GPR15–C10ORF99 functional pairing initiates colonic Treg homing in amniotes
2021; Springer Nature; Volume: 23; Issue: 3 Linguagem: Inglês
10.15252/embr.202153246
ISSN1469-3178
AutoresJingjing Song, Huaping Zheng, Jingwen Xue, Jian Liu, Qian Sun, Wei Yang, Fang Liu, Xiangyin Xiang, Kai He, Younan Chen, Jingqiu Cheng, Wei Li, Jin Jin, Juergen Brosius, Cheng Deng,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle23 December 2021Open Access Transparent process GPR15–C10ORF99 functional pairing initiates colonic Treg homing in amniotes Jingjing Song Jingjing Song Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Huaping Zheng Huaping Zheng orcid.org/0000-0002-0716-8987 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Jingwen Xue Jingwen Xue orcid.org/0000-0002-5972-2329 Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Jian Liu Jian Liu Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Qian Sun Qian Sun orcid.org/0000-0002-9532-3042 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Wei Yang Wei Yang Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Fang Liu Fang Liu Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Xiangyin Xiang Xiangyin Xiang Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Kai He Kai He Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, and Guangdong Provincial Key Laboratory of Single Cell Technology and Application, Southern Medical University, Guangzhou, China Search for more papers by this author Younan Chen Younan Chen orcid.org/0000-0002-0455-2473 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Jingqiu Cheng Jingqiu Cheng Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Wei Li Wei Li orcid.org/0000-0002-9585-2884 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Jin Jin Jin Jin MOE Laboratory of Biosystem Homeostasis and Protection, and Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Juergen Brosius Juergen Brosius orcid.org/0000-0003-1650-2059 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Cheng Deng Corresponding Author Cheng Deng [email protected] orcid.org/0000-0002-5296-8879 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Jingjing Song Jingjing Song Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Huaping Zheng Huaping Zheng orcid.org/0000-0002-0716-8987 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Jingwen Xue Jingwen Xue orcid.org/0000-0002-5972-2329 Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Jian Liu Jian Liu Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Qian Sun Qian Sun orcid.org/0000-0002-9532-3042 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Wei Yang Wei Yang Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Fang Liu Fang Liu Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Xiangyin Xiang Xiangyin Xiang Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Kai He Kai He Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, and Guangdong Provincial Key Laboratory of Single Cell Technology and Application, Southern Medical University, Guangzhou, China Search for more papers by this author Younan Chen Younan Chen orcid.org/0000-0002-0455-2473 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Jingqiu Cheng Jingqiu Cheng Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Wei Li Wei Li orcid.org/0000-0002-9585-2884 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Jin Jin Jin Jin MOE Laboratory of Biosystem Homeostasis and Protection, and Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Juergen Brosius Juergen Brosius orcid.org/0000-0003-1650-2059 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Search for more papers by this author Cheng Deng Corresponding Author Cheng Deng [email protected] orcid.org/0000-0002-5296-8879 Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Search for more papers by this author Author Information Jingjing Song1,2,†, Huaping Zheng1,†, Jingwen Xue2, Jian Liu2, Qian Sun1, Wei Yang2, Fang Liu2, Xiangyin Xiang1, Kai He3, Younan Chen1, Jingqiu Cheng1, Wei Li1, Jin Jin4, Juergen Brosius1 and Cheng Deng *,1,2 1Institutes for Systems Genetics, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China 2Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China 3Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, and Guangdong Provincial Key Laboratory of Single Cell Technology and Application, Southern Medical University, Guangzhou, China 4MOE Laboratory of Biosystem Homeostasis and Protection, and Life Sciences Institute, Zhejiang University, Hangzhou, China † These authors contributed equally to this work *Corresponding author. Tel: +86 13405855560; E-mail: [email protected] EMBO Reports (2022)23:e53246https://doi.org/10.15252/embr.202153246 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Regulatory T lymphocyte (Treg) homing reactions mediated by G protein-coupled receptor (GPCR)–ligand