2 covered the same been sequenced, and annotation of protein-encoding regions in region as PHI pattern 1, but was derived from the 100% consensus sequence these genomes, it would be unlikely that a putative unrecognized by allowing for residues with similar physicochemical properties for those receptor for IL-18 would be an entirely undiscovered protein, but amino acids not conserved at the 100% level across the four proteins: Y-W-[ILV]- [ACFGHIKLMRTVWY]-[ACDEGHKNQRST]-X(0,7)-[ACDEGHKNQRST]- instead would be a known receptor that has not been recognized as [CDWHKNQRST]-[ACFGHIKLMRTVWY]-X-[DE]-[CDEHKNQRST]-L- being related to IL-18BP or involved in IL-18 action. We therefore [ACDGNPSTV]-[ACDGNPSTV]-[ACDEGHKNQRST]-X(0,2)-[ACFGHIKLM used bioinformatics approaches to examine the potential rela- RTVWY]-X-E. PHI pattern 3 extended farther in the N- and C-terminal tionship between IL18BP and genes encoding known transmem- directions to span through two key cysteine residues necessary for disul- phide bridge formation and includes residues involved in IL-18 binding close brane receptors. to the more C-terminal cysteine (see Fig. 1): C-X-[GA]-[CD]-X(5,6)-[VF]-S- X(2)-Y-W-X(5,12)-[FT]-X-[DE]-X-L-X(5,7)-E-X(2)-[TY]-X(8,10)-[NT]- Materials and Methods X(5)-L-X(4,5)-[TD]-X(7)-[LF]-X-C-V-L-X-[DT]-[PL]-X(2)-[VP]-X(5,6)-L. Downloaded from These PHI patterns were first checked for validity by conducting PHI- Identification of mammalian IL-18BP and IL-1R family BLAST searches using human IL-18BP as a query and limiting results to members Chordopoxvirinae (taxid: 10241) to verify that they did indeed identify poxviral IL-18BPs. Searches were then performed limited to Homo sapiens To identify transmembrane receptors that could putatively be related to (taxid: 9606) using these PHI patterns and each of the proteins used to IL-18BP, mammalian species with protein records of IL-18BP were generate the consensus sequence in Fig. 1 as query terms; although Yaba- identified from National Center for Biotechnology Information and like disease virus NP_073399.1 was used to generate the consensus se- Ensembl databases by searching for “IL-18BP” or “IL-18 binding protein” http://www.jimmunol.org/ quence, this is not shown in Table II, as the PHI pattern 1 is not present in in April–May 2015. Additional proteins were identified by examining the NP_073399.1. gene tree of Ensembl IL18BP entries across various species to identify proteins showing similarity to known IL-18BPs but that did not contain the IL-1R family multiple sequence alignment above search terms in their naming schemes. Identified proteins (n = 86) are listed in the Supplemental Table I. Given that human IL-18BP isoform Members of the IL-1R family were identified from National Center for a (IL-18BPa; NP_766630.2) shows the highest affinity for inhibition of Biotechnology Information and Ensembl databases for the same list of human IL-18 (3), human IL-18BPa and those IL-18BP isoforms showing species for which IL18BP records were identified. Accession numbers the highest similarity to human IL-18BPa were identified to proceed with for IL-1R family members are shown in Supplemental Table I. Proteins one isoform per species for subsequent basic local alignment search tool with $10% unknown residues (X) were excluded from alignments. (BLAST) searches for related proteins within each respective species. A To examine conserved residues in each IL-1R family member across quality control measure was adopted where proteins with strings of un- multiple species, proteins were aligned by multiple sequence comparison by by guest on September 28, 2021 known residues (X) amounting to .10% of the protein sequence were log expectation and passed through the MView visualization tool available excluded (quality control in Supplemental Table I), which effectively ex- from the European Bioinformatics Institute to generate consensus se- cluded an IL-18BP sequence from only one species, the hyrax (Procavia quences for each family member across species. Eighty percent consensus capensis). sequences were aligned manually using the alignment of human IL-1R Given that it is possible for different genes to acquire similar functions family members by multiple sequence comparison by log expectation, via convergent evolution, the likelihood of identified proteins being truly position-specific iterative Coffee, and M-Coffee (19) as guides. Codes for homologous was assessed by aligning sequences to assess whether iden- amino acids with similar physicochemical properties are those generated tified sequences exhibited similarity across the length of the protein, and by by the MView tool, originally defined by Taylor (20), with the exception annotating the chromosomal positions of the genes encoding each of the of the code for negatively charged amino acids: o, alcoholic; l, aliphatic; identified proteins (gene synteny columns in Supplemental Table I). a, aromatic; c, charged; h, hydrophobic; n, negative; p, polar; +, positive; s, small; u, tiny; t, turnlike. Conserved cross-mammalian intron/exon Evaluation of mammalian IL-18BP similarity to other proteins boundaries were derived by from six species: human, aardvark, armadillo, platypus, as examples from the Boreoeutheria, Afrotheria, Xenarthra, and BLAST searches were conducted to identify proteins or nucleotides within Monotremata taxonomic groups, respectively, and opossum and Tasmanian the same organism showing similarity to IL-18BP. BLAST searches for devil as examples of Metatheria (Supplemental Fig. 2). related nucleotide or protein sequences were performed in July 2015 and were limited to the species of the query sequence. Protein BLAST searches Assessment of the evolutionary origin of IL18BP were performed using the blastp algorithm against the refseq protein database, using default algorithm parameters. Searches for related nucleotide To establish when in evolutionary history IL-18BP first appeared, searches sequences were performed against the nr/nt nucleotide collection database for nonmammalian proteins were undertaken by conducting BLAST with discontiguous megablast, using default algorithm parameters. Results searches, using human IL-18BP as a query sequence, against reference with an E-value up to 5 were included in analysis, with hits on IL-18BP sequence proteins of nonmammalian taxonomic groups, for example, isoforms excluded. BLAST rankings were assigned to results, where one Sauropsida, Actinopterygii, and Aves, using an E-value of 1 as a cut-off for equals the BLAST result with the lowest E-value, and multiple isoforms potential IL-18BP homologs. Identified proteins were then used as a BLAST were excluded so that subsequent rankings of two, three, and such represent query sequence against human reference sequence proteins to assess whether the next highest protein or nucleotide product of a different gene. The they showed greatest similarity to human IL-18BP, using an E-value of 1 as an ranking and E-value of BLAST results were compared with two-tailed t test upper limit. The gene synteny of potential orthologs was identified, and on the using Microsoft Excel version 14.0.7166.5000 (32-bit). basis of BLAST results and gene synteny, 25 proposed IL-18BP orthologs in nonmammalian species were identified (Fig. 3, Supplemental Table I). Pattern hit–initiated BLAST searches for proteins exhibiting motifs of key IL-18BP amino acid residues Results Human (3, 13), ectromelia virus (14, 15), Yaba-like disease virus (16), and Examination of receptors showing similarity to IL-18BP Molluscum contagiosum virus (17) IL-18BPs have been subject to experi- mentation to identify residues involved in binding to human IL-18. The To pursue the hypothesis that IL-18BP may be related to a trans- respective protein sequences (NP_001034748.1, NP_671531.1, NP_073399.1, membrane receptor, two strategies were adopted. First, we identified and NP_044005.1) were aligned by Expresso (18) multiple sequence all available mammalian IL18BP records and assessed IL18BP gene The Journal of Immunology 3 synteny across species (Supplemental Table I) and used the protein have identified specific amino acid residues that influence their and mRNA products from these genes in BLAST searches for function by assessing the inhibition or binding of human IL-18 (3, related proteins or nucleotide sequences within each species to 13–16). These studies provide largely congruous results identify- determine whether consistent similarity with a transmembrane re- ing a number of key regions across human and viral IL-18BPs. We ceptor could be identified. The rationale for this approach was that therefore used this information to derive amino acid motifs re- if IL18BP had arisen via gene duplication, then comparing search sponsible for IL-18 binding or inhibition (Fig. 1, Supplemental results for related nucleotide sequences or their protein products in Fig. 1) to search for other human proteins exhibiting these motifs. those species with a copy of IL18BP would provide a cross-species Krumm et al. (16) have previously demonstrated that Yatapox view of similarity across different evolutionary trajectories. The disease-like virus IL-18BP inhibits IL-18 action by a unique second approach was to use available evidence on residues in IL- mechanism involving the formation of IL-18BP dimers, unlike 18BP that confer the ability to bind or inhibit human IL-18 to guide those of other viral or human IL-18BPs. By focusing on residues PHI-BLAST searches for related human proteins sharing similar conserved at the 70% level (effectively excluding one of the four binding motifs. This second approach would therefore focus spe- IL-18BPs assessed), a motif of key amino acids involved in cifically on regions of IL-18BP necessary for IL-18BP function, binding site A (14, 16) was generated for use in a PHI-BLAST with the first approach examining similarity across the length of (PHI pattern 1). A second PHI pattern was generated from the IL18BP nucleotide and protein sequences. same span of amino acids allowing for ambiguities to match the 100% consensus sequence (PHI pattern 2). A longer PHI pattern BLAST searches for similar proteins was derived from the 70% consensus sequence to include two key

