Identification of Two Nonrearranging IgSF in Chicken Reveals a Novel Family of Putative Remnants of an Antigen Receptor Precursor This information is current as of September 28, 2021. Yanbin Fu, Zhi Yang, Jinwei Huang, Xueqian Cheng, Xifeng Wang, Shiping Yang, Liming Ren, Zhengxing Lian, Haitang Han and Yaofeng Zhao J Immunol published online 15 February 2019 http://www.jimmunol.org/content/early/2019/02/14/jimmun Downloaded from ol.1801305

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 15, 2019, doi:10.4049/jimmunol.1801305 The Journal of Immunology

Identification of Two Nonrearranging IgSF Genes in Chicken Reveals a Novel Family of Putative Remnants of an Antigen Receptor Precursor

Yanbin Fu,*,1 Zhi Yang,*,1 Jinwei Huang,*,1 Xueqian Cheng,* Xifeng Wang,† Shiping Yang,* Liming Ren,* Zhengxing Lian,‡ Haitang Han,* and Yaofeng Zhao*

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane , and their recombinant products formed a monomer and a

disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in Downloaded from response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of

the immune system of jawed vertebrates. The Journal of Immunology, 2019, 202: 000–000. http://www.jimmunol.org/

he V(D)J recombination-mediated adaptive immunity relies on the gross diversity in the V domains of TCRs and Igs, and arose ∼600 million years ago (MYA) during a narrow each of these V domains is generated by a process called somatic T period when the common ancestor of jawed vertebrates, gene rearrangement (3). The V, D, and J gene segments are so- placoderms, had just evolved from agnathans (1, 2). Unlike innate matically assembled in a split germline configuration by the RAG immunity, which features the presence of a limited number of products, RAG-1/2, and this process results in the combinatorial germline-encoded receptors or cytokines, the adaptive immune diversity that is responsible for a substantial proportion of the system (AIS) exhibits extraordinary flexibility in generating the diversity in V regions (4–8). The AIS appears to be conserved in rearranging Ag receptor repertoire. This enormous repertoire all jawed vertebrates, ranging from cartilaginous fishes (including by guest on September 28, 2021 sharks, skates, and chimaeras), which are the most evolutionarily ancient extant species, to eutherian mammals. However, the absence *State Key Laboratory of Agrobiotechnology, College of Biological Science, National Engineering Laboratory for Animal Breeding, China Agricultural University, Beijing of a RAG recombinase-based defense system in jawless vertebrates 100193, People’s Republic of China; †Key Laboratory of Animal Ecology and Conser- has been undoubtedly confirmed; instead, their adaptive immune vation Biology, Institute of Zoology, Chinese Academy of Science, Beijing 100101, ‡ responses of these jawless fishes depend on the variable lymphocyte People’s Republic of China; and Laboratory of Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, receptors (9). People’s Republic of China The origin of the AIS has always been intriguing but remains 1Y.F., Z.Y., and J.H. contributed equally to this work. obscure. It has been widely suggested that both transposable el- ORCIDs: 0000-0001-8454-1071 (X.C.); 0000-0001-5631-3549 (S.Y.); 0000-0001- ement pollution and nonrearranging Ig superfamily (IgSF) mem- 8418-4916 (Z.L.); 0000-0003-4499-8610 (Y.Z.). bers contribute vastly to the first step in the development of adaptive Received for publication September 27, 2018. Accepted for publication January 22, immunity (2, 10–12). The V(D)J recombinase complex compo- 2019. nent RAG-1 is thought to be derived from transposase, which is This work was supported by the National Natural Science Foundation of China encoded by autonomously jumping transposons dating back to (31472085, 31272433). prokaryotes (13). Despite notable research progress, including the The sequences presented in this article have been submitted to the National Center for Biotechnology Information’s GenBank (http://www.ncbi.nlm.nih.gov/) under ac- finding that transposons initiated their invasion into an intact cession numbers MK349013 and MK349014. V exon of IgSF and the indication that transposons gave rise to the Address correspondence and reprint requests to Prof. Yaofeng Zhao and Dr. Haitang TCR and BCR genes, there is scarce information regarding the Han, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, rare IgSF members, which are hypothesized to be the evolutionary China Agricultural University, Beijing 100193, People’s Republic of China. E-mail addresses: [email protected] (Y.Z.) and [email protected] (H.H.) precursors of the rearranging Ag receptors seen today (8, 14, 15). The online version of this article contains supplemental material. All IgSF members contain Ig domain(s). In general, Ig domains can be classified into four types based on differences in their structure Abbreviations used in this article: AIS, adaptive immune system; BAC, bacterial arti- ficial ; bursa of F., bursa of Fabricius; C, constant; CHIR, chicken Ig-like and sequence: V-set, C1-set, C2-set, and I-set (16). The Ag receptor receptor; EGFP, enhanced GFP; F, forward; IgSF, Ig superfamily; IP, immunoprecipi- V regions belong to the V-set type, whereas the C2-set includes most tation; ITSM, immunoreceptor tyrosine-based switch motif; LP, leader peptide; LRC, leukocyte receptor cluster; MYA, million years ago; PAMP, pathogen-associated mo- Ig domains in Fc receptors and clusters of differentiation molecules, lecular pattern; PDB, Data Bank; PIGR, poly-Ig receptor; PRARP, putative and the I-set constitutes cell adhesion molecules and several other remnants of an Ag receptor precursor; R, reverse; SIRP, signal-regulatory protein; protein families (17). The C1-set is mainly found in the constant (C) w.p.i., week postinfection. regions of Igs, TCRs, and the MHC (or MHC-linked) molecules that Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 are exclusively present in gnathostomes. Thus, it has been speculated