interactions play a central role in maintaining intestinal immune homeostasis by restraining inappropriate immune responses in the gastrointestinal tract. However, the origin of Treg homing to the colon remains mysterious. Here, we report that the C10ORF99 peptide (also known as CPR15L and AP57), a cognate ligand of GPR15 that controls Treg homing to the colon, originates from a duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. Evolutionary analysis and experimental data indicate that the GPR15–C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes and their expression patterns are positively correlated with herbivore diet in the colon. With the first herbivorous diet in early amniotes, a new biological process (herbivorous diet short-chain fatty acid-C10ORF99/GPR15-induced Treg homing colon immune homeostasis) emerged, and we propose an evolutionary model whereby GPR15–C10ORF99 functional pairing has initiated the first colonic Treg homing reaction in amniotes. Our findings also highlight that GPCR–ligand pairing leads to physiological adaptation during vertebrate evolution. SYNOPSIS The GPR15–C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes. Their expression patterns are positively correlated with herbivore diet, suggesting that this functional pairing initiates the first colonic Treg homing reaction in amniotes. C10ORF99 originated via duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. The GPR15–C10ORF99 pair is functionally conserved to mediate the first colonic Treg homing reaction in amniotes. GPR15–C10ORF99 expression patterns in the colon correlate with an herbivore diet in amniotes. Introduction G protein-coupled receptors (GPCRs) comprise the largest membrane protein family (with > 800 members encoded in vertebrate genomes) and play normal physiological roles in cell proliferation, survival and motility, and are associated with multiple diseases (Kroeze, 2003). Dysfunctional GPCRs contribute to many human diseases and are targeted, directly or indirectly, by 50–60% of all therapeutic agents (Pierce et al, 2002; Kroeze, 2003). Chemokines are small secreted proteins that direct the systemic trafficking and microenvironmental homing of various cell types in health and disease (Kroeze, 2003; Zlotnik & Yoshie, 2012; Zabel et al, 2015). Chemokines signal through class-A GPCRs and participate in homeostatic and inflammation-induced immune cell trafficking (Zlotnik & Yoshie, 2012; Zabel et al, 2015). Particularly, chemokines and their cognate GPCRs control the recruitment of various types of lymphocytes from the blood, contributing to the systemic organization of the immune system (Kim et al, 2013; Ocon et al, 2017). For example, the homing of CCR9-positive regulatory T lymphocytes (Tregs) to the small intestine is activated by CCL25; Treg homing to CXCR4-positive bone marrow cells is activated by CXCL12; and CCR4 activated by CCL17 is associated with Treg homing in the heart (Ding et al, 2012; Castan et al, 2017). GPR15 has been considered an orphan GPCR and an HIV co-receptor with sequence similarity to leucocyte-chemoattractant receptors (Adamczyk, 2017; Suply et al, 2017). Recently, GPR15 was highlighted as a colon-specific Treg-homing receptor (Kim et al, 2013; Nguyen et al, 2014). A GPR15–GFP knock-in model revealed selective GPR15 expression by colon Tregs under homeostatic conditions and GPR15-mediated Treg recruitment to the colon (Kim et al, 2013). CCR9 functions similarly by promoting Treg recruitment to the small intestine (Johnson et al, 2010; Perrigoue et al, 2014). GPR15L (encoded by human C10ORF99 and mouse 2610528A11Rik) was identified as the cognate ligand of GPR15 (Yang et al, 2015; Suply et al, 2017). C10ORF99 is mainly expressed by epithelial cells in the gastrointestinal tract (particularly the colon and rectum) and attracts Treg subsets in a GPR15-dependent manner (Kim et al, 2013). Moreover, colonic C10ORF99 expression is developmentally determined and affected by inflammation and the microbiota (Ocon et al, 2017). Therefore, GPR15–C10ORF99 signalling is important for recruiting specialized T lymphocytes to the colon and sites of cutaneous inflammation (Ocon et al, 2017; Suply et al, 2017), and the colon exhibits a different immune regulation than the small intestine. Animal microbiota in the gastrointestinal tract have coevolved with the host (Mazmanian et al, 2005; Ley et al, 2008; Kamada et al, 2012), and their coexistence reflects an equilibrium established with the host immune system (Hooper et al, 2012; Kim et al, 2013). The colon harbours significantly more microbiota than the small intestine (Geuking Markus et al, 2011; McGuckin et al, 2011; Kuhn & Stappenbeck, 2013) and higher Treg frequencies (Atarashi et al, 2016). Humans and mice rely on colonic bacteria to break down undigestible