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Evolution of the Pentraxin Family: The New Entry PTX4 This information is current as of April 12, 2017. Yeny Martinez de la Torre, Marco Fabbri, Sebastien Jaillon, Antonio Bastone, Manuela Nebuloni, Annunciata Vecchi, Alberto Mantovani and Cecilia Garlanda J Immunol published online 31 March 2010 http://www.jimmunol.org/content/early/2010/03/31/jimmun ol.0901672 http://www.jimmunol.org/content/suppl/2010/03/31/jimmunol.090167 2.DC1 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Email Alerts Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 Supplementary Material Published March 31, 2010, doi:10.4049/jimmunol.0901672 The Journal of Immunology Evolution of the Pentraxin Family: The New Entry PTX4 Yeny Martinez de la Torre,* Marco Fabbri,* Sebastien Jaillon,* Antonio Bastone,† Manuela Nebuloni,‡ Annunciata Vecchi,* Alberto Mantovani,*,x and Cecilia Garlanda* P entraxins (PTXs) are a superfamily of multifunctional conserved proteins that are characterized by a cyclic multimeric structure and by the presence in their carboxylterminal of an ∼200 aa-long conserved domain, called pentraxin domain. In addition, all the members of this family share an 8 aalong conserved sequence (HxCxS/TWxS, in which x is any amino acid) in the pentraxin domain, called pentraxin signature (1). Some pentraxins, together with collectins and ficolins, constitute the humoral arm of innate immunity and behave as functional ancestors of Abs by mediating agglutination, complement activation, and opsonisation (2). C-reactive protein (CRP), which, together with serum amyloid P (SAP) component (APCS), constitutes the short pentraxin arm of the superfamily, was the first fluid-phase pattern recognition molecule to be identified and named after its ability to bind in a calcium-dependent manner the C-polysaccharide of Streptococcus pneumoniae (2). CRP and SAP are acute-phase proteins *Laboratorio di Immunologia e Infiammazione, Istituto Clinico Humanitas, Istituto Di Ricovero e Cura a Carattere Scientifico, Rozzano; †Mario Negri Institute for Pharmacological Research; and ‡Pathology Unit, L. Sacco Department of Clinical Sciences and xDepartment of Translational Medicine, University of Milan, Milan, Italy Received for publication May 27, 2009. Accepted for publication March 3, 2010. This work was supported by Associazione Italiana per la Ricerca sul Cancro, Ministero Istruzione Università e Ricerca (RBLA039LSF_007), European Commission (project MUGEN, LSHG-CT-2005-005203), Cariplo (Project Nobel), and European Research Council (project HIIS). The name PTX4 was approved by the HUGO Gene Nomenclature Committee on March 25, 2010. Address correspondence and reprint requests to Dr. Alberto Mantovani and Dr. Cecilia Garlanda, Istituto Clinico Humanitas, Via Manzoni 113, I-20089, Rozzano, Milan, Italy. E-mail addresses: [email protected] and cecilia.garlanda@ humanitasresearch.it The online version of this article contains supplemental material. Abbreviations used in this paper: C, pentraxin domain; CRP, C-reactive protein; EMBL, European Molecular Biology Laboratory; FP, female protein; GPR, G-protein–coupled receptor; Mptx, mucosal pentraxin; MS, mass spectrometry; N, N-terminal domain; NCBI, National Center for Biotechnology Information; NJ, neighbor-joining; NP or NPTX, neuronal pentraxin; PTX, pentraxin; SAP, serum amyloid P. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901672 that regulate innate resistance to microbes and the scavenging of cellular debris, conserved from mammals to arthropods (1). In Limulus polyphemus, different forms of CRPs and SAP are normal and abundant constituents of the hemolymph and are involved in recognizing and destroying pathogens (3–5). PTX3 and subsequently other long pentraxins were identified in the 1990s as inducible genes or molecules expressed in specific tissues (e.g., neurons, spermatozoa) (6–8). Long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxyl-terminal pentraxin domain and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in inducing stimuli and ligand-recognition ability. In particular, PTX3 behaves as a soluble pattern recognition receptor playing a nonredundant role in innate immunity against selected pathogens (9–11); it also has a nonredundant role in female fertility due to its structural role in the extracellular matrix (12, 13). PTX3 has also been observed to have a regulatory role on inflammation by acting as a feedback mechanism of inhibition of leukocyte recruitment (14). The long pentraxins identified after PTX3 include guinea pig apexin (15, 16), neuronal pentraxin (NP or NPTX) 1 (17, 18) and NP2, also called NPTX2 or NARP (19, 20), and NPTX receptor, which is the only member associated to the cell through a transmembrane domain (21–23) (see below). NPTXs have been shown to be involved in the excitatory synaptic remodeling (21). NPTX2 has been implicated in long-term neuronal plasticity as well as dopaminergic nerve cell death (24) and NPTX1 in hypoxia-ischemia– and amyloid-b–induced neuronal death (25, 26). The pentraxin domain has also been found in multidomain proteins, such as in the extracellular protein polydom [which includes an N-terminal von Willebrand factor A domain, 2 hyalin repeat domains, 10 epidermal growth factor repeats, 34 complement control protein domains, and a single pentraxin domain (27)] and in a few adhesion G-protein–coupled receptors (GPRs), in particular GPR144, GPR112, and GPR126 (28) (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de/; Prosite, www.expasy.org/prosite/database). The function of these proteins has not been defined yet, nor has the role of the pentraxin domain in multidomain proteins. Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 Pentraxins (PTXs) are a superfamily of multifunctional conserved proteins, some of which are components of the humoral arm of innate immunity and behave as functional ancestors of Abs. They are divided into short (C-reactive protein and serum amyloid P component) and long pentraxins (PTX3 and neuronal pentraxins). Based on a search for pentraxin domain-containing sequences in databases, a phylogenetic analysis of the pentraxin family from mammals to arthropods was conducted. This effort resulted in the identification of a new long pentraxin (PTX4) conserved from mammals to lower vertebrates, which clusters alone in phylogenetic analysis. The results indicated that the pentraxins consist of five clusters: short pentraxins, which can be found in chordate and arthropods; neuronal pentraxins; the prototypic long pentraxin PTX3, which originated very early at the divergence of the vertebrates; the Drosophila pentraxin-like protein B6; and the long pentraxin PTX4 discovered in this study. Conservation of flanking genes in mammalian evolution indicates maintenance of synteny. Analysis of PTX4, in silico and by transcript expression, shows that the gene is well conserved from mammals to lower vertebrates and has a unique pattern of mRNA expression. Thus, PTX4 is a new unique member of the pentraxin superfamily, conserved in evolution. The Journal of Immunology, 2010, 184: 000–000. 2 The present study was designed as a search for pentraxin domain-containing sequences in different databases. We found that based on phylogenetic analysis, the pentraxin superfamily consists of five distinct clusters. This effort led to the identification of a new long pentraxin (PTX4) conserved from mammals to lower vertebrates, which clusters alone in phylogenetic analysis. Materials and Methods Bioinformatics Human tissues and cells Human normal tissue and bone marrow total RNA was purchased from Applied Biosystems (FirstChoice Total RNA; Foster City, CA). Monocytes, lymphocytes, NK cells, and polymorphonuclear cells were isolated from fresh buffy coats of healthy donors (Centro Trasfusionale Ospedale Niguarda, Milan, Italy) using Ficoll (Biochrom, Berlin, Germany) and Percoll (Amersham Biosciences, Uppsala, Sweden) as described (35). Monocytes were incubated with the b form of pro-IL-1 (100 ng/ml; Dompè, L’Aquila, Italy) for 4 or 24 h and T lymphocytes with 100 U/ml PHA for 48 hours. B cells were prepared from tonsils as described (36). HUVECs were obtained as described (6) and stimulated with the b form of pro-IL-1 (20 ng/ml) or LPS (100 ng/ml) for 4 h. Cells were plated at 106 cell/ml, 3 ml/well. Two to three donors were tested for each condition. Murine tissues and cells C57BL/6 mice (Charles River Laboratories, Calco, Italy) were used for ptx4 expression studies. When indicated, mice were injected i.p. with 30 mg/kg LPS (Escherichia coli O55:B5; Sigma-Aldrich, St. Louis, MO) and sacrificed after 6 or 24 h. Mouse peritoneal macrophages and bone marrow-derived dendritic cells were generated and treated as described (37). Leukocytes and the stromal compartment of the thymus and spleen were separated by passing the tissue through a cell strainer (Falcon, BD Biosciences, San Jose, CA). Procedures involving animals and their care conformed to institutional guidelines in compliance with national (4D.L. N.116, G.U., supplement 40, 18-2-1992) and international law and policies (EEC Council Directive 86/ 609, OJ L 358,1,12-12-1987; National Institutes of Health Guide for the Care and Use of Laboratory Animals, U.S. National Research Council 1996). All efforts were made to minimize the number of animals used and their suffering. Quantitative real-time PCR Tissues were homogenized, and total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA). cDNA was synthesized from 1 mg total RNA after DNase treatment by High Capacity cDNA archive kit (Applied Biosystems). The following primers were designed with Primer Express software (Applied Biosystems): mouse pentraxin (mPTX)3 (sense, 59-ACGAAATAGACAATGGACTTCATCC-39, antisense, 59-AGTCGCATGGCGTGGG-39); mPTX4 (sense, 59-TCATCAAGCAGCCCCACC39, antisense, 59-TTGCAAATGTTTCCTGGTCCT-39); b-actin (sense, 59TCACCCACACTGTGCCCATCTACGA-39, antisense, 59-CAGCGGAACCGCTCATTGCCAATGG-39); hPTX3 (sense, 59-CGAAATAGACAATGGACTCCATCC-39, antisense, 59-CAGGCGCACGGCGT-39); and hPTX4 (sense, 59-TCCGGAGATTCCAGGAGGT-39, antisense, 59-TGCTGGCGATGTTCTGCA-39). Quantitative real-time PCR was performed using the Sybr Green PCR Master Mix (Applied Biosystems) in a 7900HT fast real-time PCR system (Applied Biosystems). Data were analyzed with the DDCT method (Applied Biosystems, Real-Time PCR Applications Guide). A standard curve for each reference gene was generated using serial dilutions of a reference sample (tissue cDNA from three control mice). mRNA levels were determined from the appropriate standard curve. Data were normalized by b-actin expression. Analysis of all samples was performed in triplicate. Statistical analyses were carried out with GraphPad Prism software (version 4; GraphPad, San Diego, CA). Differences were evaluated with Dunnett’s multiple comparison test (one-way ANOVA analysis). PTX4 cloning, expression in E. coli, and purification. Murine cDNA form bone marrow and spleen and putative exons from genomic DNA were sequenced by Primm (Milan, Italy). Full-length PTX4 cDNA was amplified from total cDNA of mouse thymus and human small intestine using Phusion High-Fidelity DNA polymerase (New England Biolabs, Beverly, MA) with specific primers (mPTX4 sense: 59GAATTCATGAGGTGCTTGAAGAAGAAGAC-39, antisense: 59-CTCGAG TTATGGACACTGCTCCAGGCAGG-39; and hPTX4 sense: 59-GAATTCATGGGTTGCTCGTGGAGG-39, antisense: 59-CTCGAGTCAGGGACAGCGTTCCAG-39) containing EcoRI and XhoI restriction sites, respectively, and cloned into the pGEX-4T-1 expression vector (Amersham Biosciences). E. coli BL21 (DE3) cells were transformed with the recombinant plasmids. Expression of the fusion protein was induced with 1 mM isopropyl-b-D-thiogalactopyranoside at 20˚C overnight. PTX4-GST fusion proteins were extracted and purified by GSTrap-FF affinity chromatography, according to the manufacturer’s protocol (Amersham Biosciences). Purified proteins were analyzed by 10% SDS-PAGE under reducing conditions and analyzed by Western blotting using anti-GST polyclonal Ab (Amersham Biosciences). In-gel digestion, MALDI-TOF/TOF mass spectrometry analysis, and protein identification. The murine purified protein was