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801305 2 DESCENDANTS OF AN ANCESTRAL Ag RECEPTOR that the C1-set originated at the same time as the rearranging immune 10% FCS (Gemini Bio-Products), 2% chicken serum (Life Technologies, system and that these coevolved (18–22). Therefore, any invariant Thermo Fisher Scientific), 10 mM HEPES (Life Technologies, Thermo 3 receptors with a V-C1 core can be regarded as relatively immemorial, Fisher Scientific), and 1 penicillin-streptomycin (MacGene) in a CO2 incubator (Thermo Fisher Scientific) at 37˚C. The Marek disease virus- with an emergence time equal to that of jawed vertebrates. transformed lymphoblastoid cell line MDCC-MSB1 was propagated at As mentioned above, there is scarce information regarding the 37˚C in RPMI 1640 medium (Life Technologies, Thermo Fisher Scien- precursors of rearranging Ag receptors. To address this issue, at- tific), supplemented with 10% FCS and 13 penicillin-streptomycin, and tention was automatically drawn to hagfishes and lampreys, which the chicken fibroblast cell line DF-1 was propagated at 37˚C in DMEM (Life Technologies, Thermo Fisher Scientific), supplemented with 10% are the existing jawless organisms closest to the early jawed FCS and 13 penicillin-streptomycin. vertebrate ancestors. Pancer et al. (23) identified a prototypic DNA and RNA extraction, amplification of full transcripts by TCR (TCR-like) in lamprey that is preferentially expressed in RACE, spleen cell subset isolation, Southern blotting, and lymphocyte-like cells, and this receptor was later proven to pos- analyses of V region variations sess inhibitory signaling potential in vitro by recruiting the protein tyrosine phosphatases (SHP)-1 and SHP2 (24). A closer relative to The total RNA from chicken spleen was extracted using the RNeasy Mini the lamprey TCR-like receptor was found in hagfish, and this Kit (QIAGEN), and first-strand cDNAs were synthesized with SuperScript finding gave rise to the polymorphic multigene families called II Reverse Transcriptase (Invitrogen) and gene-specific primers 1 (GSP1) for chicken PRARP1 (59-GTG TTC ATA TTT CTG CGT TCC-39) and ITAM-containing IgSF receptors (NICIR) and agnathan-paired chicken PRARP2 (59- CAC GGT GCC ATT GCC AAA CTT C-39). The receptors resembling Ag receptors (APAR) (25, 26). Addition- cDNA purification, TdT tailing, and the following PCRs were conducted ally, several IgSF members, which have structures that are rela- using the 59 RACE System for RACEs (Invitrogen) according to the tively similar to that of Ag receptors and have been denoted V and manufacturer’s instructions. For the 39 RACE, the cDNA was acquired Downloaded from using Moloney murine leukemia virus reverse transcriptase (Promega), the C domain-bearing protein (VCP) and V region-containing chitin- oligo(dT)20 primer, and forward (F) primers for chicken PRARP1 (F1: 59- binding protein (VCBP), were found in an early chordate, am- AGC CCG GAT TCC TCA CGT TCA-39, F2: 59-AAC GGG AGG ACT phioxus (Branchiostoma belcheri), but their V domains share little CCG GCT TTT-39) or chicken PRARP2 (F1: 59- CGT GGA ACC TGG sequence identity with those of TCR or Ig (27, 28). Overall, the GGC TGC AGT G-39, F2: 59- GTC GCC CCA ACT GCT TCT GAA above receptors have the following commonalities that make C-39). Four-week-old chicken splenocytes treated with ammonium- chloride-potassium lysing buffer were used for lymphoid and myeloid them top candidates for studying ancestral TCR/Ig genes: 1) one cell isolations. Fluorescent cell sorting was performed on BD Influx (BD http://www.jimmunol.org/ or multiple nonrearranging V-set Ig domains in the distal mem- Biosciences), and data were analyzed with FlowJo software. For T and brane and signaling motifs or charged residues in the cytoplasmic B cell sorting, ∼106 splenocytes were labeled with 1 mg FITC-conjugated tail, which associates with adaptor molecules (e.g., FcRg- anti-chicken CD3 mAb (Clone CT-3) and 0.2 mg PE-conjugated anti–Bu-1 mAb (Clone AV20) (both from SouthernBiotech), respectively (31, 32). The chain, DAP12); and 2) preferential expression in lymphocyte-like monocytes/macrophages were sorted as double-positive cells (KUL01+/ cells or leukocytes and potential function as an immune regulator. MHC II+) by labeling with 0.2 mg PE-conjugated KUL01 mAb and 1 mg The gnathostome lineages also contain a few IgSF members, FITC-conjugated anti–MHC II mAb (both from SouthernBiotech) for per such as the signal-regulatory proteins (SIRP) family, which might million cells (33). The following primers spanning were used in the have originated from a precursor of the rearranging Ag receptors subsequent RT-PCR analysis: chicken PRARP1 (F: 59-TGA GCC CTT TGA GTT TGG GG-39, reverse [R]: 59-ACC ATT GCA TAC TCG GTC TCC-39) by guest on September 28, 2021 (29, 30). It is possible that some other gene relics survived the and chicken PRARP2 (F: 59-TCT GTG CGA ACA CCA TCG TC-39,R: prototypical RAG transposon-mediated disruption of the “big 59-TGA TGG GAC AGG AGC GT-39). To analyze the expression of these bang” event that resulted in the emergence of the AIS and per- genes in primary cells and cell lines, PCR using 23 Taq Master Mix sisted throughout the subsequent evolutionary process of jawed (CWBIO) was performed with the following temperature program: pre- denaturation at 95˚C for 2 min, 35 cycles of denaturation at 95˚C for 30 s, vertebrates. In this study, we describe the cloning and character- annealing at a specific temperature for 30 s and extension at 72˚C for 30 s, ization of two IgSF genes that display typical inhibitory features and a final extension of 7 min at 72˚C. Genomic DNA from the chicken in chickens and can be classified into a novel gene family, named liver was isolated according to the routine phenol-chloroform method, and putative remnants of an Ag receptor precursor (PRARP) based on the obtained genomic DNA was digested with various restriction enzymes, the presence of nonrearranging VJ segments, a V-C1 structure, and separated by 0.8% agarose gel electrophoresis, transferred to a positively charged nylon membrane (Roche), and hybridized. The PRARP V exon- the results of phylogenetic analyses. The PRARP multigene family specific probes were labeled with digoxigenin-11–dUTP using a PCR can be found in birds, reptiles, amphibians, and bony fish but not Digoxigenin Probe Synthesis Kit (Roche) and the following PCR primers: in mammals. The comprehensive characterization of PRARP PRARP1 F, 59-GGG GCT GCC CCA CAC TCA GA-39,R,59-TCC CAA family members has allowed us to introduce this novel discovery TGC TCA CCT GTG AC-39; and RRARP2 F, 59-TCC CCG CAG CGC TGC TGA AG-39,R,59-TCC CCC CAC TCC TTC CTA CCT C-39. regarding the descendants of an ancestral Ag receptor and to de- Hybridization and final detection were conducted using the DIG-High velop an evolutionary model for this novel family. Prime DNA Labeling and Detection Starter Kit II (Roche) according to the manufacturer’s instructions. For the analysis of V region variation, PCR products spanning the entire V exons of chicken PRARP were directly Materials and Methods sequenced, and the nucleotide mutations were tallied based on DNA se- Animals and cell lines quencing chromatograms. Commercial White Leghorn chickens (hatching eggs, specific pathogen- DT40 cell stimulation, chicken immunization, and free grade) were purchased from Merial Vital Laboratory Animal Tech- quantitative RT-PCR nology (Beijing, China) and used to determine their expression pattern and for vaccination simulations at the ages of 1 d, 1 mo, and 33 mo. All the The DT40 cell cultures in the six-well plates (∼5 3 106 cells/well) of the ex- animal experiments performed in this study were certificated by the Ani- perimental groups were incubated with (1) 100 ng/ml Escherichia coli O111:B4 mal Care and Use Committee of China Agricultural University. Blood LPS (Beyotime Biotechnology) for 0, 3, 6, 12, or 24 h or (2) 10 mg/ml poly(I:C) samples from eight chicken breeds (including Chinese native breeds (Sigma-Aldrich) for 6, 8, 12, or 24 h. The control groups were given the buffer Chahua, Taihe Silky fowl, Wenchang, Beijing Youji, Gamecock, Tibetan used to dissolve the above-mentioned pathogen-associated molecular fowl, and the commercial breeds White Leghorn and Arbor Acres broiler) patterns (PAMPs). At the end of the incubation period, the cells were used in the gene polymorphism analysis were generous gifts from Prof. Hu harvested in a 15-ml conical tube by gentle centrifugation, washed with (China Agricultural University, Beijing). The White Peking duck bacterial PBS, and lysed with the MagZol Reagent (Magen BioSciences). Total artificial chromosome (BAC) genomic library deposited in the labora- RNA extraction and single-strand cDNA synthesis were performed as tory was used for BAC clone screening. The chicken B lymphoblast cell described above. The gene-specific quantitative primers are as follows: line DT40 was maintained in RPMI medium modified with 2.05 mM chicken PRARP1 F, 59-ATG AGG AGA GCG AAG GGA TT-39,R,59- L-glutamine (HyClone; GE Healthcare Life Sciences), supplemented with CCA AAC TCA AAG GGC TCA GA-3; chicken PRARP2 F, 59-CGT CCT The Journal of Immunology 3

CTT CGT GGA ACC TG-39,R,59-TGT CGG AAC CAC AGC ACT TT- PBS with Tween 20 and incubated with a Cy3-labeled goat anti-mouse IgG 3; chicken cathelicidin-B1 F, 59-GCA TCT GGG AGT GGT TGA ATG-39, (H+L) secondary Ab (Beyotime Biotechnology) for 1 h at room temper- R, 59-AGG CAG AAG GGA CGT TTA TT-39; IFN-b F, 59-GCC CAC ature. The cells were resuspended in mounting medium and placed at the ACA CTC CAA AAC ACT G-39,R,59-TTG ATG CTG AGG TGA GCG center of a clean microscope slide, which was subsequently covered with a TTG-39; and GAPDH F, 59-CCT CTC TGG CAA AGT CCA AG-39,R,59- coverslip. The samples were then observed using the DeltaVision OMX SR CAT CTG CCC ATT TGA TGT TG-39. For chicken immunization, the imaging system (GE Healthcare Life Sciences). chicks were immunized with the inactivated bivalent avian influenza virus vaccine H5N1 strain Re-6 and H9N2 strain Re-2 (Qingdao Yebio Bioen- Three-dimensional structure modeling and gineering) at the age of 2 wk or with an inactivated avian colibacillosis sequence alignments vaccine mixed with strain EC24, 30, 45, and 50 (Huahong) at the age of 1 wk, according to the manufacturers’ instructions. The immune-related The secondary structure and transmembrane region were predicted online tissues from the virus and bacterial groups were finally collected at 1, 2, (https://www.predictprotein.org/) and using the TMpred program in the and 4 wk postimmunization and at 1 and 4 wk postimmunization, re- ExPASy Bioinformatics Resource Portal. The most suitable model tem- spectively. Chicken EF1A1 was selected as the internal control gene: F, 59- plate for three-dimensional structure construction was found using the AAC CAC CCT GGC CAA ATC AGT-39,R,59-TGC CGG AAC GAC SWISS-MODEL program (http://swissmodel.expasy.org/) (35). Because of ab GAT CAA TCT-39. A quantitative RT-PCR was performed using Light- a high-sequence similarity with the TCR -chain V regions or IgH/IgL Cycler 480 SYBR Green I Master mix (Roche) with the above-mentioned C regions, template structures of human MR1–diclofenac in complex with primers and the following protocol: 95˚C for 5 min and 40 cycles of 95˚C human mucosal-associated invariant A-F7 TCR ( for 10 s, 60˚C for 10 s, and 72˚C for 10 s. The relative expression levels of [PDB]: 5U1R) and of the C regions from Ig were selected for homology chicken PRARP mRNA were calculated using the 22OOCt method by modeling (Table I). The protein structure visualizations were prepared with comparing the values with the internal control values. PyMOL software (36). The DNA and protein sequence editing, align- ments, and comparisons were performed using the DNASTAR Lasergene