BLAST searches for proteins or nucleotide sequences similar to cysteines involved in Ig-like domain folding and residues toward Downloaded from IL18BP were performed across 85 species. Searches for similar the C-terminal end of IL-18BPs involved in binding sites A, B, mRNA sequences produced hits on nuclear mitotic apparatus 1 and C (PHI pattern 3). These PHI patterns were used in PHI-BLAST (NUMA1) and ring ﬁnger protein 121 (RNF121) variants, which searches using each of the four viral and human IL-18BPs used in neighbor and/or overlap IL18BP in many species, clone cDNA mutation studies as query sequences to identify other human proteins sequences, or chromosomal sequences corresponding to the po- containing similar binding motifs (Table II).

sition of IL18BP, with few hits on other nucleotide sequences. Searches with PHI pattern 1 and human IL-18BPa, EVM013, or http://www.jimmunol.org/ Searches for similar protein sequences produced hits in 84 of the MC054L as query sequences produced hits on IL-18BP isoforms, 85 species assessed, with 72 (86%) of these producing hits on IL-1R IL-1RAPL1, also known as IL-1R8, IL-1R9 (all three query se- accessory protein–like (IL-1RAPL)2 (also known as IL-1R9 and quences), and kinesin-like protein KIF20B (human IL-18BPa and X-linked IL-1RAPL2, from the IL1RAPL2 gene); in 54 species (64%) EVM013 query sequences) (Table II). Queries with PHI patterns 2 IL-1R9 was the hit with the highest E-value. A lower degree of and 3 produced a more limited set of results, including only IL-18BP similarity was observed with IL-1R2, with hits in 19 species (23% isoforms. of those with BLAST hits). Other members of the IL-1R family Thus, both of the above approaches highlighted the similarity of appeared in BLAST search results in five or fewer species (#10% the protein products of IL18BP and IL1R9, and they show that of those with BLAST hits). No proteins outside the IL-1R family many of the residues that have been experimentally verified to by guest on September 28, 2021 featured as prominently as did IL-1R9 across BLAST results. facilitate binding or inhibition of IL-18 by IL-18BPs are also Across all mammalian species a greater degree of similarity was found in proteins encoded by IL1R8 and IL1R9 (henceforth observed with the protein products of IL1R9 than IL1R2 (Table I). IL-1RAPL proteins). These results suggest that IL18BP is evolu- There were a greater number of IL-18BPs producing hits on the tionarily related to IL1R9. Given that KIF20B (also known as IL-1R9 protein sequence from their respective species than IL- cancer/testis Ag 90, M-phase phosphoprotein 1) encodes an 1R2 proteins, with hits on IL-1R9 having a higher mean position intracellular protein (21), that the region of similarity with IL-18BP in BLAST results, with a mean (SD) BLAST ranking of 1.49 identified in PHI-BLAST searches does not occur within an Ig-like (0.99) for IL-1R9 and 2.26 (1.45) for IL-1R2, p = 0.007, and a domain, and that this protein does not feature prominently in lower mean E-value, with a mean (SD) E-value of 0.58 (1.01) for BLAST searches across species for proteins similar to IL-18BP, it IL-1R9 and 1.25 (1.48) for IL-1R2, p = 0.022. was deemed unlikely that there would be an evolutionary relationship between IL18BP and KIF20B, and this similarity was not PHI-BLAST searches for conserved binding motifs pursued further. A number of Chordopoxvirinae produce viral IL-18 binding pro- To further examine the similarity of the protein products of teins. Mutation analyses of human or viral IL-18 binding proteins IL1R8, IL1R9, and IL18BP across evolution, we identified records

Table I. BLAST hits for proteins similar to IL-18BP in mammalian species

Hits on IL-1RAPL2 Proteins Hits on IL-1R2 Proteins

n (% of Species with BLAST Hits, n = 84) Mean E-Value (SD) n (% of Species with BLAST Hits, n = 84) Mean E-Value (SD) Totals 72 (86) 0.58 (1.01) 19 (23) 1.25 (1.48)

BLAST Rank Distribution of Hits

n (% of IL-1RAPL2 Hits) n (% of IL-1R2 Hits)