run in a 10% SDS-PAGE and identified according to standard protocols following in gel tryptic digestion. Briefly, Coomassie blue-stained gel bands were manually excised from gel, destained overnight with 40% ethanol in 25 mM ammonium bicarbonate, and washed with increasing concentrations of acetonitrile in distilled water. Gel slices were incubated with 10 mM dithiothreitol in 100 mM ammonium bicarbonate at 56˚C for 30 min to reduce disulfide bridges. Thiol groups were alkylated upon reaction with 55 mM iodoacetamide in 100 mM ammonium bicarbonate at room temperature in the dark for 20 min. Tryptic digestion was carried out overnight with 10 ng/ml sequencing modified bovine trypsin (Roche, Basel, Switzerland) at 37˚C in 5% acetonitrile in 25 mM ammonium bicarbonate. The reaction was stopped by adding trifluoroacetic acid (0.1% final). A total of 0.6 ml tryptic digest was loaded on an Opti-Tof 384 Well Insert (Applied Biosystems) and air-dried; before mass spectrometric analysis, 0.6 ml matrix a-cyano-4-hydroxycinnamic acid was added, and the sample was air-dried. The remaining tryptic digest was desalted, concentrated with C18 ZipTip pipette tips (Millipore, Bedford, MA) and cocrystallized on the insert with the matrix before mass spectrometric analysis. The stock solution of matrix was prepared as saturated solution in 50% acetonitrile containing 0.1% trifluoroacetic acid, and diluted 1:1 with 50% acetonitrile containing 0.1% trifluoroacetic acid before mixing with the sample. Peptide mass fingerprinting and mass spectrometry (MS)/MS analysis was done on a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems). The mass spectra were internally calibrated with trypsin autolysis fragments. The five most abundant precursor ions out of the exclusion mass list (ions from human keratin and trypsin) were selected for MS/MS analysis. The combined MS and MS/MS data were submitted by the GPS Explorer version 3.6 software (Applied Biosystems) to the MASCOT database search engine (version 2.1; Matrix Science, Boston, MA) and searched with the following parameters: Swissprot 55.2x database over all Mus musculus protein sequences deposited, no fixed Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 Sequences were retrieved from the Swiss-prot (www.ebi.ac.uk/swissprot/), National Center for Biotechnology Information (NCBI) (http://ncbi.nlm. nih.gov), European Molecular Biology Laboratory (EMBL; www.ebi.ac. uk/embl/), Ensembl (www.ensembl.org), DNA Databank of Japan (www. ddbj.nig.ac.jp/), and University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu/) database using the sequence retrieval system and/or basic local alignment search tool (BLAST) (29). Multiple sequence alignments were carried out using clustal w (30). Phylogenetic trees were constructed on the basis of amino acid difference (p-distance) by the neighbor-joining (NJ) method (complete deletion) using Molecular Evolutionary Genetics Analysis version 3.1 and 4 (31). Reliability of the tree was assessed by bootstrapping, using 1000 bootstrap replications. Information on the organization of PTX3 and PTX4 genes as well as their chromosomal location was retrieved from the Ensembl (www. ensembl.org/) and NCBI (http://ncbi.nlm.nih.gov) databases. Signal peptide predictions were carried out using SignalP 3.0 (32). Calculation of pairwise amino acid identities was carried out using the SIM Alignment tool (33). The NetNGlyc 1.0 software (www.cbs.dtu.dk/services/NetNGlyc/) was used to determine PTX4 potential glycosylation sites. The search of conserved domains was performed with reversed positionspecific (RPS)-BLAST at NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi) using the full-length PTX4 protein sequence as query. The SCWRL3.0 program was used for prediction of protein side-chain conformations (34). The species analyzed were: Homo sapiens (human); Pan troglodytes (chimpanzee); Macaca mulatta (Rhesus macaque); Mus musculus (mouse); Rattus norvegicus (rat); Cavia porcellus (guinea pig); Mesocricetus auratus (golden hamster); Cricetulus migratorius (grey hamster); Canis familiaris (dog); Bos taurus (cow); Sus scrofa (pig); Oryctolagus cuniculus (rabbit); Monodelphis domestica (opossum); Gallus gallus (chicken); Xenopus laevis and tropicalis (African clawed toad); Danio rerio (zebrafish); Takifugu rubripes (pufferfish); L. polyphemus (horseshoe crab); Anopheles gambiae and Anopheles aegypti (mosquito); Drosophila melanogaster (fruit fly); and Ciona intestinalis and savignyi (sea squirt). THE NEW PENTRAXIN PTX4 The Journal of Immunology 3 Table I. Accession number of pentraxins selected in this study retrieved from NCBI or Ensembl databases Table I. (Continued ) Gene Gene Species Database Identification PTX4 Database Identification NM_001017413 ENSXETG00000012244 ENSTRUG00000012858 NM_001013658 XM_372607 ENSMUSG00000044172 XM_220237 ENSMODG00000016175 ENSCAFG00000019558 ENSBTAG00000005148 ENSDARG00000038072 ENSXETG00000009128 Y17570 M. musculus R. norvegicus M. domestica C. familiaris B. taurus D. rerio X. tropicalis b6 Drosophila melanogaster AGAP005038- Anopheles gambiae PA XM_558416 The pentraxins used to generate the phylogenetic tree are in boldface. modifications, as possible modifications carboamidomethylation of cysteine and oxidation of methionine, 1 missed trypsin cleavage, and a mass tolerance of 60.1 D for the peptide mass values and of 60.3 D for the MS/ MS fragment ion mass values. A protein was regarded as identified if MASCOT protein score, based on combined MS and MS/MS data, was above the 5% significance threshold for the database (score .51) (38). Results Phylogenetic analysis of the pentraxin family To understand the relationship among the known short and long pentraxins and their evolution, we performed a phylogenetic analysis looking for conserved sequences in mammals, lower vertebrates, arthropods, and nematodes. All available orthologous sequences of known short and long pentraxins were retrieved from various sequence databases by extensive systematic BLAST searches (Table I). Orthologous molecules have been found so far for the short pentraxin