dietary components such as fibres (Remely et al, 2013). Short-chain fatty acids (SCFAs) are bacterial fermentation products with variable concentrations (50–100 mM) in the colonic lumen (Cummings et al, 1987). In mice, SCFAs and the SCFA receptor (GPR43) may help induce colonic Treg homing (Perrigoue et al, 2014). Tregs utilize GPR15 (inducible by SCFAs) for colonic homing, and SCFAs and GPR43 are required to generate and expand colonic Tregs (Perrigoue et al, 2014). Moreover, SCFAs induce GPR15 expression, hence contributing to colonic Treg homing (Perrigoue et al, 2014). Interestingly, an herbivorous diet increases SCFA contents and microbial metabolites in the colon (Perrigoue et al, 2014). These data suggest that an herbivorous diet promotes SCFA- and GPR15-dependent colonic Treg homing in humans and mice. GPCR–ligand pairs typically show high binding affinities and conserved functions in vertebrates and invertebrates (Strotmann et al, 2011; Vogel et al, 2013). Consequently, polypeptide ligands (i.e. apelin, angiotensin, cholecystokinin) from different vertebrates (even humans and fishes) can cross-stimulate GPCRs, and these orthologous receptor–ligand pairs control similar physiological functions across all vertebrates (de Mendoza et al, 2014; Hu et al, 2017). The small intestine is conserved across all vertebrates, and receptor–ligand (CCR9–CCL25)-dependent Treg homing is conserved to maintain physiological functions in vertebrates (Devries et al, 2006; Perrigoue et al, 2014). However, the emergence of the colon in tetrapods and the origination of colonic Treg homing remain mysterious. Here, we studied the origin and functional evolution of the GPR15–C1ORF99 pairing in vertebrates to gain insight into early colonic immune function in tetrapods. Results GPR15 evolved differently in fishes and amniotes Vertebrate amino acid sequences of the GPR15, GPR25, apelin receptor (APJ), angiotensin (AGT) receptor 1 (AGTR1) and bradykinin receptor B2 (BDKRB2) were downloaded from the NCBI and Ensemble databases. A consensus neighbour-joining tree was built for the apelin/angiotensin/bradykinin receptor family (including mammals, birds, reptiles, amphibians and fishes), using MEGA 7.0.26 (JTT + G + I; bootstraps, 500; cut-off for the condensed tree, 20%) (Fig 1A). An ancestral gene, AGTR-like, from Ciona intestinalis served as an outgroup (Fournier et al, 2012). A similar tree was derived using maximum likelihood analysis, implemented in MEGA 7.0.26 (Fig EV1A). Only GPR25 was still considered as an orphan GPCR; bradykinin (KNG1), AGT, apelin and C10ORF99 are endogenous peptide ligands for BDKRB2, AGTR1, APJ and GPR15 respectively (Fig 1A; Yang et al, 2015; Suply et al, 2017). GPR25, AGTR1 and BDKRB2 were highly conserved among all vertebrates tested (Fig 1A and B). However, GPR15 orthologues were detected in mammals, birds, three kinds of fishes and reptiles, but was presumably deleted in amphibians and most fishes. Regarding fish GPR15, Latimeria chalumnae belongs to the Sarcopterygii clade, and Lepisosteus oculatus and Scleropages formosus belong to the Actinopterygii clade, which did not undergo a third round of whole-genome duplication (Bian et al, 2016). GPR15 was not detected in other fishes, particularly those who underwent a third round of whole-genome duplication. Figure 1. Evolution of the apelin/angiotensin/bradykinin peptide receptor subfamily Phylogenetic tree of the apelin, angiotensin and bradykinin receptor, Scale (0.1). The Ciona intestinalis AGTR-like gene served as an outgroup. The ω parameters of two fish branches are shown. Scale: The unit of evolutionary distance is the number of amino acid substitutions at each position. Conservation of the apelin/angiotensin/bradykinin peptide receptor subfamily among all vertebrates except for the GPR15–C10ORF99 pair. *Indicates that these three fish species did not undergo a third round of whole-genome duplication during fish evolution. Result of rapidly evolving genes (REGs). This calculates the χ2 critical value and P-value for conducting the likelihood ratio test. The genes with P < 0.05 were considered. GPR15 underwent positive selection in fishes. The parameters for each branch are shown; the dN/dS ratio was calculated with the whole protein-coding region. **P < 0.01, *P < 0.05, NA: not available. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Phylogenetic tree of the apelin/angiotensin/bradykinin peptide receptor subfamily showing rapidly evolving genes and positive selection in other GPR15 family receptors The evolutionary history was inferred using the maximum likelihood method implemented in MEGA7.0.26 (Sudhir et al, 2016). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2,000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree, Scale (0.2). Result of rapidly evolving genes (REGs) analysis of apelin/angiotensin/bradykinin peptide receptor subfamily, χ2 test. The positive selection results of apelin/angiotensin/bradykinin peptide receptor subfamily were shown. NA, not available. Scale: The unit of evolutionary distance is the number of amino acid substitutions at each position. Branch numbers indicate bootstrap. ω ratio: nonsynonymous (dN)/synonymous (dS) substitution rate ratio. Download figure Download PowerPoint After removing gaps, indels and stop codons from the alignment, we obtained 981-base pair sequences for the GPR15 open-reading frame (ORF) sequences. We used the same orthologous genes and tree topology to identify rapidly evolving genes (REGs) in the GPR15 receptor family. The fish GPR15 genes were found to evolve rapidly (Fig 1C). The same REGs were identified in other receptors in the same family (Fig EV1B). No other GPCRs from the same family underwent rapid evolution (Fig EV1B). REGs can evolve neutrally or under positive selection. A branch-site model was utilized to assess whether GPR15 homologues underwent positive selection in different species. The GPR15 sequences of two lineages of fishes (Latimeria chalumnae and Lepisosteus oculatus/Scleropages formosus) had a large nonsynonymous (dN)/synonymous (dS) substitution rate ratio (branch site dN/dS of ω >> 1; Fig 1A and D), which was highly significant (likelihood ratio tests [LRTs], P < 0.05; Fig 1D). No other branches exhibited this ratio (Fig 1A and D). Residues that underwent positive selection differ in both fish lineages (Fig 1D, Dataset EV1). The same branch-site model was utilized to detect whether other receptors in the same family underwent positive selection as well (Fig EV1C). No other GPCRs from the same family underwent positive selection (Fig EV1C). The C10ORF99 ligand originated from CDHR1 gene duplication in amniotes To determine the origination of the GPR15–C10ORF99 receptor–ligand relationship, the gene synteny of vertebrate chromosomes containing these genes was analysed (Fig 2A). GPR15 exists in various vertebrate species (but not amphibians and most fishes) and shows conservation with its flanking gene. GPR15 localizes closely with CLDND1 on the same chromosomes of all vertebrates, although the corresponding amphibian and most fish chromosomes lack both GPR15 and C10ORF99 (Fig 2A). Similarly, the gene encoding the C10ORF99 ligand, and the flanking genes CDHR1, GHITM, LRIT2 and LRIT1, are highly conserved in amniotes, but C10ORF99 is absent from both fishes and amphibians (Fig 2A). BLAST searches detected the C10ORF99 gene among several non-protein-coding RNAs of reptiles and birds, and these sequences were conserved, particularly in regions encoding mature peptides and both cystine linkages of C10ORF99 (Dataset EV2–EV4). Interestingly, the signal peptide of CDHR1 in amniotes, but not of other vertebrates (fishes and amphibians), share similarity with the signal peptide encoded by C10ORF99 (an upstream gene; (Fig 2A–C and Dataset EV5 and EV6). Moreover, aligning C10ORF99 and CDHR1 of Chelonia mydas showed that CDHR1 genes shared large nucleotide similarities with C10ORF99, including 56% similarity between their 5′-untranslated regions (UTRs); 61% similarity between the protein coding region of exon 1, which encodes the signal peptide for both C10ORF99 and CDHR1; 59% similarity between intron 1; 81% similarity between CDHR1 intron 6 and C10ORF99 intron 1, 45% similarity between CDHR1 exon 7 and C10ORF99 exon 2, 80% similarity between CDHR1 intron 8 and C10ORF99 3′-UTRs, 93% similarity between CDHR1 intron 11 and C10ORF99 3′-UTRs respectively (Fig 2C, Dataset EV5). But C10ORF99 shared no significant similarity with CDHR1 of fishes or amphibians (Fig 2C). The CDHR1 genes also shared high similarity (5′-UTR, the protein coding region of exon1 and intron 1) with C10ORF99 in other reptiles, birds and mammals including humans, based on alignments with sequences from Homo sapiens, Mus musculus, Phascolarctos cinereus, Coturnix japonica, Nothoprocta perdicaria, Dromaius novaehollandiae, Alligator mississippiensis, Pelodiscus sinensis and Chelonia mydas (Fig 2B and Dataset EV6). The protein coding region of exon 1 of CDHR1 and C10ORF99, encoding their signal peptide, shared relatively high conservation of protein sequences and nucleic acid sequences (Fig 2B and C, Dataset EV5), and CDHR1 and C10ORF99 also had similar 5′-UTRs, which was consistent with the high colonic expression of both CDHR1 (expression data from NCBI database) and C10ORF99. In contrast, exon 2 of C10ORF99 exhibits a different ORF compared with CDHR1 exon 7, although they share similarity in nucleic acid sequences (Dataset EV5). Therefore, we conclude that C10ORF99 originated from reptile CDHR1 gene. This process encompassed exon deletions, co-optation of a novel exon (3) and