1 54 (75) 7 (37) 2 8 (11) 6 (32) 3 5 (7) 3 (16) 4 3 (4) 1 (6) 5 2 (3) 1 (6) 6 0 1 (6) 4 IL1R9 AND IL18BP BINDING MOTIFS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Generation of PHI-BLAST query patterns from human and viral IL-18BPs and comparison of IL-18BP consensus and IL-1 receptor family member consensus sequences. (A) Amino acids in human and three viral IL-18BPs responsible for inhibition or binding of human IL-18 as shown across six studies: Esteban and Buller (15), Krumm et al. (14) (ectromelia virus EVM013), Xiang and Moss (13), Kim et al. (3) (human IL-18BPa), Xiang and Moss (28) (M. contagiosum MC054L), and Krumm et al. (16) (Yaba-like disease virus 14L). EVM013 and IL-18BPa lines are duplicated to highlight results of two studies per protein. Binding sites (A–C) are deﬁned by Krumm et al. (14, 16). PHI-BLAST query patterns derived from consensus sequences are aligned below. Supplemental Fig. 1 shows additional detail of annotation. (B) Cross-species 80% consensus sequence alignment for IL-18BP, IL-1R8, and IL-1R9 derived from 85 species. Highlighting in IL-18BP shows binding sites (A–C) from Krumm et al. (14, 16). Gray highlighting in IL-1R8 and IL-1R9 consensus sequences shows residues identical to IL-18BP. Alternating blue/black coloring shows exon boundaries, with red amino acids encoded across exons. (C) Cross-species 80% consensus sequence alignment of IL-18BP and IL-1R family members. highlighted as per (B). IL-1R family member amino acids aligning with identical residues in IL-18BP are highlighted gray; only IL-1R8 and IL-1R9 exhibit sequences similar to IL-18BP binding site (A). Exon boundaries are largely conserved across IL-1R family and IL-18BP. for each of the IL-1R family members across the same range of analysis of IL-18 sequences across the same 85 species shows a 85 mammalian species with identiﬁed copies of IL18BP passing high degree of conservation of residues conferring the ability for quality control (Supplemental Table I). We generated cross-species IL-18 to interact with IL-18BP (Fig. 2A) or the IL-18R subunits consensus sequences of each IL-1R family member to highlight (Fig. 2B) as well as sites of enzymatic cleavage (Fig. 2C) (4, 14, conserved residues and used alignment from six species across 16). This analysis suggests that a putative interaction of IL-18 with the mammalian class to derive conserved intron/exon boundaries IL-1RAPL proteins via similar functional epitopes as IL-18BP is (Supplemental Fig. 2). Cross-mammalian 80% consensus se- likely to be conserved, given the high degree of conservation of quences for the IL-1R family members and IL-18BP were then IL-18 and IL-1RAPL proteins. aligned (Fig. 1). This analysis demonstrated that the key binding motif exhibited in PHI pattern 1, which covers most of the resi- IL18BP gene formation dues involved in binding site A, is conserved in the protein The results above suggested an evolutionary relationship between products of IL18BP, IL1R8, and IL1R9 (Fig. 1B), but absent from IL18BP and IL1R9. To assess the timing and formation of IL18BP, the protein products of other IL-1R family genes (Fig. 1C). we performed BLAST searches to identify the current evolution- In order for a conserved ligand–receptor or ligand–binding ary spread of proteins showing similarity to human IL-18BP and protein relationship to exist across evolution, the ligand itself must mapped their gene synteny to assess whether they possess similar also retain key features to allow protein interaction. A similar chromosomal locations as other IL18BP orthologs. Some potential The Journal of Immunology 5

Table II. PHI-BLAST hits using PHI pattern 1 amino acid motif derived from viral and human IL-18BP sequences to search for human proteins containing similar motifs

Maximum Query Query Sequence Accession Description Score Cover (%) E-Value Accession No. Human IL-18BPa NP_001034748.1 Hits above threshold IL-18BP isoform c 303 87 3.00E-91 NP_005690.2 IL-18BP isoform d 219 65 6.00E-66 NP_766632.2 IL-1RAPL2 11.6 60 0.003 NP_059112.1 Hits below threshold IL-1RAPL1 3 39 0.99 NP_055086.1 Kinesin-like protein KIF20B 1.5 11 2.8 NP_057279.2 isoform 2 EVM013 NP_671531.1 Hits above threshold IL-18BP isoform c 17.7 71 4.00E-05 NP_005690.2 IL-18BP isoform a 17.7 71 4.00E-05 NP_001034748.1 IL-18BP isoform d 11.2 42 0.003 NP_766632.2 Hits below threshold Kinesin-like protein KIF20B 1.7 21 2.4 NP_001271188.1 isoform 1 Kinesin-like protein KIF20B 1.7 21 2.4 NP_057279.2

isoform 2 Downloaded from IL-1RAPL2 1.7 20 2.4 NP_059112.1 IL-1RAPL1 1.5 47 2.8 NP_055086.1 MC054L NP_044005.1 Hits above threshold IL-18BP isoform a 55.6 46 1.00E-16 NP_001034748.1 IL-18BP isoform c 52.5 39 1.00E-15 NP_005690.2 IL-18BP isoform d 19.9 20 8.00E-06 NP_766632.2 Hits below threshold IL-1RAPL1 6.1 37 0.12 NP_055086.1 http://www.jimmunol.org/ IL-1RAPL2 4.2 23 0.44 NP_059112.1 See Materials and Methods for full PHI pattern sequence. In the search using human IL-18BPa as a query sequence, hits on IL-18BPa have been excluded. orthologs identified in these searches, for example, in Coelecanth type 2–like) was identified in a Chondrichthyes species (Callorhin- and a number of Actinopterygii species, showed greater similarity chus milii). This potential ortholog has a protein sequence of 181 aa, to human IL1R2 than IL18BP when used as query sequences to similar to other identified IL18BP orthologs, and exhibits amino acids search for similar human proteins. However, they are included as similar to binding site A on IL-18BP (Fig. 1). However, this protein presumed IL18BP orthologs on the basis of shared gene synteny showed greater similarity to human IL-1R2 isoforms (with an E-value by guest on September 28, 2021 (Fig. 3). One potential IL18BP ortholog (XP_007884022.1; IL-1R of 9 3 e212 for similarity to XP_011510110.1) than human IL-18-BP