CRP and SAP, the long pentraxin PTX3, and NP1, NP2, and NPR in human, mouse, rat, opossum, chicken, but also in lower vertebrates, such as zebrafish, pufferfish, and frog (Xenopus) (Ensembl and NCBI database). In the rat, a short pentraxin, called Mptx (NM_001037642), has been described; Mptx is a colon pentraxin for which the expression is downregulated by dietary heme (39). According to our analysis, Mptx is different from other short pentraxins and has putative orthologs in the mouse (NM_025470) and human (XM_001131442). Hamster female protein (FP) is a short pentraxin with close homology to SAP, which is preferentially expressed in females at high constitutive levels and is differentially regulated in different hamster species during pregnancy (40). In arthropods, orthologs of the short pentraxins CRP and SAP and a long pentraxin XL-PXN1 have been found in L. polyphemus. As the L. polyphemus genome sequence is still incomplete, the existence of other pentraxins cannot be excluded. Among insects, in D. melanogaster and Anopheles spp., we found multidomain proteins containing a pentraxin domain, which are not related to the vertebrate long pentraxins (Y17570 in D. melanogaster, XM_558415 in A. gambiae and AAEL011440 in A. aegypti). Finally, we did not find putative orthologs of short or long pentraxins in C. elegans or in the ancient chordate Ciona spp. We found multidomain proteins containing a pentraxin-domain in C. elegans (W02C12.1 in Chromosome IV, NCBI: AAB37995) and in C. intestinalis (ENSCING00000010582). Pentraxins coding sequences or amino acid sequences were aligned using the ClustalW algorithm and then uploaded into Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 Homo sapiens M11880 Pan troglodytes ENSPTRT00000002803 Macaca mulatta ENSMMUT00000012470 Oryctolagus M13497 cuniculus Cavia porcellus S60422 Mesocricetus auratu S56005 Monodelphis ENSMODT00000012826 domestica Mus musculus X17496 Rattus norvegicus M83176 Canis familiaris ENSCAFT00000018706 Bos taurus ENSBTAT00000018469 Xenopus laevis L08166 Xenopus tropicalis ENSXETT00000027727 CRP1(1.4) Limulus polyphemus M14024 CRP3(3.3) L. polyphemus M14025 CRP4(1.1) L. polyphemus M14026 FP Cricetulus M31610 migratorius SAP (APCS) H. sapiens D00097 P. troglodytes ENSPTRT00000002799 M. mulatta ENSMMUT00000009360 M. domestica ENSMODT00000012843 M. auratus L22024 M. musculus M23552 R. norvegicus X55761 C. porcellus S60421 Sus scrofa NM_213887 B. taurus ENSBTAT00000026133 Gallus gallus ENSGALG00000022137 X. tropicalis ENSXETT00000052578 L. polyphemus AY066022 Mptx R. norvegicus NM_001037642 NP1 (NPTX1) H. sapiens NM_002522 P. troglodytes ENSPTRT00000017905 M. mulatta ENSMMUT00000001825 M. domestica ENSMODT00000002976 M. domestica ENSMODT00000002979 M. musculus NM_008730 R. norvegicus U18772 C. familiaris ENSCAFG00000005689 B. taurus ENSBTAT00000011769 X. tropicalis ENSXETT00000042466 Danio rerio ENSDART00000066418 Takifugu rubripes NEWSINFRUT00000135826 NP2 H. sapiens U29195 ENSPTRT00000035972 P. troglodytes ENSMMUT00000016767 M. mulatta M. musculus AF318618 R. norvegicus NM_001034199 M. domestica ENSMODT00000038226 C. familiaris ENSCAFT00000024339 B. taurus ENSBTAT00000010374 G. gallus ENSGALT00000005641 X. tropicalis ENSXETT00000051833 D. rerio ENSDART00000055071 T. rubripes NEWSINFRUT00000135347 Apexin C. porcellus U13234 NPR (NPTX2) H. sapiens NM_014293 M. musculus NM_030689 R. norvegicus NM_030841 X. tropicalis ENSXETT00000015097 D. rerio ENSDART00000078201 D. rerio ENSDART00000059181 T. rubripes NEWSINFRUT00000151340 XL-PXN1 X. laevis L19881 PTX3 H. sapiens X63613 M. musculus NM_008987 R. norvegicus ENSRNOT00000016541 M. domestica ENSMODG00000015690 (Table continues) CRP Species G. gallus X. tropicalis T. rubripes H. sapiens 4 molecular evolutionary genetics analysis (Molecular Evolutionary Genetics Analysis version 3.1) (41). We used different algorithms for the construction of phylogenetic trees: the maximumparsimony method and the NJ method. NJ trees were constructed on the basis of the following distances: the uncorrected proportion of amino acid difference (p) and the Poisson-corrected proportion of amino acid differences. The results obtained are shown in Fig. 1, representing the tree for selected short and long pentraxins. Similar results were obtained aligning coding sequences and amino acid sequences. The overall topology of the pentraxin family tree consists of five major distinct clusters containing nearly all the vertebrate pentraxins and, in a separate clade, the invertebrate pentraxins. THE NEW PENTRAXIN PTX4 Phylogenetic analysis of the C- and N-terminal domains of long pentraxins FIGURE 1. Phylogenetic analysis of short and long pentraxins. Accession numbers of all available orthologous sequences of known short pentraxins (CRP, SAP, hamster FP, rat Mptx), long pentraxins (NP1, NP2, NPR, apexin, PTX3, PTX4, Xenopus PXN1) and pentraxin domain-containing sequences (Drosophila and Anopheles pentraxins) used to generate this NJ tree are reported in Table I. The five clusters identified are marked with circles. The name of species analyzed is reported as follows: anogam, A. gambiae (mosquito); capo, C. porcellus (guinea pig); crimi, C. migratorius (grey hamster); drome; D. melanogaster (fruit fly); gaga, G. gallus (chicken); hosa, H. sapiens (human); lipo, L. polyphemus (horseshoe crab); meau, M. auratus (golden hamster); mumu, M. musculus (mouse); orycu, O. cuniculus (rabbit); rano: R. norvegicus (rat); xela: X. laevis (African clawed toad). The amino acid sequence identity among all the members of the long pentraxins is relatively high in the carboxyl-pentraxin domain and ranges from 28% between human PTX3 and NP1 to 68% between human NP1 and NP2, according to an analysis of multiple sequence alignments performed with ClustalW (1.82). By contrast, a lower level of sequence similarity is found in the amino-terminal domain of the subfamily members; in particular, the amino-terminal sequence of PTX3 shows only 10% identity with the human NP1 N-terminal domain sequence; however, the amino acid identity in the amino-terminal domain among the NPTXs