coding sequence changes by frame-shift yielding a new gene product, namely the C10ORF99 mature peptide (encoded by exon 2 and exon 3, Fig 2C). Figure 2. The C10ORF99 ligand originated in amniotes via CDHR1 gene duplication Synteny for chromosomal regions containing the GPR15–C10ORF99 pair. The pair and their neighbouring genes in vertebrate genome fragments are shown. Deep red shading represents exon 1 of the CDHR1 gene of amniotes. Deep grey colour represents exon 1 of the CDHR1 gene of amphibians and fishes. Light red represents exon 1 of the C10ORF99 gene. 3'rd WGD indicates the fishes that underwent a third round of whole-genome duplication. Amino acid sequence alignment of C10ORF99 and CDHR1 signal peptides. Molecular evolution of C10ORF99 from CDHR1. Regions in CDHR1 corresponding to regions in the C10ORF99 gene and nucleotide sequence similarities are given. The locations of the protein-coding regions of exons and as well as some intron locations are marked, and protein-coding regions of exon sequences are bold and capitalized. Coding means protein coding region in exon. Download figure Download PowerPoint GPR15–C10ORF99 functional pairing in amniotes Five prototypical vertebrate GPR15 homologues were selected for functional pairing experiments, including human and mouse GPR15 (hGPR15 and mGPR15 respectively), Japanese quail GPR15 (qGPR15), painted turtle GPR15 (tGPR15), Latimeria chalumnae/coelacanth GPR15 (cGPR15) and Scleropages formosus/Asian bony tongue GPR15 (aGPR15). Stimulation of intracellular calcium by GPR15 receptors requires ligand binding to stimulate Gq signalling (Yang et al, 2015; Suply et al, 2017). Human C10ORF99 (hC10ORF99) and Japanese quail C10ORF99 (qC10ORF99), featuring conserved mature peptides of C10ORF99 in mammals and reptiles/birds, respectively (Dataset EV2), were synthesized for receptor–ligand function assays (Suply et al, 2017; Fig 3A–G). Both hC10ORF99 and qC10ORF99 can interchange to stimulate hGPR15, mGPR15, qGPR15 and tGPR15 (Fig EV2A). The hC10ORF99 was utilized to activate cells transfected with the empty vector plasmid pcDNA3.1-V5-His (Fig 3A) or the pcDNA3.1-V5-His containing a gene encoding one of the mammalian GPR15, and qC10ORF99 was utilized to activate cells transfected with the pcDNA3.1-V5-His containing a gene encoding one of the reptile/bird/fish GPR15. The neurotensin (NTS) peptide was used to stimulate the human neurotensin receptor 1 (NTSR1) as a positive control for Gq-signalling activation (Fig 3A; Slosky et al, 2020). A remarkable increase in Ca2+ ions was observed in cells expressing hGPR15 (highest stimulation, 10 nM), mGPR15 (highest stimulation, 10 nM), tGPR15 (highest stimulation, 100 nM) and qGPR15 (highest stimulation, 10 nM) (Fig 3B–E), but not cGPR15 or aGPR15 (Fig 3F and G), when stimulating with qC10ORF99 (even with high dose-1 μM). YM.254890, a well-known inhibitor of the Gq-signalling pathway, was used to confirm GPR15 receptor–ligand stimulation (Schrage et al, 2015; Tang et al, 2019). The Gq-signalling pathway was stimulated by hGPR15, mGPR15, tGPR15 and qGPR15, but inhibited by YM.254890 (Fig 3H). Figure 3. GPR15–C10ORF99 functional pairing in amniotes A–G. The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of the indicated GPR15 receptors (human, mouse, Japanese quail, painted turtle, coelacanth and Asian bony tongue GPR15). A plasmid-encoding human NTSR1 and the parental pcDNA plasmid were transfected as positive and negative controls respectively. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm respectively). Calcium RLU (relative light unit) fold was calculated using no stimulation data as standard value. H. The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in human, mouse, Japanese quail, painted turtle. YM.254890 inhibits Ca2+ ion levels in cells transfected with GPR15 receptors of vertebrates. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm respectively). I, J. The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in coelacanth, Asian bony tongue and their mutants. K, L. The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulating GPR15 in Japanese quail and its mutants. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm respectively). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. The cellular levels of Ca2+ ions after C10ORF99 stimulation of the GPR15 receptor A, B. The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of the indicated GPR15 receptors cGPR15 and its mutants (A), aGPR15 and its mutants (B). C. GPR15 from different species but not fishes could be activated by both hC10ORF99 and qC10ORF99. The calcium fold levels were calculated by fluorescence intensity (excitation/emission wavelength: 490/520 nm). Download figure Download PowerPoint Several point mutations
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