FIGURE 2. Alignment of 80% mammalian consensus sequence of IL-18 with human IL-18 showing key binding sites and protein features. (A) Highlighted on human IL-18 and the respective aligned residues in the consensus sequences are binding sites (yellow, light orange, cyan) for binding ectromelia virus IL-18BP; from Krumm et al. (14, 16). (B) Binding sites for human IL-18 binding to the human IL-18 receptor complex are marked along with the respective aligned residues in the consensus sequence according to Kato et al. (4). Site I, red, site II, orange; site III, blue. (C) Sites of IL-18 variation or modification are marked: spl var, amino acids predicted to be missing in a splice variant identified in the rat (49) and mouse (50); iso2, amino acids missing in isoform 2 found in many species; chym-p16 and chym-p20, sites of cleavage of human IL-18 with mast cell chymase (51); myr, potential myristoylation site for generating membrane-bound human IL-18 (52, 53); casp-1, casp-3, and PR-3, sites of cleavage of human IL-18 with caspase-1, caspase-3, and proteinase-3, respectively. The figure was derived in part from Omoto et al. (51). 6 IL1R9 AND IL18BP BINDING MOTIFS isoforms (with E-values of 1 3 e24 for similarity to human IL-18BP current records, these analyses point to the emergence of IL18BP isoforms a and c; NP_005690.2, NP_001034748.1), and it also has a from a common Euteleostomi ancestor (Fig. 3), occurring ap- chromosomal location neighboring MAP4K4 and genes showing proximately during the Cambrian era. If the identified protein in similarity to IL-1R family members, which reflects the gene synteny C. milii (XP_007884022.1) is regarded as an IL18BP ortholog, of human IL1R2 on chromosome 2. On this basis, it was regarded this would place the evolutionary origin somewhat earlier, at a as more closely related to IL1R2. Additional searches in other taxo- common Gnathostomata ancestor. nomic groups, including Cyclostomata, Tunicata, Cephalochordata, Hemichordata, Echinodermata, Protostomia, Platyhelminthes, Discussion Cnidaria, Placozoa, Porifera, and Fungi, failed to identify further Identifying gene duplicates is often based on mathematical potential IL18BP orthologs. likelihood-based assessments of nucleotide and protein sequence Among the identified IL18BP orthologs (Fig. 3, Supplemental similarity, further informed by characteristic features of duplica- Table I), the only mammalian species exhibiting a different chro- tion, such as chromosomal location (e.g., clusters of similar genes mosomal location for IL18BP is the platypus, Ornithorhynchus such as the IL-1 ligand and receptor families) or the presence of anatinus, where IL18BP is surrounded by the genes TNFRSF19, poly-A tails (as evidence of retrotransposition) as well as similarity MIPEP, C1QTNF9, and SPATA13, all of which tend to occur on of known biological functions (22). However, delineating the the same chromosomal stretch in other species (e.g., chromosome process of gene duplication is difficult due to the necessity to infer, 13 in humans, chromosome 14 in mice). In Aves and a number of from existing gene sequences, events that may have occurred in fish species (Actinopterygii) chromosomal translocations appear the distant evolutionary past. Genetic changes in the postduplica- to have occurred, resulting in differing positions of NUMA1 (Aves), tion period may obscure obvious similarities between present day Downloaded from or NUMA1 not being located in the same chromosomal region duplicates. (Actinopterygii). In this study, we propose that IL18BP has evolved via a pre- The protein similarity and common gene synteny neighboring viously unrecognized duplication of IL1RAPL2. There are a number RNF121 and/or NUMA1 of identified IL-18BPs suggests that these of converging pieces of evidence in support of this hypothesis: are true orthologs arising from a common ancestor. Based on 1) IL-18BP shows little resemblance to the proteins that form http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Gene synteny of IL18BP across species with known copies of IL18BP. The taxonomic structure of species with identiﬁed IL18BP orthologs indicates a likely Euteleostomi ancestor. Percentages indicate similarity to human IL-18BPa (100%). Genes indicated are: *, Aves species possess one or more variable genes in this location, which have been omitted for simplicity; CNKSR2, connector enhancer of kinase suppressor of ras 2–like; C1QTNF9, gene showing similarity by BLAST comparison with complement C1q and TNF-related protein 9 in the human; MIPEP, mitochondrial intermediate peptidase; NLRP3, NACHT, LRR, and PYD domains–containing protein 3; NUMA1; PPP5C, protein phosphatase 5, catalytic subunit; PPP5D1, PPP5 tetratricopeptide repeat domain containing 1; RNF121; RRM1, ribonucleoside-diphosphate reductase large subunit–like; SPATA13, genes showing similarity by BLAST comparison with spermatogenesis-associated protein 13 in the human; TNFRSF19, TNFR superfamily, member 19; TRPC2-like, gene showing similarity by BLAST comparison with short transient receptor potential channel 2–like in the human. The Journal of Immunology 7 the IL-18R, a heterodimer of IL-18Ra (from the IL18R1 gene) testicular expression (27). The C-terminal sequences of IL-18BPs and IL-18Rb (from IL18RAP), suggesting that it did not arise from different species are highly variable, where some species from their duplication; 2) IL-18BP contains an Ig-like domain, (e.g., pig and guinea pig) exhibit longer C-terminal tails, similar to similar to all members of the IL-1R family; 3) BLAST searches the longer C-terminal tail of M. contagiosum virus IL-18BP, which for proteins similar to IL-18BP across the range of mammalian confers the ability of this viral IL-18BP to bind cell membranes species with known IL18BP genes consistently produce hits on (28). This C-terminal variation may therefore represent differing the IL-1RAPL2 protein in that respective species; 4) PHI-BLAST ways in which orthologs previously encoding transmembrane and searches for human proteins exhibiting amino acid motifs neces- cytosolic domains have been truncated in response to selection sary for the binding and inhibitory function of human and viral pressures. IL-18BPs produce hits on IL-1RAPL proteins; 5) these binding There is currently only one study that has attempted to verify motifs are largely preserved in IL-18BP and the third Ig-like do- whether IL-18 acts as a ligand for IL-1RAPL receptors (29). mains of IL-1RAPL proteins across the range of species with However, this used NF-kB luciferase activity as a marker of receptor known IL-18BPs, but not in other members of the IL-1R family; activation, and subsequent research suggests that IL-1RAPL receptors and 6) exon boundaries of a key region encoding the Ig-like do- are incapable of activating NF-kB (30). As such, whether IL-18 mains of IL18BP and wider IL-1R family are preserved. These may function as a ligand for IL-1RAPL receptors has not been ﬁndings suggest that although IL-18BP has been regarded as a thoroughly investigated. member of a separate family of secreted proteins (23), it should be Until recently, the IL-1RAPL proteins were orphan receptors, regarded as a member of the IL-1R family, and they suggest that although studies have now shown that these are able to form

IL-18 may function as a ligand for IL-1RAPL receptors. transynaptic interactions with protein tyrosine phosphatase re- Downloaded from A ﬁnal piece of evidence necessary to infer that IL1R9 is the ceptor d (PTPRD), with presynaptically localized PTPRD binding parent gene in a duplication event would be to show a wider to the extracellular domain of postsynaptic IL-1RAPL receptors to evolutionary spread of IL1R9 than IL18BP. Based on current regulate the strength of glutamatergic synapses (31, 32). If IL-18 reference sequences, IL1R9 appears to have a similar evolutionary were to act on IL-1RAPL receptors to bring about similar effects spread to IL18BP, and there is an absence of evidence to show it as PTPRD binding, or interfere with PTPRD–IL-1RAPL binding,