is higher and ranges between 28% and 38%, suggesting the existence of subclasses of molecules among the long pentraxins. The sequence similarity between NP1, but also NP2, and PTX3 at the N-terminal level is restricted to the extreme N terminus; this characteristic and the longer size of NP1 and NP2 suggest the presence of a third domain localized between the N-terminal and the pentraxin domains (8). To better understand the evolution and biology of the N- and Cterminal domains of long pentraxins, we performed a second analysis using separately the sequences of the two domains of each Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 The first cluster includes the short pentraxins, CRP and SAP, which originated diverging from the common ancestor of all pentraxins and can be found in chordates (mammals) as well as in arthropods (Limulus) and X. laevis XL-PXN1, which is a long pentraxin (Fig. 1). Because Limulus pentraxins evolved earlier in the pentraxin evolution, they appear on the branch before mammalian short pentraxins, forming a separate clade. The second group includes all the NPTXs that cluster as a subclass of long pentraxins found in mammals as well as in lower vertebrates (Fig. 1). According to the length of branches, among the NPTXs, NPR is the oldest that diverged from a common ancestor of NPTXs; subsequently NP2 and finally NP1 appeared. Human, murine, and rat orthologs of apexin have not been identified so far; it has been suggested that NP2 is the apexin ortholog because of sequence similarity, even if the acrosomal localization is restricted to guinea pig apexin and has not been described for NP2 (19). Accordingly, in our analysis, apexin clustered with the NPTXs and in particular with NP2. The third cluster includes only PTX3, for which the sequence has been identified in mammals as well as in birds (G. gallus) (Fig. 1) and in the most ancient vertebrate T. rubripes (pufferfish) (not shown). PTX3 originated directly from the common ancestors of the pentraxins very early in the evolution of pentraxins, at the divergence of vertebrates. In an attempt to find human and murine orthologs of apexin (see below), we found a new long pentraxin, which we named PTX4. The fourth cluster includes PTX4 and its orthologs in mammals (Fig. 1), Xenopus and D. rerio (zebrafish) (not shown). Also, PTX4 originated very early in the pentraxin evolution, directly from the common ancestor of all of the pentraxins. Finally, the last cluster is represented by D. melanogaster B6 (or CG3100-RA) protein, a 558 aa-long protein containing a pentraxin domain (Y17570), for which the biological function is unknown, and A. gambiae AGAP005038-PA (Fig. 1). B6 protein originated from the common ancestor of pentraxins. BLAST analysis of the B6 sequence versus the human database did not suggest the existence of a putative human ortholog of B6. The NJ tree generated in Fig. 1 shows the lack of relationship among the four groups of long pentraxins identified in this analysis and suggests that these subfamilies originated and evolved independently by fusion events between the gene encoding the ancestral pentraxin domain and other unrelated sequences. The Journal of Immunology 5 pentraxin. The results shown in Fig. 2 indicate that for NPTXs, the C-terminals and the N-terminals of all orthologs form two separate groups. In contrast, the N-terminal domains of PTX3 of each species cluster with the entire molecule because of the low levels of sequence identity among the orthologous N-terminals, whereas the C-terminal domains cluster with the orthologs of other species. The same clusterization occurs for PTX4 N- and C-terminal domains. As expected, short pentraxins cluster together. These results further support the hypothesis that in long pentraxins, the Nterminal domain evolved independently of the pentraxin domain. In particular, N-terminal domains of each NPTX are evolutively close compared to the N-terminal of PTX3 and PTX4, which present sequence divergence among orthologs. The search of conserved domains in the N-terminal portion of long pentraxins using reversed position-specific basic local alignment search tool at NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi) did not reveal significant relationship or similarity among the members of the subfamily or with other known proteins. Identification of a new pentraxin: PTX4 In an attempt to find new pentraxin domain-containing proteins, and in particular the murine and human counterpart of guinea pig apexin (accession number U13236, http://ncbi.nlm.nih.gov), we used the apexin amino acid sequence to search for undefined pentraxins in databases. This analysis led to the identification of the murine clone IMAGE: 1294272 39 in the Expressed Sequence Tags database, deposited in the NCBI database under the accession number AI563675 (http://ncbi.nlm.nih.gov) and defined as “similar to SW:APEX_CAVPO P47970 APEXIN PRECURSOR.” This partial nucleotide sequence was used as bait for searches in murine, rat, Table II. PTX4 orthologs in vertebrates Chromosome/ Scaffold Database Identification (Protein) Amino Acids Identitya versus Human (1) Similaritya versus Human (1) Identitya versus Human (2) Similaritya versus Human (2) Homo sapiens 16 Mus musculus 17 ENSP00000293922 (1) XP_372607 (2) ENSMUSP00000055984 Sequenced from cDNAb ENSRNOP00000022697 ENSMODP00000020216 473 478 482 478 478 492 100 88.5 57.7 58.9 57.2 49.0 100 88.9 67.9 65.4 68.5 60.5 88.5 100 64 69 63.3 53.2 88.9 100 75 76.2 74.8 64.7 478 468 478 482 61.1 58.8 33.1 35.5 69.4 67.9 50.3 51.0 68.8 66.2 35.5 38.5 77.5 75.9 52.9 55.3 Species Rattus norvegicus 10 Monodelphis 6 domestica Canis familiaris 6 ENSCAFP00000028929 Bos taurus 25 XP_602381 Danio rerio 3 ENSDARP00000055499 Xenopus tropicalis Scaffold_702 ENSXETP00000020030 Sequences listed under Database Identification (Protein) were retrieved from Ensembl (www.ensembl.org) and EMBL-European Bioinformatics Institute (www.ebi.ac.uk/). a Identity and similarity were compared to the two human sequences reported: ENSP00000293922 (1) and XP_372607 (2). b Mouse protein predicted according to sequenced cDNA from thymus. Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 FIGURE 2. Phylogenetic analysis of the pentraxin and N-terminal domains of the short and long pentraxins (in human, mouse, and rat). The sequences of the two domains were retrieved from NCBI (see Table I for accession number). C-terminal domains of long pentraxins represented by a continuous circle; Nterminal domains represented by a dotted circle. Orthologous N-terminal sequences cluster together in the case of NPTXs, whereas orthologous N-terminal sequences of PTX3 and PTX4 cluster with the entire molecule. C, pentraxin domain; N, N-terminal domain. 6 THE NEW PENTRAXIN PTX4 human, caw, dog, opossum, zebrafish, pufferfish, and Xenopus genome database for similar yet unknown sequences. This search led to the identification of a new long pentraxin, consisting of ∼470 aa, different from apexin, that we call PTX4, conserved in all mentioned species (Tables I, II). Human, murine, and rat PTX4 show ∼30% identity at the pentraxin domain level with other long pentraxins and 10% identity at the N-terminal level. In multiple sequence alignment of PTX4 sequences, identity ranges from 31–48% among vertebrates and from 49–86% among mammals. Information about PTX4 nucleotide and protein sequences was gathered by several different databases (NCBI, EMBL, Ensembl, DNA Databank of Japan, and University of California Santa Cruz Genome Bioinformatics). Conservation of synteny in PTX3 and PTX4 Analysis of the predicted PTX4 The comparison of the genomic organization of human, murine, rat, and opossum PTX4 revealed a well-conserved gene consisting of three exons. The exons are almost of identical length and all introns contain well-recognizable 59 donor (gt) and 39 splice acceptor (ag) sites (Fig. 4). Identity and similarity between human and other species sequences are reported in Table II. For human PTX4, two alternative in silico transcripts (NM_001013658 [www.ensembl.org] and XM_372607 [EMBLEuropean Bioinformatics Institute; www.ebi.ac.uk/) (Tables I, II), which differ in the first exon, are proposed in several databases. In particular, two alternative possibilities were described for the start FIGURE 3. Comparison of the syntenic blocks around PTX3 (A) and PTX4 (B) in vertebrates: order, orientation, and chromosome location are reported. FIGURE 4. Analysis of PTX4 gene. Comparison of the genomic organization of the human, mouse, rat, and opossum PTX4 genes. Boxes represent exons. Exon sizes are indicated within the boxes; intron sizes are given underneath the introns. The three nucleotide residues surrounding each splice site are shown; coding residues are represented by capitals. The actual splice donor and acceptor residues are indicated in boldface. The two alternative human sequences (1, NM_001013658 and 2, XM_372607) are reported. Accession numbers for PTX4 genes are reported in Tables I and II. codon and thus for the first exon; second and third exons are the same in the two sequences (Fig. 4). Identity and similarity between PTX4 orthologs in vertebrates and the XM_372607/XP_372607 (www.ebi.ac.uk/) sequence is higher compared to NM_001013658/ ENSP00000293922 (www.ensembl.org) (Table II). Conservation in evolution among mammalian PTX4s and the presence of a putative signal peptide only in the sequence XM_372607 (data not shown) suggest that the correct human PTX4 ortholog is XM_372607. Moreover, we failed to amplify the total cDNA or the first exon using primers designed on the NM_001013658 sequence, whereas we amplified a full-length PTX4 cDNA from human small intestine total cDNA using primers designed on the XM_372607 sequence. The complete nucleotide sequence of murine ptx4 (ENSMUSG00000044172, www.ensembl.org) consists of a 74 bp 59 untranslated region and an open reading frame of 1449 bp with a TGA stop codon at position 1521. The predicted murine protein sequence is 482 aa-long (Fig. 5). A significant alignment was found between the C-terminal portion of the PTX4 protein sequence, from position 269–468, and the C-terminal portion of the pentraxin family members. These residues constitute a pentraxin domain; the pentraxin signature typical of the family (HXCXS/ TWXS/T) differed for an amino acid in position 5 of the signature, with an isoleucine replacing the serine or threonine. Similarly, in rat Mptx and in human and rat NPR, the amino acid in position 5 of the signature is replaced. In the murine sequence (ENSMUSG00000044172), the first methionine at nucleotide position 74 is immediately followed by a typical signal peptide sequence (Fig. 5), as predicted according to the analysis performed with SignalP 3.0, with a cleavage site between the amino acids (serine-glutamine) at position 25 and 26 of the amino terminus. This putative signal peptide sequence suggests that this protein belongs to a family of classically secreted proteins. Concerning the human PTX4, a predicted signal peptide is present in the amino acid sequence XP_372607 but not in ENSP00000293922. The NetNGlyc 1.0 computer analysis of the murine amino acid sequence showed the presence of three potential N-linked Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 The relationship of PTX3 and PTX4 orthologs was also addressed analyzing the syntenic regions in different species. Fig. 3 shows the order and orientation of PTX3 and PTX4 and adjacent genes in human, mouse, rat, opossum, frog (X. laevis), and chicken. For PTX3, in Xenopus (scaffold S-50), chicken (chromosome 9), and opossum (chromosome 7), the gene order is SHOX2, VEPH1, PTX3, CCNL1 (Fig. 3A). Further chromosomal rearrangements appear in the mouse (chromosome 3 E1) and rat (chromosome 2q31) involving both order and orientation, which are conserved in human (chromosome 3q25.32). For PTX4, in opossum, the gene order is CLCN7, PTX4, TELO2, IFT140, TMEM204 (chromosome 6), and it is maintained in human (chromosome 16p13.3), whereas the entire chromosomal region changes orientation in rat (chromosome 10q12) and mouse (chromosome 17A3.3) (Fig. 3B). The Journal of Immunology 7 Expression of PTX4 mRNA in human and murine tissues