Vector constructions, stable transfection, and establishment of software suite (37). Multiple sequence alignments were performed using Downloaded from overexpressing cell lines the ClustalW or ESPript (http://espript.ibcp.fr) programs (38). The pIRES2–enhanced GFP (EGFP) (BD Biosciences, Clontech) plasmids Phylogenetic analyses encoding a Kozak consensus (59-GCC ACC-39) upstream of the leader peptides (LP) of chicken PRARP1 and chicken PRARP2, as well as a Myc Phylogenetic and molecular evolutionary analyses were conducted using and a FLAG epitope tag immediately downstream from the predicted Molecular Evolutionary Genetics Analysis 6.0 (39) based on the neighbor- chicken PRARP1 and chicken PRARP2 LP cleavage sites, respectively, joining method with 1000 bootstrap replicates. FigTree version 1.4.1 was used to view the trees. The accession numbers of the sequences used in the were constructed. Briefly, DNA fragments from full-length cDNA were http://www.jimmunol.org/ amplified with the Q5 High-Fidelity DNA Polymerase (New England construction of the phylogenetic tree obtained in this study are listed in BioLabs) and then inserted into the backbones to generate plasmids termed Supplemental Table II. pIRES2-EGFP-N-Myc-PRARP1 and pIRES2-EGFP-N-FLAG-PRARP2. Statistical analysis For stable transfection, DT40 cells were transfected using the DMRIE-C reagent (Invitrogen) with linearized pIRES2-EGFP-N-Myc-PRARP1, lin- The column bar graphs obtained after cell stimulation represent the earized pIRES2-EGFP-N-FLAG-PRARP2, or both of the above plasmids results from three independently repeated experiments, and the bars in equal amounts according to the manufacturer’s recommended protocol. indicate the means with SDs. The immunization data were obtained The cells were harvested at 48 h posttransfection, plated in Cell Plaza from five chickens per group, and the bars indicate the means 6 SEMs. (Genloci Biotechnologies), and screened with 800 mg/ml geneticin The statistical significance of the differences among the groups was cal- (Sigma-Aldrich). After 2 wk of selection and propagation, single GFP+ culated using t test with a two-tailed p value, and the confidence intervals colonies were screened with BD Influx (BD Biosciences). Three cell lines were set to 95%. by guest on September 28, 2021 with high expression levels, designated DT40-Myc-PRARP1, DT40- FALG-PRARP2, and DT40-Co, were established and used in the subse- quent experiments. Results Identification of two nonrearranging, inhibitory IgSF members Immunoprecipitation, Western blotting, immunofluorescence, in chicken and superresolution imaging A pair of intact exons encoding nonrearranging V Ig domains was 3 7 Approximately 5 10 cells were harvested, washed once with ice-cold accidentally found from a Basic Local Alignment Search Tool PBS, and lysed for 40 min on ice with a Western and immunoprecipitation (IP) cell lysis buffer (Beyotime Biotechnology) containing 1% Triton search against the chicken genome using a TCR sequence. RACE X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, Na3VO4, EDTA, leupeptin, and RT-PCR were thus performed to obtain full transcripts of these and 1 mM PMSF. The insoluble components were removed by centrifu- two putative genes. Two mature transcripts, termed PRARP1 gation at 12,000 3 g and 4˚C for 30 min, and 5 mg of either mouse mAb (the reasons for this nomenclature are described later in this anti-Myc 9B11 or anti-FLAG 9A3 (Cell Signaling Technology) was added article) and PRARP2, were predicted to encode two structur- to the supernatant. After an overnight incubation at 4˚C under gentle ro- tation, 30 ml of protein G-coupled magnetic beads (New England BioLabs) ally similar transmembrane IgSF proteins, both with a single was added to the supernatant, and the mixture was incubated with constant N-terminal Ig V-set domain proximal to hydrophobic signal pep- rotation for 4 h. The beads were immobilized with a magnetic rack and tides (Fig. 1B). The deduced polypeptide PRARP1 (306 residues) thoroughly washed three times with lysis buffer. The immunoprecipitates consists of a nonsplit IgV domain (spanning residues 26–129) were eluted by incubating the beads in 30 mlof33 SDS sample loading buffer at 70˚C for 5 min and separated by 8% SDS-PAGE. The gels were with the sequence GDGTRLVV in the C terminus that obviously transferred on a 0.45-mm polyvinylidene difluoride membrane (Milli- corresponds to a TCR or Ig J-segment consensus sequence poreSigma) and blocked with 5% non-fat dry milk in TBS containing (GxGTxLxV, in which x refers to any amino acid), a structurally 0.05% Tween-20. The membranes were then probed with anti-Myc or anti- FLAG diluted 1:1000 in a blocking buffer and then incubated with an HRP-conjugated goat anti-mouse IgG second Ab (ZSGB Biotech). The Table I. Information about templates used for conducting homologous immunoblotted proteins were visualized with ECL reagents (Thermo modeling Fisher Scientific). The immunofluorescence of the cells in suspension was performed according to standard procedures (34). Briefly, ∼106 cells were transferred Sequence from each well to a centrifuge tube, centrifuged at 800 3 g for 3 min, and PDB ID Species Description Similarity (%) Coverage (%) washed once with PBS. The cells were then fixed by incubation with ab- 1igy.1.B Mouse g1 CH1 33 70 solute ice-cold methanol for 5 min at 220˚C and washed twice with PBS. 4i18.1.B Human g1 CH1 34 93 The cells were permeabilized with 0.3% Triton X-100 in PBS and then 5vod.1.G Human k CL 32 73 blocked with 2% BSA in PBS containing 0.1% Tween-20 for 1 h at room 5n2k.1.A Human l CL 33 62 temperature. The cells were incubated with the primary Ab at 1:500 di- lution in blocking solution overnight at 4˚C, then washed three times with ID, identification. 4 DESCENDANTS OF AN ANCESTRAL Ag RECEPTOR Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Identification of two invariant IgSF members in chicken. (A) Sequence alignment of the chPRARP1/duPRARP1 and chPRARP2/duPRARP2 V domains with those of TCR b-chains and TCR a-chains in various species. The top bold lines indicate the predicted locations of (a–g) b-strands. The canonical cysteine residues involved in the formation of intrachain disulfide bonds are indicated in yellow, and the additional pair of cysteines in chPRARP2/duPRARP2 that are thought to generate a second intrachain disulfide bond are topped by red circles. The residues highlighted in pink are frequently found in the V domains of TCR b-chains and TCR a-chains (69), and the black and gray shading indicates conserved percentages of 100 or $80 in those areas, respectively. The inverted triangles indicate the potential N-glycosylation sites (NxT/S). The accession numbers are listed in Supplemental Table II. (B) Kyte-Doolittle hydropathy analyses of the chicken PRARP1 and chicken PRARP2 gene products (top) and schematic presentation of their structure (bottom). The diagram shows the signal peptide (SP; vertical striped bar), the extracellular domain (ECD), the transmembrane region (TM; shaded bar), the cytoplasmic tail (CYT), and the ITIM or ITSM motif (spotted bar). (C) Physical map of the chicken PRARP1 (top) and chicken PRARP2 (bottom) gene loci. The boxes indicate exons, and the slash shading indicates the protein-encoding areas. A dotted line referring to 1.9 kb indicates the distance between the two genes. The lengths of the exons and introns are drawn to scale and are indicated by numbers. (D) Southern blotting of the chicken PRARP1 and PRARP2 genes. Approximately 6 mg of genomic DNA was loaded in each lane after digestion with various restriction enzymes. The V domain exons were used as digoxin-labeled probes. The restriction enzymes used are indicated at the top of each lane. (E) The homology models of chPRARP V domains show structural similarities with the TCR b-chains and TCR a-chains domains. A structural model of the chPRARP1 V domain (in cyan) is shown superimposed on a homologous TCR b-chains Ig domain (in yellow, PDB identification: 5U1R.E), and the chPRARP2 V domain (in green) is shown superimposed on a homologous TCR a-chains Ig domain (in magenta, PDB identification: 5U1R.D). The arrow indicates the protruding CDR3 region in 5U1R.E. (F) Analyses of the amino acid variations of chPRARP V domains. The alternative amino acid changes were calculated as described in the Materials and Methods section. The corresponding amino acid positions are shown on the x-axis, and the framework region or CDR positions are marked on the top. ch/chick, Gallus gallus (chicken); dolp, Tursiops truncatus (dolphin); du/mal, Anas platyrhynchos (duck/mallard); hu, Homo sapiens (human); mou, Mus musculus (house mouse); trout, Oncorhynchus mykiss (rainbow trout). The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. Expression patterns of PRARP genes in chickens. (A) Quantitative RT-PCR analyses of chicken PRARP in 1-d-old chicks by guest on September 28, 2021 (top panel), 1 mo-old chickens (middle panel), and 33-mo-old chickens (lower panel). The data for each tissue are representative of three individuals. The vertical bars show the SEMs of the mean. The EF1A1 gene was used as an internal control. (B) Isolation of T and B cells and monocytes/macrophages from chicken spleen. T cells were sorted as CD3+ cells, B cells were sorted as Bu-1+ cells, and monocytes/macrophages were sorted as KUL01+ MHC class-II+ cells. (C) RT-PCR analyses of the expression of chicken PRARP genes in T and B cells and monocytes/macrophages. The expected size of each PCR product is shown on the right. The chicken EF1A1 gene amplification served as an internal control. indeterminate region (spanning residues 130–238), a transmem- some 25 (spanning from position 3.752 to 3.760 Mb based on brane region lacking a charged residue (spanning residues 239– the Reference RGCg6a Primary Assembly in National Center 255), and a 52-residue cytoplasmic tail. PRARP2 (296 residues) for Biotechnology Information) and are thus organized in an is composed of a V-set Ig domain fused to a J-like sequence opposite transcriptional direction. The two genes share 22.2% (GNGTVLLV), which is also a structurally indeterminate region, amino acid identity. Each gene was encoded by five exons followed by an uncharged transmembrane region and a 48-residue spanning ∼2.7 kb (Fig. 1C). All the exon– boundaries cytoplasmic tail. The J-like portion of each receptor V domain examined followed the “gt-ag” rule of conservation (Supplemental includes a characteristic diglycine bulge, which makes the G Fig. 1). strands more reminiscent of a true J region (40, 41). Moreover, in accordance with the immunoreceptor tyrosine-based switch motif Both PRARP1 and PRARP2 in chickens are single-copy genes (ITSM; consensus TxYxxV/I) and ITIM consensus sequences that exhibit low polymorphism (I/L/VxYxxI/L/V), a tyrosine residue that can likely be phos- As IgSF members usually show high variations in either gene phorylated was found to be embedded in the TEYAMV sequence number or sequence diversity (polymorphism), Southern blotting of the PRARP1 cytoplasmic tail and the LLYAHL sequence of using probes corresponding to the V exon was conducted to de- the PRARP2 cytoplasmic tail, respectively. Conserved residues in termine whether PRARP1 and PRARP2 are present in the genome the V domains of TCR b-chains or TCR a-chains were also ob- as single or multiple copies and, more importantly, whether there served in these domains of PRARP1 and PRARP2 (Fig. 1A). are highly homologous genes present in split forms as observed Homologous modeling showed that the predicted structures of in nurse shark NS4 Ig L chain loci (42). The results revealed a the PRARP1 and PRARP2 V domains well resembled those of the single restriction fragment in each lane, demonstrating that human TCR b-chains and TCR a-chains, respectively, with the both PRARP1 and PRARP2 were present as single-copy genes exception that the loop corresponding to CDR3 of TCR b-chains and that only the preassembled configuration of these genes can was less protruding in PRARP1 (Fig. 1E). be found in the whole genome (Fig. 1D). Moreover, genomic The analysis of the gene loci revealed that the two genes are fragments of V regions amplified from 80 individuals of eight organized in a head-to-head manner on the chicken chromo- different chicken breeds were subjected to sequencing for 6 DESCENDANTS OF AN ANCESTRAL Ag RECEPTOR