predates IL18BP: it is found, for example, in Actinopterygii such IL-18 action on IL-1RAPL receptors could form a mechanism of http://www.jimmunol.org/ as the tilapia (Oreochromis niloticus), cod (Gadus morhua), and regulating glutamatergic synaptic input. There is recent evidence zebrafish (Danio rerio) and throughout Mammalia, suggesting the to suggest this may be the case. A recent study by Francesconi same evolutionary spread and origin as shown for IL18BP in et al. (33) reported that IL-18 reduces food intake by actions in the Fig. 3. However, it is likely that future genome builds will alter the bed nucleus of the stria terminalis. The authors concluded that this known evolutionary spread of IL1R9 genes, as in all species with is mediated via IL-18Ra, as these effects were not observed in an identified copy the IL1R9 gene is large with multiple exons and IL-18Ra2/2 mice. Unusually, the mechanism identified involves large intronic regions, making genome builds with long contigs presynaptic effects of IL-18, which causes a reduced excitatory necessary to map its evolutionary spread. For example, the human input to g-aminobutyric acid–ergic neurons in the bed nucleus of IL1R9 gene was first identified in 2000 and spans ∼1000 kb (24). the stria terminalis. Of note, the highly related cytokine IL-1 has by guest on September 28, 2021 Similarly, the zebrafish IL1R9 (ENSDARG00000037553) spans recently been shown to exert CNS-specific effects via a splice ∼950 kb, suggesting that similarly large sizes would be expected variant of IL-1R1 (IL-1R3), the extracellular portion of which for other IL1R9 genes if they exist beyond the evolutionary spread consists only of the third Ig-like domain (34), and a CNS-specific of IL18BP genes depicted in Fig. 3. splice variant of IL-1RAP (IL-1RAcPb), which shares similarity Circumstantial evidence supporting the hypothesis that IL1R9 is to IL-1RAPL receptors (35–38). The IL-1RAcPb protein forms the parent gene of IL18BP comes from considering possible trans-synaptic interactions with PTPRD (35), similar to IL-1RAPL mechanisms of duplication. It is likely that at the proposed time of receptors. Thus, the proposal that IL-18 may activate IL-1RAPL duplication IL1R9 was encoded across a similarly large chromo- receptors to give rise to CNS effects involving PTPRD has prece- somal region as it is in most species today. Compared to the size of dent in discoveries of IL-1 ligand and receptor function, and would IL18BP genes, it is unlikely that complete gene duplication took further the similarity of these two cytokines. The findings of place, and even in the case of partial gene duplication the size of Francesconi et al. (33) could be interpreted as suggesting central the intronic regions surrounding a key exon in IL1R9 encoding the IL-18 functions in a similar manner to central IL-1 and involves a PHI pattern 1 motif in binding site A would necessitate the sub- splice variant of the a-chain of the IL-18R forming a heterodimer sequent deletion or transposition of a large amount of genetic with IL-1RAPL proteins to regulate glutamatergic input. material to arrive at present day IL18BP gene sizes. Therefore, The crystal structure of the IL-18/IL-18R complex has recently duplication of DNA sequences seems unlikely. One possible mech- been described (39), and some key features of this structure show anism of duplication is that IL18BP was formed via retrotrans- the potential for similar ligand–receptor interaction between IL-18 position of an ancestral IL1R9 mRNA sequence; present day and IL-1RAPL proteins: residues in IL-1RAPL proteins align to human IL1R9 mRNA spans 2985 bp (NM_017416.1) whereas the Trp249(Ra) and Glu253(Ra) on the acidic surface of IL-18Ra, which human IL18BP gene (gene ID 10068) spans 4010 bp, such that the bind Lys53 in IL-18, and an asparagine in IL-1RAPL receptors formation of IL18BP as a retrogene of IL1R9 is plausible, with closely aligns with Asn297(Ra), the mutation of which was found to the proviso that subsequent intronization must have occurred. reduce IL-18Ra affinity to IL-18 by a third (39) (an asparagine is Retrogene formation has been shown to involve an excess of retro- also highly conserved in this position in IL-18BP, see Fig. 1B). transposition from the X chromosome to autosomes (25), with Furthermore, the Ig-like domain in IL-18BP was found in this retrogenes more likely to be found close to other genes or within study to show similarity to the third Ig-like domain in IL-1R9, and introns (26), and often exhibiting expression in the testis (26). the IL-18/IL-18R crystal structure shows that binding involves Note that all of these are the case for the hypothesis of IL18BP only the second and third Ig-like domains of the IL-18R complex. being a retrogene of IL1R9: this would involve duplication from The biological role of MHb-derived IL-18 is unknown, and the the X chromosome to an autosome, IL18BP in very close proximity only report of its regulation is a study that observed an increase in to and on the same strand as RNF121, and IL18BP exhibiting MHb IL-18 following an acute stress in rats (6). The MHb and IPN 8 IL1R9 AND IL18BP BINDING MOTIFS have been shown to play a role in anxiety and nicotine addiction 19. Taly, J.-F., C. Magis, G. Bussotti, J.-M. Chang, P. Di Tommaso, I. Erb, J. Espinosa-Carrasco, C. Kemena, and C. Notredame. 2011. Using the T-Coffee (40–42). Aside from the central effects of IL-18 on food intake package to build multiple sequence alignments of protein, RNA, DNA sequences described above, other reported effects of central IL-18 action and 3D structures. Nat. Protoc. 6: 1669–1682. include changes in motor activity and anxiety-related behaviors 20. Taylor, W. R. 1986. The classification of amino acid conservation. J. Theor. Biol. 119: 205–218. (43) and hippocampal long-term potentiation in vitro (44). How- 21. Abaza, A., J.-M. Soleilhac, J. Westendorf, M. Piel, I. Crevel, A. Roux, and ever, there is currently little evidence regarding mechanisms by F. Pirollet. 2003. M phase phosphoprotein 1 is a human plus-end-directed which central IL-18 mediates these effects. Additionally, although kinesin-related protein required for cytokinesis. J. Biol. Chem. 278: 27844–27852. 22. Katju, V. 2012. In with the old, in with the new: the promiscuity of the dupli- constitutive IL-18 production within the brain appears to be cation process engenders diverse pathways for novel gene creation. Int. J. Evol. largely anatomically demarcated to the MHb, following hypoxic- Biol. 2012: 341932. ischemic injury IL-18 expression is upregulated and widespread 23. Dinarello, C. A., D. Novick, S. Kim, and G. Kaplanski. 2013. Interleukin-18 and IL-18 binding protein. Front. Immunol. 4: 289. in the brain (45). The possibility of IL-18 action on IL-1RAPL 24. Jin, H., R. J. Gardner, R. Viswesvaraiah, F. Muntoni, and R. G. 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Lines depicting Ectromelia virus IL-18BP (EVM013) and human IL-18BPa have been duplicated to highlight the results of two studies on each protein: Esteban and Buller, 2004 (15) and Krumm et al., 2008 (14) for EVM013; and Xiang & Moss, 2001 (13) and Kim et al. 2000 (3) for human IL-18BPa. Residues on other proteins are derived from the publications: Molluscum contagiosum MC054L - Xiang and Moss, 2001 (17); Yaba-like disease virus 14L - Krumm et al., 2012 (16). Below aligned sequences are depicted the PHI Patterns 1, 2, and 3; see methods for full sequences of PHI Patterns 1 through 3. Xn signifies the number of X residues entered into PHI BLAST queries. Alternate aa signifies where the option of more than one amino acid was entered into PHI BLAST queries by representing alternate amino acids vertically aligned – e.g. the code [GA] entered into a PHI BLAST search is depicted as a G in the PHI Pattern, with an A at the same point below. A simplified version of this figure is shown as Figure 1. See Key for further information.

1 [ . . . . : . . . 80 1 MC054L 100.0% MARGGKSGPRPWPDLLSHVRLVIVLVA------LLFLYSRAC-EL---EIS-- 2 EVM013 Esteban & B. 9.9% MR------ILFLIAFM----YGC------VH------SYVNAVETKCPNL---DIV-- 3 EVM013 Krumm et al. 9.9% MR------ILFLIAFM----YGC------VH------SYVNAVETKCPNL---DIV-- 4 14L_YLDV 10.6% MKTQ------IIILLL------I------YFVQDSKNECVKTRSVNIH-- 5 IL18BPa Kim et al. 20.1% MTMRHN-----WTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPAL---EVTWP 6 IL18BPa Xiang 20.1% MTMRHN-----WTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPAL---EVTWP consensus/100% Mt...... llhlhh...... l...tptC.th...pl... consensus/70% Mpht...... lllLlsh. h.. .. .hV.sspppCspL -Is