PTX4 mRNA expression was analyzed by real-time PCR on commercially available human cDNA from different tissues in normal conditions. As shown in Fig. 6, PTX4 was expressed at low levels in most tissues analyzed. Expression was higher in selected tissues, such as small intestine, testes (Fig. 6), and bone marrow (not shown). We further analyzed PTX4 expression in endothelial cells and leukocytes, in particular monocytes, resting and PHAactivated peripheral blood lymphocytes, tonsil B lymphocytes, neutrophils, and NK cells (not shown). In endothelial cells, monocytes, neutrophils, and lymphocytes, PTX4 expression was FIGURE 5. Analysis of murine ptx4 gene and protein sequence. Predicted nucleotide and protein sequence of mouse ptx4 (ENSMUSG00000044172; www.ensembl.org) are shown. Amino acids are numbered from 1–482 and nucleotides from 1–1563. The potential signal peptide starting with the first methionine is in italics with the putative cleavage site underscored (Q). The pentraxin domain is underlined, the laminin G domain is double underlined, and the COG4372 domain is underlined with a dotted line. The 8 aas that constitute the pentraxin consensus signature are in boldface and italics. The two cysteine residues that are conserved in all members of the pentraxin family are shaded (C300, C364). Asterisks indicate the end of first and second exons. The potential N-glycosylation sites are indicated in boldface. Nucleotide and amino acid differences found sequencing cDNA from tissues are in grey and boldface. glycosylation sites at the amino acid positions 91 (NQS), 277 (NTS), and 458 (NVT) (Fig. 5). A fourth potential N-glycosylation site was predicted at the position 202 (NPT), but future additional confirmatory evidence is needed because a proline (P) occurs just after the asparagine (N) residue, and this makes it highly unlikely for the asparagine to be glycosylated, presumably due to conformational constraints. In the human sequence, the glycosylation site in position 91 (NRS) is conserved, suggesting conservation of glycosylation. The search of conserved domains using reversed positionspecific basic local alignment search tool at NCBI (www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi) with the murine full-length ptx4 protein sequence as query indicates the presence of the pentraxin FIGURE 6. Expression of human PTX4 mRNA by real-time PCR in normal tissues. Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 domain at position 269 with an e-value of 8e-36, a laminin G domain starting at position 311 with an e-value of 1e-3. A further conserved domain is present in the N-terminal portion, spanning aa 59–188, called COG4372 and found in uncharacterized myosin-like domain-carrying proteins conserved in bacteria (e-value 7e-3). According to structural analysis performed with SCWRL3.0 (34), the cysteins in positions 300 and 364 could form disulphide bridges. We sequenced the murine ptx4 cDNA obtained from bone marrow and spleen of C57BL/6 mice and the putative three exons from 129Sv genomic DNA. Compared to the deposited sequences (ENSMUSG00000044172 and XM_128459), we observed few relevant differences, which are reported in Fig. 5. Among these are those changing the amino acid sequence in positions 116 (R instead of Q), 187 (S instead of R), 202 (T instead of N), 437 (G instead of R), and 448 (F instead of L). Finally, a nucleotide modification in position 1511 introduces a stop codon, indicating that the protein is 478 and not 482 aa long. The modification in position 202 eliminates the predicted potential N-glycosylation site (NPT). Interestingly, our sequence exactly overlaps with that of the commercially available I.M.A.G.E. consortium cDNA clone BC118508 (ID 40106397). Concerning the human PTX4, the sequence of the cDNA obtained from human small intestine using primers designed on the XM_372607 human sequence perfectly overlaps with the in silico deposited XM_372607 sequence. 8 THE NEW PENTRAXIN PTX4 very low, and it was not induced by IL-1 or LPS (endothelium, monocytes, and neutrophils) or PHA (lymphocytes). Murine ptx4 expression was analyzed by real-time PCR in several tissues in normal conditions and posttreatment with LPS and in leukocytes. As shown in Fig. 7A, ptx4 was expressed at low levels in all tissues analyzed and was not induced by LPS. On the contrary, ptx4 expression was downmodulated in the liver (p , 0.01), lung, heart, and spleen. The only exception is the thymus, where we observed a significant ptx4 induction by LPS (p , 0.05). In dendritic cells and peritoneal macrophages, stimulation with TNF-a or LPS downmodulated ptx4 expression (not shown). In spleen, liver, and thymus, which are the organs expressing higher murine ptx4 levels, we compared ptx3 and ptx4 relative expression upon stimulation with LPS (Fig. 7B). The results obtained indicate divergence in regulation of these two genes by LPS, because in basal conditions, ptx4 expression is higher than ptx3 expression and is not induced by LPS treatment apart from the thymus, whereas ptx3 is always upregulated. In the spleen and thymus, we further analyzed the cellular and stromal compartments of these organs separately and observed that ptx4 relative expression is higher in the stroma than in lymphocytes (p , 0.01) (Fig. 7C). were analyzed by SDS-PAGE, and a protein with an apparent m.w. of ∼75 kDa was observed (Fig. 8). Given that the mass contribution from the GST tag, present in both chimeric proteins, is 26 kDa, the observed immunoreactive bands at ∼75 kDa are likely to correspond to the PTX4-GST fusion protein. Moreover, little or no signal was detected without isopropyl-b-D-thiogalactopyranoside induction (Fig. 8). Murine ptx4-GST was purified by GSTrap-FF affinity chromatography followed by in gel tryptic digestion, Expression of human and murine PTX4 protein FIGURE 8. Expression of recombinant human and murine PTX4. Human and murine PTX4 cDNA were amplified from small intestine and thymus, respectively, and cloned into pGEX-4T1 vector and expressed in E. coli. The bacterial lysates were analyzed by Western blotting using the polyclonal