polymorphic analyses. Only ,10 variation sites leading to amino acid changes were identified, and these were mostly concen- trated in biallelic framework regions (Fig. 1F). Thus, both the PRARP1 and PRARP2 genes appear to show minimal poly- morphism in chickens. Spatiotemporal expression patterns of PRARP1 and PRARP2 in chickens The tissue distributions of PRARP1 and PRARP2 transcripts were determined by quantitative RT-PCR. The IgL and TCR a-chains expression levels among various tissues were also determined for comparison (data not shown). In 1-d-old chicks, IgL and TCR a-chains were highly expressed in the bursa of Fabricius (bursa of F.) and thymus, respectively. In 1-mo-old chickens, IgL and TCR a-chains expression was substantially enhanced in the spleen and intestine, and lymphocytes were migrating from the central lymphoid tissues to the periphery. Interestingly, a similar tendency was also observed for PRARP1 and PRARP2 (Fig. 2A, top and

middle panels), indicating that both receptors are very likely Downloaded from specifically expressed in B and T lymphocytes, and this finding was further confirmed in vitro by RT-PCR using the chicken B and T lymphocyte cell lines DT40 and MDCC-MSB1, respectively (Supplemental Fig. 2C). As a control, no transcript of these two genes was detected in the fibroblast cell line DF-1. An analysis of 33-mo-old chickens indicated that the expression of both PRARP1 http://www.jimmunol.org/ and PRARP2 was stabilized in these chickens older than 1 mo of age (Fig. 2A, lower panel). Primary peripheral immunocytes from 4-wk-old chicken splenocytes, including T and B lymphocytes and monocytes/ macrophages, were isolated to further confirm the above con- clusion. Well-characterized mAbs directed toward chicken CD3 and Bu-1 Ags were used for T and B cell sorting using flow cytometry, respectively, whereas the combinational use of mAbs

against KUL01 and MHC II molecules allowed a precise iso- by guest on September 28, 2021 lation of monocytes/macrophages (Fig. 2B). Primers designed to span the introns of chicken PRARP were used for the RT- PCR analysis of RNA from ∼0.2 million freshly isolated cells of the above-mentioned types. The amplification of chicken EF1A1 served as a control (Fig. 2C). The results showed that the PRARP1 and PRARP2 genes were dominantly expressed in primary T and B cells, which is in accordance with the con- clusion derived from the tissue analysis. Additionally, weak RNA expression of both PRARP genes was detected in mono- cytes/macrophages. Recombinant PRARP1 and PRARP2 proteins can form disulfide-linked homodimers or heterodimers as membrane proteins Three stably transfected cell lines that expressed Myc-tagged PRARP1 (DT40-Myc-PRARP1), FLAG-tagged PRARP2 (DT40- FLAG-PRARP2), and both of these proteins (DT40-Co) were biochemically characterized. Denatured cell lysates treated with

homodimers, respectively. The chPRARP1–chPRARP2 heterodimer was immunoprecipitated in the following IP experiment. (B and C) The IPs of lysates from three different stably transfected DT40 cell lines were sub- FIGURE 3. Characterization of the biochemical properties of recombi- jected to nonreduced SDS-PAGE and Western blotting analyses; nant chPRARP proteins. (A) Western blot detection of recombinant the chPRARP1 homodimer (B, lanes 1 and 3), chPRARP2 homodimer chPRARP protein in DT40 cell lysates. The PVDF membranes were in- (C, lanes 5 and 6) and chPRARP1–chPRARP2 heterodimer (B, lanes 3 and cubated with anti-Myc Ab (left panel) or anti-FLAG Ab (right panel). 6; C, lanes 3 and 6) are represented by arrows. (D) Immunofluorescence After PNGase F treatment, both proteins showed a decreased m.w. (lanes image of DT40-Myc-PRARP1 and DT40-FLAG-PRARP2 stably trans- 1 and 5). Under the nonreduced condition, a band corresponding to a m.w. fected clones. Positive signals surrounding the whole cell membrane were that was 2-fold higher than that of the monomer was observed in lanes detected with a Cy3-labeled secondary Ab and captured by the DeltaVision 3 and 7, and this band corresponded to the chPRARP1 and chPRARP2 OMX SR imaging system. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Effects of LPS and poly(I:C) on the expression levels of chPRARP1 and chPRARP2 mRNA in DT40 cells and the responses of both genes to bacterial/viral vaccination challenge. (A and B) DT40 cells were incubated with 100 ng/ml E. coli O111:B4 LPS or 10 mg/ml poly(I:C) for various times. CATH-B1 and IFN-b were selected as positive controls. The amounts of chPRARP1 and chPRARP2 mRNA were normalized with respect to those of chicken GAPDH transcripts in each sample. The values and vertical bars represent the means and SDs, respectively, from three independent experiments. (C) Amounts of chPRARP1 mRNA in the bursa of F. of the bacterial vaccine group (left panel) and in the spleen of the viral vaccine group (right panel). The data represent the results from five individuals in each group. *p , 0.05, **p , 0.01.