81 . 1 . . . . : . 160 1 MC054L 100.0% -TQVGPNGTTILTCLGCTN-HTHVSLIYWIVN------ESFPEQLDSS--LSEGRTHKHKFPNQSLTEISTNLTVGP-D 2 EVM013 Esteban & B. 9.9% ------TSSGEFHCSGCVEHMPEFSYMYWLAKDMKSDEDTKFIEHLGDG--INEDETVRTT--DGGTTTLRKVLHVTD-T 3 EVM013 Krumm et al. 9.9% ------TSSGEFHCSGCVEHMPEFSYMYWLAKDMKSDEDTKFIEHLGDG--INEDETVRTT--DGGTTTLRKVLHVTD-T 4 14L_YLDV 10.6% -VPVKETSKVVLECRGDSY-FRHFSYVYWIIGK------NKTVDQLPPNSGYRERIYLFKK-PHRCENRPRADLILTNIT 5 IL18BPa Kim et al. 20.1% EVEVPLNGTLSLSCVACSR-FPNFSILYWLGN------GSFIEHLPGR--LWEGSTSRER--GSTGTQLCKALVLEQLT 6 IL18BPa Xiang 20.1% EVEVPLNGTLSLSCVACSR-FPNFSILYWLGN------GTFIEHLPGR--LWEGSTSRER--GSTGTQLCKALVLEQLT consensus/100% ...... suph.hpC.uss..h.phShhYWlht...... tph.-pLsst..h.Et.h.hpp..tts.sp.ptsLhlt..s consensus/70% s.V..suos.LpChGCop ascFShlYWlss. spFlEpLsss lpEspTh+c+ .spstTplppsLhlss.T

PHI Pattern 1 YW-----X------FXEXL---X---E PHI Pattern 1 Xn (5,12) 1 1 (5,7) PHI Pattern 2 YWlht-X-----tphXnpLsst-XhXE PHI Pattern 3 CXGC--X---FSX-YW-----X------FXEXL---X---E-XT----X-----T--X--L--X--T PHI Pattern 3 Xn/alternate aa 1AD (5,6)V 2 (5,12) T1D1 (5,7) 2Y (8,10) N 5 (4,5)D

161 . . . 2 . . . . 240 1 MC054L 100.0% VATHSTNFSCVLVDPEQVVQRHLALTPP-GTTPPTATRTPPENADAAGARRRRRGAPTSPGAPTPPGAPTSPGAPTLPGA 2 EVM013 Esteban & B. 9.9% NKFAHYRFTCVLTTLDGVSKKEY-LAEVAHDT------IF------3 EVM013 Krumm et al. 9.9% NKFAHYRFTCVLTTLDGVSKKEY-LAEVAHDT------IF------4 14L_YLDV 10.6% DEMRNEKLTCVLIDPKDPLKESVILSKI-WNC------5 IL18BPa Kim et al. 20.1% PALHSTNFSCVLVDPEQVVQRHVVLAQL-WAG------LRA--TLPPTQEALPSSHSS------6 IL18BPa Xiang 20.1% PALHSTNFSCVLVDPEQVVQRHVVLAQL-WAG------LRA--TLPPTQEALPSSHSS------consensus/100% sthtp.phoCVLhs.cts.pcph.Ls...hss...... consensus/70% sth+sppFoCVLlDP-pVlp+clhLupl ass ......

PHI Pattern 3 --X----FXCVLXDPX-V--X---L PHI Pattern 3 Xn/alternate aa 7 L1 1TL2 P 5,6

Key: 1 MC054L: Annotations derived from Xiang and Moss, J Virol (2001), 75(20): 9947–9954 4 YLDV: Annotations derived from Krumm et al., PLoS Pathog (2012), 8(8): e1002876 TPPTA... amino acids in italics: C-terminal dispensible for IL-18 binding C cysteines in bold, italics: cysteines mutated for chrystal formation profound effect on IL-18 binding cysteines forming intramolecular bond smaller effect on IL-18 binding cysteines forming intramolecular bond cysteine forming intermolecular bond for dimer formation 2 EVM013: Annotations derived from Esteban and Buller, Virology (2004), 323: 197–207 residues involved in dimer formation L Leucine in italics; protein unstable when mutated Site A slight increase in Kd for IL18. Site B reduced binding to human IL18 but not murine IL18 Site C influenced binding ability, principally through increased Kd. cysteines, protein unrecoverable when mutated 5 IL18BPa: Kim et al., PNAS (2000), 97(3): 1190–1195 mutation to alanine had little difference on binding to human or murine IL18 residues forming electrostatic interactions with IL-18 mutation eliminated binding to human and murine IL18 residues forming hydrophobic interactions with IL-18

3 EVM013: Annotations derived from Krumm et al., PNAS (2008), 105(52): 20711–20715 6 IL18BPa: Xiang and Moss, J Biol Chem (2001), 276(20): 17380–17386 The same color scheme as used by the authors in Figure S2, PNAS, 105: 20711-21715 has been retained: T bold, italics; a tyrosine mutation was introduced at this point for all assays Site A mutation resulted in a large change in binding affinity Site B mutation resulted in a smaller change in binding affinity Site C Supplemental Figure 2: A - J show alignments of IL-1 receptor family proteins from up to six species across the mammalian class used to derive consensus intron-exon boundaries depicted in Figure 1. Alternating black and blue shading represents amino acids encoded by different exons, red shows amino acids encoded across exons. Below these six species the aligned 80% consensus sequences derived from all Mammalian species are shown, as they appear in Figure 1, highlighted with consensus intron-exon boundaries derived from the six species above. See Materials and Methods for ambiguous amino acid codes shown in consensus sequences. A Human TRPVIVSPANETMEVDLGSQIQLICNVTGQLSDIAYWKWNGSVIDEDDPVLGEDYYSVENPANKRRSTLITVLNISEIESRFYKHPFTCFAKNTHGIDAAYIQLIYPVT-NFQKH Opossum KLPTILAPENKTMEVELGSRLLLDCNFTVGKNQYPYWSWNGSYADID-PNIAWFTPQIPNLTKTNPSTEICRLNVSKVQSKHYLYPFICSVKNGAGEVSAYIALIPPVP-DYKIY T. Devil NIPKILSPENKTMEVELGSQLLLDCNFTAQRNDVQYWTWNGSY-AYNEPNVTWSELLVPNLNNLNTVTRVSRLVILKVESRHYLYPFLCWVSNDAGIVTTYIALKPRVV-DARMY Platypus DIPKILYPVNSSVEVELGSQFVVDCNVSGMSIDLVYWTWNRK--AINDPKVFMEYLESEEHSETRPVTVISRINISEVGSRHYLHPFVCLASSTYGYTEAYIALKPPTP-DFRAC Aardvark NRNEICHPSNDTLILLLGSQTQLICNVTGQ--SFVQWWWNGSYIDDDDPVLAEEYEIVENPTRKRQ-TIILMLNISEVESRFYLYPFTCTAMNGLGKNEAYVQLIHPVH-DCQNH Armadillo TMPVIVRPINETMEVELGSQAQLICNVTGLVTDTVFWKWNGS--DIDDPLLEEDFKTMDIPSTKKWKTLIVMLNISQVESRFYLYPFTCLAMNMAGENAAYIRLIHPAAQDFQKH