anti-GST Ab. The observed 75-kDa immunoreactive bands are likely to correspond to the PTX4-GST fusion proteins (52.3 kDa plus 26 kDa for human PTX4 and 53.1 kDa plus 26 kDa for murine ptx4). To produce the putative proteins, murine and human PTX4 cDNA were amplified from total cDNA of mouse thymus and human small intestine and cloned into the pGEX-4T1 vector and expressed in E. coli. Human and murine PTX4 have a predicted m.w. of 52.339 Da and 53.084 Da, respectively. The bacterial lysates Downloaded from http://www.jimmunol.org/ by guest on April 12, 2017 FIGURE 7. Expression of murine ptx4 mRNA by real-time PCR. A, ptx4 expression in murine tissues in normal conditions and after LPS injection. Mice were injected i.p. with LPS (30 mg/kg) and analyzed 6 and 24 h later. B, Comparison of ptx4 and ptx3 expression in selected organs in basal conditions and after LPS treatment (30 mg/kg). C, Comparison of ptx4 expression in the stromal compartment and in leukocytes of the thymus and spleen. Leukocytes and the stromal compartment were separated by passing the tissue through a cell strainer. Error bars indicate the standard deviation of three replicate samples. Results are representative of one out of three independent experiments. Asterisks indicate a significant difference. pp , 0.05; ppp , 0.01, one-way ANOVA analysis. The Journal of Immunology peptide mass fingerprinting, and MS/MS analysis. MALDI-MS of purified murine ptx4 resulted in the detection of 18 peptides for which the molecular masses are reported in the Supplemental Table I. Three of these peptides were successfully sequenced by MALDI-MS/MS. The 18 peptides identified in our analysis (178 aa residues) represent 39% of the entire primary structure of the protein, and the combined MASCOT protein score of MS and MS/ MS analysis was 98 [protein scores .51 are significant (p , 0.05)], making certain the identification of murine ptx4 (ENSMUSP00000055984). Using the polyclonal Abs directed against three murine ptx4 peptides, we performed immunohistochemistry and found that ptx4 is indeed present in liver as expected (not shown). Discussion Analysis of the new entry PTX4, in silico and by transcript expression, shows that the gene is well conserved among mammals. For human PTX4, two alternative cDNA sequences that differ in the first exon have been published in databases. We failed to amplify the PTX4 cDNA using primers designed on the NM_001013658 sequence, whereas we amplified the PTX4 cDNA from small intestine using primers designed on the XM_372607 sequence. The sequence of the amplified cDNA suggests that, at least in this tissue, the transcribed PTX4 corresponds to XM_372607. Moreover, identity and similarity between PTX4 orthologs in vertebrates and this latter sequence (XM_372607/XP_372607) is higher compared to NM_001013658/ENSP00000293922. Finally, a predicted signal peptide for human PTX4 is present in XP_372607 but not in ENSP00000293922. Collectively, these data suggest that the human PTX4 corresponds to XM_372607/XP_372607. Whether an alternatively spliced form corresponding to NM_001013658 exists in particular conditions has to be determined. Finally, PTX4 has a unique pattern of mRNA expression. In particular, the results suggest that expression of PTX4 is distinct from that of other members of the family. For instance, unlike NPTXs, PTX4 expression is low in the brain. Unlike CRP and SAP, in spite of expression in the liver, it does not behave as an acute phase gene (1). The high expression in the stroma of thymus and spleen is unique among pentraxins. Thus, PTX4 is a new unique member of the pentraxin superfamily, conserved in evolution. Further studies are needed to define its function. Acknowledgments We thank Alfredo Cagnotto, Istituto Mario Negri, for the generation of peptides. Disclosures The authors have no financial conflicts of interest. References 1. Pepys, M. B., and G. M. Hirschfield. 2003. C-reactive protein: a critical update. J. Clin. Invest. 111: 1805–1812. 2. Garlanda, C., B. Bottazzi, A. Bastone, and A. Mantovani. 2005. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu. Rev. 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The results discussed in this paper indicate that based on phylogenetic analysis, the pentraxin superfamily consists of five distinct clusters: short pentraxins, which can be found in chordate and arthropods and originated diverging from the common ancestor of all pentraxins; NPTXs, a subgroup of long pentraxins, with NPR being the first to diverge from a common ancestor; the prototypic long pentraxin PTX3, which originated very early at the divergence of vertebrates; Drosophila B6, a long pentraxin localized near PTX3 and PTX4 in the phylogenetic tree; and the long pentraxin PTX4 present in mammals, Xenopus, and zebrafish, which was discovered in the context of this study. The short pentraxins CRP and SAP originated diverging from the common ancestor of all pentraxins. Both can be found in chordates as well as in arthropods, suggesting that the duplication event that gave rise to these highly homologous proteins possibly occurred very early in the evolution. However, this phylogenetic analysis supports previous studies that proposed that the duplication of CRP or SAP, followed by sequence divergence and evolution of function, occurred independently along the chordates and arthropods rather than in a common ancestor (42). In fact, the arthropod (Limulus) CRP and SAP sequences emerge as a monophyletic group, thus suggesting their strict homology, and cluster together rather than with the orthologous mammalian CRP and SAP. The monophyly of each of the CRP and SAP clades of vertebrates is also unequivocal, confirming that the gene duplication event leading to their generation occurred before the divergence of the vertebrates analyzed in this study. Interestingly, X. laevis XL-PXN1, which is a long pentraxin, clusters with the short pentraxins, possibly because of the low level of homology between its N-terminal domain and that of the other long pentraxins. 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