N-glycosidase F were blotted and detected with anti-Myc or anti- under nonreduced conditions, and an ∼70-kDa band, which is FLAG Ab. Two single bands, corresponding to the PRARP1 and consistent with the molecular mass of a PRARP1 homodimer, PRARP2 monomers with molecular masses of ∼38 and ∼40 kDa, was observed in the lanes for the DT40-Myc-PRARP1 and DT40-Co respectively, were detected under both reduced and nonreduced cell lines (Fig. 3B, left panel). Additionally, an ∼80-kDa band, conditions (Fig. 3A). Treatment with PNGase F reduced the mo- whichishighlylikelytobethePRARP1–PRARP2hetero- lecular weights of both monomers to ∼35 kDa, which is close to dimer, was also detected with the DT40-Co cell line (Fig. 3B, the predicted molecular mass. As expected, the glycosylation level left panel). Only the heterodimer band was observed in the IP of PRARP2 was higher than that of PRARP1 because the se- with anti-Myc Ab and the Western blotting analysis with anti- quence of PRARP2 contains four N-glycosylation sites (NxT/S), FLAG Ab (Fig. 3B, right panel). In contrast, the use of anti- whereas only one site was found in PRARP1. Notably, a doublet FLAG Ab for both IP and Western detection resulted in the with a molecular mass of 70–80 kDa was also observed under detection of the PRARP2 homodimer and the PRARP1–PRARP2 nonreduced conditions, indicating that the two proteins are likely heterodimer in the DT40-Myc-PRARP2 and DT40-Co cell lines, able to form homo- and heterodimers. respectively (Fig. 3C, right panel). Only the heterodimer was To confirm the above speculation, for all three cell lines, IP with observed in the anti-Myc blot of the DT40-Co cell line (Fig. 3C, an anti-Myc Ab was performed for both IP and Western detection left panel). Further verifications were also performed by Western 8 DESCENDANTS OF AN ANCESTRAL Ag RECEPTOR blotting detection under reduced conditions (Supplemental The PRARP1 and PRARP2 homologs might date back to as Fig. 2A, 2B). early as teleost but are not found in mammals The subcellular localization of these two proteins was verified by We further investigated whether the homologs of PRARP1 and either anti-Myc or anti-FLAG Ab staining followed by Cy3-labeled PRARP2 could be found in other animals (Supplemental Table I). goat anti-mouse IgG secondary Ab staining. As expected, both Interestingly, nine pairs of the genes in another bird, the White PRARP1 and PRARP2 were mainly expressed on the cell surface Peking duck (Anas platyrhynchos f. domestica), clustered together as membrane proteins (Fig. 3D). as confirmed by sequencing the BAC clone 1304A21 screened Both inhibitory receptors can be upregulated by poly(I:C) from a genomic BAC library previously constructed in our labo- stimulation in vitro and respond to vaccine immunization ratory (43) (Fig. 5). The amino acid percentage identities among challenges in vivo the nine-copy duck PRARP1 (duPRARP1) V domains varied from Because these receptors showed invariant features common 78.0 to 98.9%, but the identities of the duPRARP2 V domains to some innate immune IgSF receptors (e.g., chicken Ig-like showed slightly increased fluctuation, from 64.2 to 99.0% receptors [CHIRs]), we questioned whether some PAMPs can (Supplemental Fig. 2E, 2F). A similar finding was obtained in a stimulate the expression of PRARP1 and PRARP2. The cultured reptile, the Chinese alligator (Alligator sinensis), which contains DT40 cells were incubated with 100 ng/ml LPS or 10 mg/ml poly at least eight pairs of genes stretching across ∼240 kb on scaffold (I:C), and the relative expression levels were analyzed by quan- 838_1 of the ASM45574v1 assembly. In amphibians, multiple titative RT-PCR. The expression of both receptor genes reached copies on contigs 2941, 2939, and 3103 (GenBank assembly ac- cession GCA_000004195.3), which equally matches their reptile their highest level after 24 h of incubation with poly (I:C), but Downloaded from no notable changes were obtained with LPS stimulation (Fig. 4A, counterparts, were identified in the tropical clawed frog (Xenopus 4B). Furthermore, to simulate challenges through vaccine immu- tropicalis). A careful examination of the whole genome of a lobe- nization, White Leghorn chicks were injected with an inactivated finned fish, the “living fossil” coelacanth (Latimeria chalumnae), avian colibacillosis vaccine (mixed E. coli strain EC24, 30, 45, revealed only one homolog (Fig. 5). However, despite intensive and 50) or an inactivated bivalent avian influenza virus vaccine Basic Local Alignment Search Tool searches, no orthologous gene (H5N1 and H9N2) according to the manufacturer’s instructions. was confirmed in either the most primitive jawed vertebrates or http://www.jimmunol.org/ The same bulk of sterile liquid PBS was used in the control mammals (Supplemental Fig. 2D). Nonetheless, a potential can- groups. Because PRARP1 and PRARP2 show similar expres- didate for the PRARP homolog in the whale shark (Rhincodon sional patterns as demonstrated by the above-described data, typus), which shared an amino acid sequence identity of 34% with we only analyzed the response of PRARP1.TheE. coli. vaccine the coelacanth PRARP, was selected for further multiple sequence induced an ∼2.5-fold increase in the PRARP1 mRNA level in alignments (Fig. 6A, 6B). Unexpectedly, all these predicted pro- the bursa of F. at 1 w.p.i. (weeks postinfection) compared with teins, with the exception of those in birds, contain a C1-set that found in the control chicks treated with the same dose of Ig domain following the V domain (Fig. 6B, 6C). The structures PBS (Fig. 4C, left panel). Although the expression level in the of the C regions found in the coelacanth, the clawed frog, and the

thymus at 4 w.p.i. was nearly 2-fold higher than that of the alligator were analyzed through homologous modeling against by guest on September 28, 2021 control, the difference was not statistically significant (data not templates of other C1 domain-containing proteins. The sequence shown). In the avian influenza virus group, the PRARP1 mRNA similarities and coverages of the templates with the corresponding level in the spleen increased to ∼4-fold at 2 w.p.i. and de- PRARPs met the criterion for homologous modeling (Table I). creased to a normal level at 4 w.p.i. (Fig. 4C, right panel), but The C regions in the predicted PRARP peptides showed the most no significant difference was observed in the thymus and bursa similar architecture to C1 from Ig, and the structural alignments of F. (data not shown). revealed that all predicted peptides showed an organization of

FIGURE 5. The chicken PRARP orthologs are widespread among vertebrate lineages. Chromosomal locations of the PRARP genes in different species. The arrows indicate the transcriptional orientation of the genes. Double slashes indicate gaps among contigs or the ends of the BAC clone. The “precursor” in coelacanths refers to the one-copy ancestral gene of PRARP; multiple-copy homologs are indicated by the “medium” in amphibian; and PRARP in frogs diverged completely into PRARP1 and PRARP2 at the time of emergence of the reptiles. Genes sharing syntenic relations are as follows: APH1A, aph-1 homolog A, g-secretase subunit; CA14, carbonic anhydrase 14; CIART, circadian-associated repressor of transcription; MRPS21, mitochondrial ribosomal protein S21; PRPF3, pre-mRNA processing factor 3; and RPRD2, regulation of the nuclear pre-mRNA domain containing 2. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Multiple sequence alignments of the V or C domains of PRARP orthologs among representatives of jawed vertebrates and comparative C1 domain structure analyses. (A) Multiple sequence alignment of PRARP V domains from various species. The term “chicken 1” refers to chicken PRARP1, and “chicken 2” represents chicken PRARP2. The red-boxed residues in white indicate strict identity, and the residues in red show similarity in the group. (B) Multiple alignment of PRARP C1 domains from bony fish, amphibian, reptile, and even cartilaginous fish. (C)Astructural model of each PRARP C domain is shown superimposed on a homologous IgH or IgL C1 domain. The PDB identification code and related infor- mation for each homologous template used for modeling are shown in the panel and Table I. (D) Multiple alignment of structurally indeterminate regions of chicken PRARPs with C regions of AIS components. The nearly invariant residues, namely, two cysteine residues and one tryptophan residue (or leucine residues for b2 microglobulin), are shaded in red and green, respectively. The Ig domains used in the alignment were the IgG C regions 1, 2, and 3 (gCH1–3; PDB identification: 1FC1), the TCR b-chain C region (TRB; 1BEC), the Igk C region (kCL; 1IGT), and b2micro- globulin (b2m;3D25).