IL1R1 ppPXIlSPsNEThEVsLGSplQLICNVTGphoshsYW+WNGShIpppDPlLhEnaXXlcNPXs+++sTlIshLNIStlcS+FYhaPFhChA+NsXGhssAYlQLIaPVs-sFppX B Human TIPVIISPLK-TISASLGSRLTIPCKVFLGTGTPLTTMLWWTANDTHIESAYPGGRVTEGPRQEYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVKEASSTFSWG Opossum SFPVIILPNK-TISTSLGSKLIIPCKAFFGLGPQSTMLLWWTANKTFVDNVYKEGRVKEGQHHKYLENNKSYLEAPLIFDSVEKKDLYTEFVCTARNRLGSQTLHARVKEE-PPFSWG T. Devil TIPVIITPNK-TVLTSLGSKLVIPCKAFFGLGSRSTTLLWWTANNTFLDNVYKKGRVKEGQRLEYSENNKSYIEVPLIFNSVEKKDLNTEFICTAKNRLGTQSLQARVKEE-LTFSWG Platypus IVPVIVYPNQETILTALGSKLIIPCKVFLGLGEQSSTWLRWTANNSLVTNVYKEGRVSEGERQEFSENNENYIEVPLVFDPVLKEDMYTDFKCFVHNTVGYQMLLAKVREA-PTFSWW Armadillo TIPVIISPHQ-TILASLGSRLTIPCKVFLGAGTQATTMLFWMANNSDVEVVYRGGRVTQGQRQEYSENNENYIEVPLIFNPVLREDLNTDFKCEVFNTVGFQTLRTTVKEA-STFAWE Aardvark PIPVIVSHHQ-TILASLGSKLTIPCKVFLGVGKKSTSIVWWTANDTNIDSAYPSGRVTEGPDKEYSENNENYIEVPLVFDPVAREDLNTDFKCSVTNKKIFRTLRTRVKEASSTFSWE

IL1R2 oIPVIISPhp-TIXASLGSRLTIPCKVFLGsGpXXTThLWWhANsTplnsAYXuGRVTEGXRpEYSENNENYIEVPLIFDPVhREDLphDFKCsVpNshuhQoL+TTVKEsXuoFSWt C Human VPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYT Opossum TAPDIFSPNDHITYDKEPGEELELPCKVRFPFLKDSRDEIWWTVDGRKPDE-TMNITINQSETVEIVGDKTITRILSIKKVTSDDLKRNYTCHARNDKGESVKQAIVKQIVVPPRYT T. Devil TRPEIFSPNDNIIYDKEPGEDLDLPCKVSFSFLKDSKNEIWWTIDGRKPDD-SMNITINQSETVSVIGNKMITRILSIKKVTSDDLKRNYTCHARNAKGESVKQAIVKQIVVPPRYT Platypus LPPQMLSPMDQVVYEKEPGEELLLPCEVIFTFLKDSPKEIWWTIDGKKTDD-IPDITVNQSIITSQIEDITIKKTLNIKKVSSDDLKRNYTCHARNAKGKVEKQAKVKQKVLVPRYT Armadillo SPPQIHSPNDIVIYEKEPGEELLIPCKVYFTFLKDSRNEIWWTIDGKKPDDTNTDVTFNESTTFSKTEDETKIQILSIKKVTPEDLKRSYVCHARNARGEVNKTAVVKQKVVTPRYT Aardvark LPPQMHSPNDFVVYEKEPGEELIIPCKVYFTYLKDSRNEIWWTIDGKKPED-TTEVVVNESISYSSLEDETRIRILSIKKVTPEDLKRSYVCHARSAKGEVDKAATVKQKVITPRYT

IL1RAP hPPXIaSPNDhVVYEKEPGEELLIPCpVaFoFLhDS+NElWWTIDGKKPDDhshDlTlNESlohothEDETRTQlLSIKKVTsEDLKRsYVCHARsAKGEVs+sApVKQKlXsPRYT D Human IVPVLLGPKLNHVAVELGKNVRLNCSALLNE-ED-VIYWMFGEENGSDPNIHEEKEMRIM-TPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKADMADIP Platypus VNPAILGPQIVEVEVELGKEKVLNCTVSGGDLSDFFIYWIPFPSDE------RLEEIKLNNT------Opossum V-PKLFGNMTNYVEAELGKKKTLNCTAILDG-TDGRLYWDYMEENDTNININKINSS----DSNAMTRVSSLLEIKIVKEENLNMWYNCTLSSPSITETIAYFLKKKEGPADIS T. Devil I-STLYGNSINYVEAELGKEKILNCTALLMDSSD-MIYWDKEPNNATNIIIDKKKSTPS--NSSGMLYVSNLLKIKPVKEENLNVWYNCTLITTRDPETIHFFLKKKEGPADVS Aardvark IIPVLYGPKINHVEVELGKDVELNCSALLNK-DD-VVYWSFWKENETDHNVHEEKEPKIFSTPEGKQHASILLHIRNINENNLNFQYNCTVAGGGGTDTRVFILLRKD-VVDIP Armadillo INPILFGPEVNHVEVELGKDVQLNCSALLNE-KD-IVYWNIWKENGTDPNVHEEKEIKIK-TSEGKWHASKILRIKNINENNLKFSYNCTVASEGGTDTISFILLKKEGLGDIP

IL18R1 lhPslLGPKhspVtVELGKnlpLNCSALlNn-pD-hlYWXhhccstpnPNlHEcpthphh-TsEGKhhAS+lL+IcslsEpNLNhXYNCosssptuhDTpsFlLl+KXshsDIP E Human LKPDILDPVED--TLEVELGKPLTISCKARFGFERVFNP--VIKWYIKDSDLEWEVSVPEAKSIK---STLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKE----KRGVVLL Platypus MKPDILEPIEK--TLEVELGKPFAINCTVRFGFQNSSLPPQLVEWYADD----KEPLLQKETCKL---TSTEELKICKEVRLIKVTPKDLHRTFRCFAQNANGKVVQVIQLKKKLPRKKGGTLT T. Devil SIPIILDPTHDMFTLEVELGKPLILECKIRFGFERNFNP--VIKWYVEDAKLNKEELKQENKSII---KQVEWETIHYIAYLKKVTKKDLLRNFICFAQNSVGNSTRTIKLKQ----KRGVFFI Opossum SKPTILYPTVDMLTLEVELGKPLTLDCKAQFEFERNFNP--VIKWFVDDPGLNRKELREGNKSIE---NDVEQKTIHFIAHLKEVTKKDLRRNFICFAQNSVGNTTLTIKLKQ----KKGVLFT Aardvark FKPDILVPDKD--TLEVELGKPLTLNCKARFGFETNFTP--VLKWYLNDVDQKWXVPIPEQKSILSKTSALENDIIQCVLSLKEVTKSDLHNKFTCFVKNSKGNDTRSIQLKE----KKGVFFT Armadillo SEPVILEPIKD--TLEVELGKPLTLNCRVQFGFEKDFAP--VVTWYSQDSNQKLQVLKSEQKSIK---STLKDREIQCVAHLKEVTQRDLHRKFICFAQNSIGNATQYIWLKE----KKGVVFL