“4 plus 3” b-strands (Fig. 6C). In addition, many gaps were of multiple PRARP gene copies occurred independently in the found in the sequences of chicken PRARP in comparison with different animal lineages. those of other C1-type chains, which indicated that part of the chicken PPARP C1 domain was highly degenerated or was a Phylogenetic analyses revealed that the nonrearranging VJ divergent Ig domain type compared with the corresponding genes formed a novel receptor family domains in the AIS components (Fig. 6D). This finding likely To determine whether these preassembled genes can be classified explains why the chicken PRARP cannot form a C1-set Ig into a known gene family, we selected the mammalian Fc receptors domain. poly-Ig receptor (PIGR), FcmR, and FcamR as the first options for As mentioned above, multiple copies of the PRARP1 and phylogenetic analysis because these receptors possess a V-set Ig PRARP2 genes can be found in selected species, but a given domain in their N termini and are located on the same syntenic PRARP gene clearly exhibits higher sequence identity to its chromosomal region where PRARPs are located. The results counterpart in the same species than to those from other species, showed that the PIGR, FcmR, and FcamR members were separately which suggested that the duplications that resulted in the generation clustered and formed their own branches. However, PRARP1 and 10 DESCENDANTS OF AN ANCESTRAL Ag RECEPTOR

PRARP2 clustered at a distinct branch, with a credibility value of receptor family in terms of both copy number and allelic poly- 0.93, which confirmed that PRARP1 and PRARP2 share a common morphism (50–52). In addition, the Ig domains of all CHIRs ancestral gene and do not belong to any known Fc receptor sub- can be classified as C2-type, similarly to those of the classical groups (Fig. 7). We then focused on SIRP because it is one of the Fc receptor members, such as FcgR, FcεR, and Fc receptor–like few V-C1–like receptors that share almost the same molecular (53, 54). However, there is one exception: although PIGR, FcmR, structure with PRARP and whose J portion is an integral part of the and FcamR contain V-type Ig domains and are situated on V exon in the germline. Surprisingly, the PRARP genes were more mammalian syntenic to the chicken PRARPs, our closely related to SIRP than to the other types of TCR chains from phylogenetic estimation showed that PRARPs did not cluster with various species but still formed a distinct sequence branch (Fig. 8). any of the three above-mentioned receptor families. Other chicken It has been suggested that SIRPs might have descended from the immunoregulatory IgSF receptors that were taken into consider- precursors of rearranging Ag receptors (44). We thus termed the two ation were CD200, CD300, and TREM. The identities of the nonrearranging IgSF receptors initially found in chickens and their amino acid sequences of three representative receptors ranged orthologs in other lineages as PRARPs (Fig. 9). from only 10 to 20%, and none of the three family members were found to be located on chromosome 25 (55). Discussion We then shifted attention to the SIRP family. SIRPs are composed In this study, we unveiled two IgSF genes with an intact VJ-fused of a single VJ-fused V domain and two C1-like domains, which exon and an inhibitory motif in the cytoplasmic tail that could indicates that they might have originated from the predecessors of potentially mediate signal transduction in chicken lymphoid cells TCRs and BCRs (29, 44). Previous studies discovered SIRP orthologs and myeloid cells. Based on the characteristic V-C1 structure of in chickens, which suggests a conserved role for SIRPs in modern Downloaded from their orthologs in other gnathostomes and the phylogenetic re- jawed vertebrates (55). Our phylogenetic analysis indicated that lationships with SIRP, we conclude that the newly discovered PRARPs were more closely related to the SIRP family but still genes, termed PRARPs, might also represent another multi- formed a monophyletic branch, which implies that PRARP genes gene family that originated from a precursor of rearranging can be formally incorporated into a novel gene family. Ag receptors. Germline VJ-joined genes have been previously described

Because PRARP genes were initially found in chickens and are within the TCR or BCR loci. Marsupials contain a TCRm that http://www.jimmunol.org/ located in the syntenic region of the mammalian Fc receptor uses a prejoined Vmj whose origin might be due to retro- family, their relationships with CHIR, a highly diverse family transposition (56, 57), which differs from the finding that the LP located on microchromosome 31, and those with Fc receptors portion is encoded by an independent exon for each one of the were first examined (45–49). The results clearly showed that PRARP genes. A VH3.1 fused to DH in the germline of the IgH PRARPs cannot be grouped into either CHIRs or Fc receptors locus was also observed in the opossum and was hypothesized to because PRARPs show notable differences from these two gene be caused by ectopic expression of the V(D)J recombination en- families in terms of syntenic conservation, genomic loci organi- zymatic machinery in germ cells as confirmed by the generation of zation, Ig domain composition, and gene polymorphism. For ex- germline-joined Ig VL in sharks (42, 58). Obviously, these germline ample, this study found that the PRARP genes showed minimal VJ-joined genes should have originated from the rearrangement of by guest on September 28, 2021 polymorphism, whereas CHIRs represented a highly diverse BCR- or TCR-encoding genes. However, based on the syntenic

FIGURE 7. Phylogenetic tree of PRARP and Fc re- ceptors from a panel of vertebrates. Neighbor-joining trees were constructed using the amino acid sequences of the first domain (V regions) of PRARP, Fc receptors, and human leukocyte receptor cluster (LRC) mem- bers (as an outgroup). The outgroup comprising hu- man LRC members is marked with dashed lines. The scale bar indicates the genetic distance, and bootstrap values .0.5 are shown for each node. All accession numbers of the sequences used for the construction of the trees are referenced in Supplemental Table II. The Journal of Immunology 11

core structure. In reptiles, PRARPs have completely split into PRARP1 and PRARP2, and these not only share little sequence similarity with each other but also have been organized in an opposite transcriptional direction, perhaps because of a perplexing genetic inversion. However, both the diverged PRARP1 and PRARP2 still encode V-C1 structures. In birds, the C1 domains (previously referred to as the structurally indeterminate region) of PRARP genes have degenerated to the extent that the conserved features or sites for a canonical C1 Ig domain signature can no longer be distinguished. In general, according to an analysis of over 1200 C1-set sequences involving almost all types of C re- gions in adaptive immune components, the separations between two cysteine residues in the C1-set Ig domain that form the conserved disulfide bridge were predominantly between 54 and 64 aa (59). The number of residues between the conserved cysteines in chicken PRARP1 and PRARP2 was found to equal 51 and 41, respectively, and this residue number is lower than the minimum number of amino acids required for C1 constitution

(Fig. 6D). This finding also supports the hypothesis that nucle- Downloaded from otide, exon, and even gene deletions could occasionally have oc- curred during the rapid evolution of the IgSF (60). The receptors evolved rapidly, which is characterized by differences in the copies of genes in different species and the presence or absence of in- hibitory signaling motifs (duPRARP genes have no signaling motif

in the cytoplasmic tail). The predecessor of PRARP might have http://www.jimmunol.org/ been generated at a very early stage in evolution, although no ev- ident presence of its ortholog in cartilaginous fish has been con- firmed. In reptiles, PRARP has completely diverged into two different genes. Interestingly, because of the sequence deletions, the second Ig domain of PRARP in avian species has degenerated but may still represent a divergent Ig domain as nearly all con- served amino acid residues for an Ig domain constitution, such as cysteine and tryptophan residues, are present in this region (Fig. 6D). Nearly all birds examined in this study maintained one by guest on September 28, 2021 copy of PRARP; the only exception was waterfowl duck, which has nine copies of paired receptors. In addition, an 11-nt loss was found in the last exon of duck PRARP1-7, and this frameshift mutation caused deterioration into a pseudogene. No homolog has been identified in mammals, which indicates that PRARP has been lost in mammalian genomes (Fig. 9, Supplemental Fig. 2D). It appears that, similarly to most multigene families, particularly the IgSF, the PRARP multigene family has been subject to the evo- lutionary model of birth-and-death (61, 62). FIGURE 8. Phylogenetic tree of PRARP, representative SIRP-family SIRPb was previously shown to form a disulfide-linked genes, and TCR chain sequences. Full-length TCR chains, PRARP, and homodimer, and the same finding was also obtained in our SIRPs derived from various vertebrates were subjected to a phylogenetic study (63). The overexpression of PRARP1, PRARP2, and both analysis with the Molecular Evolutionary Genetics Analysis program. The in the B cell line results in their expression as monomer, accession numbers of the sequences used in this analysis are referenced in disulfide-bonded homodimers and heterodimers, supporting Supplemental Table II. the hypothesis that all the Ig and TCR types of heterodimers diverged from a common primordial heterodimeric receptor chromosomal locations or distributions of the PRARP genes in (11). It is interesting to speculate whether such a diverse to- animal lineages, no evidence could be obtained to support the no- pology of homodimers, heterodimers, and monomers could tion that the PRARP gene originated from rearranging receptor- affect their ligand-binding specificity. The remaining question encoding genes. regarding the physiological functions of PRARP receptors re- The available information regarding the distribution of PRARPs quires to be resolved, although it is known that the functions among vertebrates allowed us to propose a model for the evolu- depend on the types of ligand(s) they virtually bind to. We tionary path of these genes (Fig. 9). First, a predecessor to PRARP propose that nonvariant ligand(s), such as PAMPs, or a self- can be traced back to at least 400 MYA based on the presence antigen, such as CD47 (the endogenous ligand to SIRPa and of a single copy in coelacanth. Although genes with an ∼30% SIRPg), are the best candidates because of the limited varia- sequence identity to the teleost homolog were found in cartilagi- tions observed in the V domains of PRARPs (64–66). Never- nous fishes, no large sequence synteny was obtained, and thus, theless, given that a clear homology was observed between whether these genes were genuine orthologs could not be proven. TCR chain V domains, we cannot rule out the possibility that In addition, PRARPs in amphibians have not undergone es- the MHC–Ag peptide complex can interact with PRARP sential changes but are present in multiple copies with the V-C1 molecules as illustrated by interactions between human Fc 12 DESCENDANTS OF AN ANCESTRAL Ag RECEPTOR

FIGURE 9. Schematic illustration of the evolutionary process of PRARP in vertebrates. The “birth-and-death” evolutionary model for PRARP is illustrated. The boxes with a dashed profile indicate the following: 1) the genes from ∼600 MYA were attacked by a transposon; 2) the C1 Ig domain had degenerated or diverged in the avian group; and 3) PRARP vanished in mammals. The green circle at the end of the C1 domain indicates the presence of an inhibitory Downloaded from motif (ITIM/ITSM), whereas the red circle in- dicates that no motif was identified. http://www.jimmunol.org/

receptor–like 6 or some leukocyte receptor cluster members 8. Schatz, D. G., M. A. Oettinger, and D. Baltimore. 1989. The V(D)J recombi- nation activating gene, RAG-1. Cell 59: 1035–1048. by guest on September 28, 2021 and MHC molecules (67, 68). 9. Pancer, Z., C. T. Amemiya, G. R. Ehrhardt, J. Ceitlin, G. L. Gartland, and In conclusion, we demonstrated in this study that a pair of VJ-intact M. D. Cooper. 2004. Somatic diversification of variable lymphocyte receptors in IgSF members, named PRARPs, are widespread among jawed ver- the agnathan sea lamprey. Nature 430: 174–180. 10. Flajnik, M. F., and M. Kasahara. 2010. Origin and evolution of the adaptive tebrates and, to a large extent, appear to be remnants of archaic Ag immune system: genetic events and selective pressures. Nat. Rev. Genet. 11: receptor genes. Further elucidation of their immunological functions 47–59. and genetic origin might provide significant insights into the evo- 11. Litman, G. W., J. P. Rast, and S. D. Fugmann. 2010. The origins of vertebrate adaptive immunity. Nat. Rev. Immunol. 10: 543–553. lution of the immune system in jawed vertebrates. 12. Flajnik, M. F. 2002. Comparative analyses of immunoglobulin genes: surprises and portents. Nat. Rev. Immunol. 2: 688–698. 13. Sakano, H., K. Hu¨ppi, G. Heinrich, and S. Tonegawa. 1979. Sequences at the Acknowledgments somatic recombination sites of immunoglobulin light-chain genes. Nature 280: We thank Prof. Ziding Zhang, China Agricultural University, for help in the 288–294. 14. Kapitonov, V. V., and J. Jurka. 2005. RAG1 core and V(D)J recombination signal homologous modeling. We also thank Prof. Jingyan Fu, China Agricultural sequences were derived from Transib transposons. PLoS Biol. 3: e181. University, for technical help in the use of high-resolution imaging system. 15. Huang, S., X. Tao, S. Yuan, Y. Zhang, P. Li, H. A. Beilinson, Y. Zhang, W. Yu, P. Pontarotti, H. Escriva, et al. 2016. Discovery of an active RAG transposon illuminates the origins of V(D)J recombination. Cell 166: 102–114. Disclosures 16. Williams, A. F., and A. N. Barclay. 1988. The immunoglobulin superfamily-- The authors have no financial conflicts of interest. domains for cell surface recognition. Annu. Rev. Immunol. 6: 381–405. 17. Harpaz, Y., and C. Chothia. 1994. Many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains. J. Mol. Biol. References 238: 528–539. 1. Thompson, C. B. 1995. New insights into V(D)J recombination and its role in the 18. Du Pasquier, L., I. Zucchetti, and R. De Santis. 2004. Immunoglobulin super- evolution of the immune system. Immunity 3: 531–539. family receptors in protochordates: before RAG time. Immunol. Rev. 198: 233– 2. Litman, G. W., M. K. Anderson, and J. P. Rast. 1999. Evolution of antigen 248. binding receptors. Annu. Rev. Immunol. 17: 109–147. 19. Du Pasquier, L. 2001. The immune system of invertebrates and vertebrates. 3. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302: Comp. Biochem. Physiol. B Biochem. Mol. Biol. 129: 1–15. 575–581. 20. Herberg, J. A., J. Sgouros, T. Jones, J. Copeman, S. J. Humphray, D. Sheer, 4. Weigert, M. G., I. M. Cesari, S. J. Yonkovich, and M. Cohn. 1970. Variability P. Cresswell, S. Beck, and J. Trowsdale. 1998. Genomic analysis of the Tapasin in the lambda light chain sequences of mouse antibody. Nature 228: 1045– gene, located close to the TAP loci in the MHC. Eur. J. Immunol. 28: 459–467. 1047. 21. Frangoulis, B., I. Park, F. Guillemot, V. Se´verac, C. Auffray, and R. Zoorob. 5. Tonegawa, S. 1976. Reiteration frequency of immunoglobulin light chain genes: 1999. Identification of the Tapasin gene in the chicken major histocompatibility further evidence for somatic generation of antibody diversity. Proc. Natl. Acad. complex. Immunogenetics 49: 328–337. Sci. USA 73: 203–207. 22. Abi Rached, L., M. F. McDermott, and P. Pontarotti. 1999. The MHC big bang. 6. Davis, M. M., Y. H. Chien, N. R. Gascoigne, and S. M. Hedrick. 1984. A murine Immunol. Rev. 167: 33–44. T cell receptor gene complex: isolation, structure and rearrangement. Immunol. 23. Pancer, Z., W. E. Mayer, J. Klein, and M. D. Cooper. 2004. Prototypic T cell Rev. 81: 235–258. receptor and CD4-like coreceptor are expressed by lymphocytes in the agnathan 7. Yancopoulos, G. D., T. K. Blackwell, H. Suh, L. Hood, and F. W. Alt. 1986. sea lamprey. Proc. Natl. Acad. Sci. USA 101: 13273–13278. Introduced T cell receptor variable region gene segments recombine in pre-B cells: 24. Yu, C., G. R. Ehrhardt, M. N. Alder, M. D. Cooper, and A. Xu. 2009. Inhibitory evidence that B and T cells use a common recombinase. Cell 44: 251–259. signaling potential of a TCR-like molecule in lamprey. Eur. J. Immunol. 39: 571–579. The Journal of Immunology 13