IL18RAP hKPDILnPlps--TLEVELGKPLTlsCpsRFGFphsFpP--Vl+WYl+DusXEhEhXsXct+slc---SThcsnlIppslhLccVTQpDLp+pFlCFsQNSlGNoTpolQLKc----KcGVVhl F Human LFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQL--NGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNP------IDHHS Platypus FHPQIKIPPNNAVEKVKLGAPVTISCSACFGKGPQEIAILKWLV--DNTVIHRFNDSRFHEYPGKMVSARGQLSCMNNTLKITQVEDNDFSSEFQCVAINQHGWTAHYLTFKKKESHSRQRSHFCCTVRRSPPNLSPSYQVHHWS Opossum MLPEITYPVNQSVIGVELGTNVSIACKVCLGRGSQTIEIVRWCIKDNKDKGQCIKDSRFHEEK-EAKNPKENLACETVNLRIPNVKKEDLSLEFYCMGLNQYGWKAHSIRIGIKKT------TDLRS T. Devil MLPEITYPVNHDVIEVELGTNVSVVCRVCLGKGMQIIEQVGWCIK-KKNIINCIKNPRFREEN-EAPKSNENLNCKTVTLRIDNVKREDLSLEFYCWALNEHGFKEHSIRIRMKKT------TDQRS Armadillo LLPVIRAPLHNDIVEVEMGKSASITCSACIGKGSRIWAAVQWRV--NGSSVSDLGEKRIYEEKGQNQSSSKELTCLNQILIISDVKEEDLSLRYDCLAYTIKGLKNHSIRLRQKNP------IDRQS Aardvark LVPVITAPLPNDTKEVEIGKTANITCSACMEKGPLFMASVKWQI--NGSDANDVGEARIHQEQVHNQSPRNELTCLKRTLRIAEVKEEDLSLKFECLAFNSRAVTIRPIRLRRKSS------IDHQN

IL1RL1 hhPVIhAPspNns+EVEIGcssslsCoACFGKGsQhhstlhWpl--NtsplpshunsRIppcptpspSXSsthsChshlLRIssV+EEDLXLpYnClAhNh+GhhpHslRLp+KpP------Xcppp G Human SVPKIIYPKNHSIEVQLGTTLIVDCNVTDTKDNTNLRCWRVNNTLVDDYYDESKRIREGVETHVSF-REHNLYTVNITFLEVKMEDYGLPFMCHAGVSTAYIILQLPAPDFRAYLIG Opossum KIPEIFSPKNNSIEVPLGSPLIVNCSIKDWKGNSHHRSWKVNNTFVD--FLNSSRIREGVETEVACGEKYIFYTVNISFSEVRQEDYDRHFTCLSGSSAAFIMLKHPAPNVQGYLIG T. Devil KEPEIIYPRNNSIEVQLGSSLIIDCNIKDGKENLNSRSWKVNDTLVD--FLNSNRIKEGIESNVSCGEKYIFYTVNISFSEVRQEDYGRPFICISGSSAAFIMLKHPAPDVRGYLIG Armadillo RVPKITYPQNNSIKVRPGSVLIVDCNITDSRENTNSRSWKVNNTLVDHYYSASRRVREGIEKWVPS-QQDKFYTVNITFLEVKMEDYGRPFVCHAGVSAAYIVLELPAPDFRAYLIG Aardvark RVPKITYPRNNSIEVKLGSTLIVDCNITDLRENTNLRCWKVNNTQVNDYYRESKRIREGYESEVSF-EQYNKYTVNITFLEVKMEDYGRPFVCHAGVSAAYIVLKLPAPDSRAYLIG

IL1RL2 plPKIhYPKNNSIEVpLGosLIVsCNITDs+nNTNXRCW+VNsTlVDXYYtnS+RIpEGhEsXsXh-pcaXhYTVNITFLEVKMEDYGhPFhCHAGVSsAYhhLphPsPDhpAYLIG H Human DKPPKLLYPMESKLTIQETQLGDSANLTCRAFFGYSGDVSPLIYWMKGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRHASVLLHKRELMYTVE Platypus DRPPKLLHPLESKLTVQEIQLGNSANLTCRAFFGYSGEVSPLIYWMKGEKFIEDLDENRVWESDIRVLKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRQASVLLHKRELMYTVE Armadillo DKPPKLLYPLESKLTIQETQLGDSANLTCRAFFGYSGDVSPLIYWMKGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRHASVLLHKRELMYTVE Aardvark DKPPKLLYPVESKLTIQETQLGDSANLTCRAFFGYSGDVSPLIYWMKGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRHASVLLHKRELMYTVE Opossum DKPPKLLYPLESKLTIQETQLGNLANLTCRAFFGYSGDVSPLIYWMKGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRHASVLLHKRELMYTVE T. Devil ------GNLANLTCRAFFGYSGDVSPLIYWMKGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRHASVLLHKRELMYTVE

IL1R8 DKPPKLLYPhESKLTIQETQLGDSANLTCRAFFGYSGDVSPLIYWMKGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLGNYSCYVENGNGRRHASVLLHKRELMYTVE I Human DKPPKPLFPMENQPSVIDVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEKFIEELAGHIREGEIRLLKEHLGEKEVELALIFDSVVEADLANYTCHVENRNGRKHASVLLRKKDLIYKIE Armadillo DKPPKPLFPMENQPSVIEVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEKFIEELAGHIREGEIRLLKEHLGEKEVELTLIFDSVVEADLANYTCHVENRNGRKHASVLLRKKDLIYKIE Aardvark DKPPKPLFPMENQPSVIDVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEKFIEELAGHIREGEIRLLKEHLGEKEVELALIFDSVVEADLANYTCHVENRNGRKHASVLLRKKDLIYKIE Platypus DKPPKPLFPMENQPSVIDVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEKFIEELEGHIREGEIRLLKEHLGEKEVELTLTFDAVQEADLANYTCHVENRNGRKHASVLLRKKDLIYKIE T. Devil DKPPKPLFPRENHPSVIDVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEKFIEELAGHIREGEIRLLREHLGEKEVELTLIFDAVVEADLANYTCHVENRNGRKHASILLRKKDLIYKIE Opossum DKPPKPLFPRENQPSVIDVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEKFIEELAGHIREGEIRLLKEHLGEKEVELSLTFDAVVEADLANYTCHVENRNGRKYASILLRKKDLIYKIE

IL1R9 DKPPKPLFPhENpPSVIDVQLGcPLNIsCKAFFGFSGESGPMIYWMKGEKFIEELAuHIREGEIRLLKEHLGEKEVELsLIFDuVVEADLANYTCHVENRNGRKHASlLLRKKDLIYKIE J Human RAPDFLSPSEDQVLRPALGSSVALNCTAWVVSGPHCSLPSVQWLKDGLPLGIGGHYSLHEYSWVKANLSEVLVSSVLGVNVTSTEVYGAFTCSIQNISFSSFTLQRAGPTSHVA T. Devil TAPNILYPSKNQVLVPTLGSKITLNCTFSVAFEGHCP-PAIQWLKDDHLLGNQSLYDIQEVFWDGVNVSERIVSSTLGVNLTSVQDYGTFTCALRNCS-TSFTLQRTDPAGHVL Armadillo GDPRFLSPPGDQVLGPALGSVVTLNCTAWAAFGPHCSLPSIHWQKDGQLLDNRSHYRLQDDSWVSANWSEAFVSSVLGVTLTGAEDYGAFSCSIWNISSPSFTLWKAGPAGHVA Aardvark GSPDFISPASIQVLGPTLSTAVTLNCTAQVVSVASCPPPSVQWLKDGLPLGNRSHYSLQEDTWVRANLSEVLVSSVLEVNLTSAEDYGVFTCLVHNVSSSPFRLWRADPPGHVA

SIGIRR puPsFLSPstsQsLtsuLGSsVsLNCTAXVsSsPpCsXPSVQWLKDGLXLGstuHasL+EXXWlpsNhSElhVSSVLslNlTpsEsaGsFoCSltNlSSsSFTLXRAGXsuHVA