25. Suzuki, T., T. Shin-I, A. Fujiyama, Y. Kohara, and M. Kasahara. 2005. Hagfish 48. Torkar, M., Z. Norgate, M. Colonna, J. Trowsdale, and M. J. Wilson. 1998. leukocytes express a paired receptor family with a variable domain resembling Isotypic variation of novel immunoglobulin-like transcript/killer cell inhibitory those of antigen receptors. J. Immunol. 174: 2885–2891. receptor loci in the leukocyte receptor complex. Eur. J. Immunol. 28: 3959–3967. 26. Haruta, C., T. Suzuki, and M. Kasahara. 2006. Variable domains in hagfish: 49. Akula, S., S. Mohammadamin, and L. Hellman. 2014. Fc receptors for immu- NICIR is a polymorphic multigene family expressed preferentially in leukocytes noglobulins and their appearance during vertebrate evolution. [Published erratum and is related to lamprey TCR-like. Immunogenetics 58: 216–225. appears in 2015 PLoS One 10: e0124530.] PLoS One 9: e96903. 27. Yu, C., M. Dong, X. Wu, S. Li, S. Huang, J. Su, J. Wei, Y. Shen, C. Mou, X. Xie, 50. Lochner, K. M., B. C. Viertlboeck, and T. W. Go¨bel. 2010. The red jungle fowl et al. 2005. Genes “waiting” for recruitment by the adaptive immune system: the leukocyte receptor complex contains a large, highly diverse number of chicken insights from amphioxus. J. Immunol. 174: 3493–3500. immunoglobulin-like receptor (CHIR) genes. Mol. Immunol. 47: 1956–1962. 28. Cannon, J. P., R. N. Haire, and G. W. Litman. 2002. Identification of diversified 51. Laun, K., P. Coggill, S. Palmer, S. Sims, Z. Ning, J. Ragoussis, E. Volpi, genes that contain immunoglobulin-like variable regions in a protochordate. Nat. N. Wilson, S. Beck, A. Ziegler, and A. Volz. 2006. The leukocyte receptor Immunol. 3: 1200–1207. complex in chicken is characterized by massive expansion and diversification of 29. van Beek, E. M., F. Cochrane, A. N. Barclay, and T. K. van den Berg. 2005. immunoglobulin-like Loci. PLoS Genet. 2: e73. Signal regulatory proteins in the immune system. J. Immunol. 175: 7781–7787. 52. Nikolaidis, N., I. Makalowska, D. Chalkia, W. Makalowski, J. Klein, and M. Nei. 30. Adams, S., L. J. van der Laan, E. Vernon-Wilson, C. Renardel de Lavalette, 2005. Origin and evolution of the chicken leukocyte receptor complex. Proc. E. A. Do¨pp, C. D. Dijkstra, D. L. Simmons, and T. K. van den Berg. 1998. Natl. Acad. Sci. USA 102: 4057–4062. Signal-regulatory protein is selectively expressed by myeloid and neuronal cells. 53. Nikolaidis, N., J. Klein, and M. Nei. 2005. Origin and evolution of the Ig-like J. Immunol. 161: 1853–1859. domains present in mammalian leukocyte receptors: insights from chicken, frog, 31. Rothwell, C. J., L. Vervelde, and T. F. Davison. 1996. Identification of chicken and fish homologues. Immunogenetics 57: 151–157. Bu-1 alloantigens using the monoclonal antibody AV20. Vet. Immunol. Immu- 54. Davis, R. S. 2007. Fc receptor-like molecules. Annu. Rev. Immunol. 25: 525–560. nopathol. 55: 225–234. 55. Viertlboeck, B. C., R. Schmitt, and T. W. Go¨bel. 2006. The chicken immuno- 32. Bernot, A., and C. Auffray. 1991. Primary structure and ontogeny of an avian regulatory receptor families SIRP, TREM, and CMRF35/CD300L. Immunoge- CD3 transcript. Proc. Natl. Acad. Sci. USA 88: 2550–2554. netics 58: 180–190. 33. Mast, J., B. M. Goddeeris, K. Peeters, F. Vandesande, and L. R. Berghman. 1998. 56. Parra, Z. E., M. L. Baker, R. S. Schwarz, J. E. Deakin, K. Lindblad-Toh, and Characterisation of chicken monocytes, macrophages and interdigitating cells by R. D. Miller. 2007. A unique T cell receptor discovered in marsupials. Proc. the monoclonal antibody KUL01. Vet. Immunol. Immunopathol. 61: 343–357. Natl. Acad. Sci. USA 104: 9776–9781. Downloaded from 34. Wang, C. C., S. S. Bajikar, L. Jamal, K. A. Atkins, and K. A. Janes. 2014. A 57. Hansen, V. L., and R. D. Miller. 2015. The evolution and structure of atypical time- and matrix-dependent TGFBR3-JUND-KRT5 regulatory circuit in single T cell receptors. In Pathogen-Host Interactions: Antigenic Variation v. Somatic breast epithelial cells and basal-like premalignancies. Nat. Cell Biol. 16: 345– Adaptations. Results and Problems in Cell Differentiation. Springer, Cham, 356. Switzerland, p. 265–278. 35. Schwede, T., J. Kopp, N. Guex, and M. C. Peitsch. 2003. SWISS-MODEL: an 58. Wang, X., and R. D. Miller. 2012. Recombination, transcription, and diversity of automated protein homology-modeling server. Nucleic Acids Res. 31: 3381– a partially germline-joined VH in a mammal. Immunogenetics 64: 713–717. 3385. 59. Smith, D. K., and H. Xue. 1997. Sequence profiles of immunoglobulin and

36. Schro¨dinger, LLC. 2015. The PyMOL molecular graphics system, version 1.8. immunoglobulin-like domains. J. Mol. Biol. 274: 530–545. http://www.jimmunol.org/ 37. Burland, T. G. 2000. DNASTAR’s Lasergene sequence analysis software. 60. Hunkapiller, T., J. Goverman, B. F. Koop, and L. Hood. 1989. Implications of the Methods Mol. Biol. 132: 71–91. diversity of the immunoglobulin gene superfamily. Cold Spring Harb. Symp. 38. Robert, X., and P. Gouet. 2014. Deciphering key features in protein structures Quant. Biol. 54: 15–29. with the new ENDscript server. Nucleic Acids Res. 42: W320–W324. 61. Nei, M., and A. P. Rooney. 2005. Concerted and birth-and-death evolution of 39. Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar. 2013. MEGA6: multigene families. Annu. Rev. Genet. 39: 121–152. molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725– 62. Eirı´n-Lo´pez, J. M., L. Rebordinos, A. P. Rooney, and J. Rozas. 2012. The birth- 2729. and-death evolution of multigene families revisited. Genome Dyn. 7: 170–196. 40. Chothia, C., J. Novotny´, R. Bruccoleri, and M. Karplus. 1985. Domain associ- 63. Liu, Y., I. Soto, Q. Tong, A. Chin, H. J. Bu¨hring, T. Wu, K. Zen, and ation in immunoglobulin molecules. The packing of variable domains. J. Mol. C. A. Parkos. 2005. SIRPbeta1 is expressed as a disulfide-linked homodimer in Biol. 186: 651–663. leukocytes and positively regulates neutrophil transepithelial migration. J. Biol. 41. DuPasquier, L., and I. Chre´tien. 1996. CTX, a new lymphocyte receptor in Chem. 280: 36132–36140.

Xenopus, and the early evolution of Ig domains. Res. Immunol. 147: 218–226. 64. Jiang, P., C. F. Lagenaur, and V. Narayanan. 1999. Integrin-associated protein is by guest on September 28, 2021 42. Lee, S. S., D. Fitch, M. F. Flajnik, and E. Hsu. 2000. Rearrangement of im- a ligand for the P84 neural adhesion molecule. J. Biol. Chem. 274: 559–562. munoglobulin genes in shark germ cells. J. Exp. Med. 191: 1637–1648. 65. Brooke, G., J. D. Holbrook, M. H. Brown, and A. N. Barclay. 2004. 43. Yang, Z., Y. Sun, Y. Ma, Z. Li, Y. Zhao, L. Ren, H. Han, Y. Jiang, and Y. Zhao. Human lymphocytes interact directly with CD47 through a novel member of the 2017. A comprehensive analysis of the germline and expressed TCR repertoire in signal regulatory protein (SIRP) family. J. Immunol. 173: 2562–2570. White Peking duck. Sci. Rep. 7: 41426. 66. Piccio, L., W. Vermi, K. S. Boles, A. Fuchs, C. A. Strader, F. Facchetti, M. Cella, 44. van den Berg, T. K., J. A. Yoder, and G. W. Litman. 2004. On the origins of and M. Colonna. 2005. Adhesion of human T cells to antigen-presenting cells adaptive immunity: innate immune receptors join the tale. Trends Immunol. 25: through SIRPbeta2-CD47 interaction costimulates T-cell proliferation. Blood 11–16. 105: 2421–2427. 45. Viertlboeck, B. C., and T. W. Go¨bel. 2011. The chicken leukocyte receptor 67. Schreeder, D. M., J. P. Cannon, J. Wu, R. Li, M. A. Shakhmatov, and R. S. Davis. cluster. Vet. Immunol. Immunopathol. 144: 1–10. 2010. Cutting edge: FcR-like 6 is an MHC class II receptor. J. Immunol. 185: 46. Kubagawa, H., P. D. Burrows, and M. D. Cooper. 1997. A novel pair of 23–27. immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc. 68. Barrow, A. D., and J. Trowsdale. 2008. The extended human leukocyte receptor Natl. Acad. Sci. USA 94: 5261–5266. complex: diverse ways of modulating immune responses. Immunol. Rev. 224: 47. Alley, T. L., M. D. Cooper, M. Chen, and H. Kubagawa. 1998. Genomic 98–123. structure of PIR-B, the inhibitory member of the paired immunoglobulin-like 69. Chothia, C., D. R. Boswell, and A. M. Lesk. 1988. The outline structure of the receptor genes in mice. Tissue Antigens 51: 224–231. T-cell alpha beta receptor. EMBO J. 7: 3745–3755.