Entry PTX4 Evolution of the Pentraxin Family

Evolution of the Pentraxin Family: The New Entry PTX4 Yeny Martinez de la Torre, Marco Fabbri, Sebastien Jaillon, Antonio Bastone, Manuela Nebuloni, Annunciata Vecchi, This information is current as Alberto Mantovani and Cecilia Garlanda of September 24, 2021. J Immunol published online 31 March 2010 http://www.jimmunol.org/content/early/2010/03/31/jimmun ol.0901672 Downloaded from

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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*

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 Downloaded from 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.

entraxins (PTXs) are a superfamily of multifunctional that regulate innate resistance to microbes and the scavenging of conserved proteins that are characterized by a cyclic cellular debris, conserved from mammals to arthropods (1). In P multimeric structure and by the presence in their carboxyl- Limulus polyphemus, different forms of CRPs and SAP are normal http://www.jimmunol.org/ terminal of an ∼200 aa-long conserved domain, called pentraxin and abundant constituents of the hemolymph and are involved in domain. In addition, all the members of this family share an 8 aa- recognizing and destroying pathogens (3–5). long conserved sequence (HxCxS/TWxS, in which x is any amino PTX3 and subsequently other long pentraxins were identified in acid) in the pentraxin domain, called pentraxin signature (1). the 1990s as inducible genes or molecules expressed in specific Some pentraxins, together with collectins and ficolins, constitute tissues (e.g., neurons, spermatozoa) (6–8). Long pentraxins have the humoral arm of innate immunity and behave as functional an unrelated, long amino-terminal domain coupled to the car- ancestors of Abs by mediating agglutination, complement acti- boxyl-terminal pentraxin domain and differ, with respect to short

vation, and opsonisation (2). pentraxins, in their gene organization, chromosomal localization, by guest on September 24, 2021 C-reactive protein (CRP), which, together with serum amyloid P cellular source, and in inducing stimuli and ligand-recognition (SAP) component (APCS), constitutes the short pentraxin arm of ability. In particular, PTX3 behaves as a soluble pattern recogni- the superfamily, was the first fluid-phase pattern recognition tion receptor playing a nonredundant role in innate immunity molecule to be identified and named after its ability to bind in against selected pathogens (9–11); it also has a nonredundant role a calcium-dependent manner the C-polysaccharide of Strepto- in female fertility due to its structural role in the extracellular coccus pneumoniae (2). CRP and SAP are acute-phase proteins matrix (12, 13). PTX3 has also been observed to have a regulatory role on inflammation by acting as a feedback mechanism of in- hibition of leukocyte recruitment (14). *Laboratorio di Immunologia e Infiammazione, Istituto Clinico Humanitas, Istituto The long pentraxins identified after PTX3 include guinea pig † Di Ricovero e Cura a Carattere Scientifico, Rozzano; Mario Negri Institute for apexin (15, 16), neuronal pentraxin (NP or NPTX) 1 (17, 18) and Pharmacological Research; and ‡Pathology Unit, L. Sacco Department of Clinical Sciences and xDepartment of Translational Medicine, University of Milan, Milan, NP2, also called NPTX2 or NARP (19, 20), and NPTX receptor, Italy which is the only member associated to the cell through a trans- Received for publication May 27, 2009. Accepted for publication March 3, 2010. membrane domain (21–23) (see below). NPTXs have been shown This work was supported by Associazione Italiana per la Ricerca sul Cancro, Min- to be involved in the excitatory synaptic remodeling (21). NPTX2 istero Istruzione Universita` e Ricerca (RBLA039LSF_007), European Commission has been implicated in long-term neuronal plasticity as well as (project MUGEN, LSHG-CT-2005-005203), Cariplo (Project Nobel), and European Research Council (project HIIS). dopaminergic nerve cell death (24) and NPTX1 in hypoxia-is- chemia– and amyloid-b–induced neuronal death (25, 26). The name PTX4 was approved by the HUGO Gene Nomenclature Committee on March 25, 2010. The pentraxin domain has also been found in multidomain Address correspondence and reprint requests to Dr. Alberto Mantovani and Dr. Cecilia proteins, such as in the extracellular protein polydom [which Garlanda, Istituto Clinico Humanitas, Via Manzoni 113, I-20089, Rozzano, Milan, Italy. includes an N-terminal von Willebrand factor A domain, 2 hyalin E-mail addresses: [email protected] and cecilia.garlanda@ repeat domains, 10 epidermal growth factor repeats, 34 comple- humanitasresearch.it ment control protein domains, and a single pentraxin domain (27)] The online version of this article contains supplemental material. and in a few adhesion G-protein–coupled receptors (GPRs), in Abbreviations used in this paper: C, pentraxin domain; CRP, C-reactive protein; EMBL, European Molecular Biology Laboratory; FP, female protein; GPR, G-protein–coupled particular GPR144, GPR112, and GPR126 (28) (Simple Modular receptor; Mptx, mucosal pentraxin; MS, mass spectrometry; N, N-terminal domain; Architecture Research Tool, http://smart.embl-heidelberg.de/; NCBI, National Center for Biotechnology Information; NJ, neighbor-joining; NP or Prosite, www.expasy.org/prosite/database). The function of these NPTX, neuronal pentraxin; PTX, pentraxin; SAP, serum amyloid P. proteins has not been defined yet, nor has the role of the pentraxin Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 domain in multidomain proteins.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901672 2 THE NEW PENTRAXIN PTX4

The present study was designed as a search for pentraxin do- Quantitative real-time PCR main-containing sequences in different databases. We found that Tissues were homogenized, and total RNA was extracted by TRIzol based on phylogenetic analysis, the pentraxin superfamily consists (Invitrogen, Carlsbad, CA). cDNA was synthesized from 1 mg total RNA of five distinct clusters. This effort led to the identification of a new after DNase treatment by High Capacity cDNA archive kit (Applied long pentraxin (PTX4) conserved from mammals to lower verte- Biosystems). The following primers were designed with Primer Express brates, which clusters alone in phylogenetic analysis. software (Applied Biosystems): mouse pentraxin (mPTX)3 (sense, 59-ACGAAATAGACAATGGACTTCATCC-39, antisense, 59-AGTCG- CATGGCGTGGG-39); mPTX4 (sense, 59-TCATCAAGCAGCCCCACC- Materials and Methods 39, antisense, 59-TTGCAAATGTTTCCTGGTCCT-39); b-actin (sense, 59- Bioinformatics TCACCCACACTGTGCCCATCTACGA-39,antisense,59-CAGCGGAACC- GCTCATTGCCAATGG-39); hPTX3 (sense, 59-CGAAATAGACAATGG- Sequences were retrieved from the Swiss-prot (www.ebi.ac.uk/swissprot/), ACTCCATCC-39, antisense, 59-CAGGCGCACGGCGT-39); and hPTX4 National Center for Biotechnology Information (NCBI) (http://ncbi.nlm. (sense, 59-TCCGGAGATTCCAGGAGGT-39,antisense,59-TGCTGGCGAT- nih.gov), European Molecular Biology Laboratory (EMBL; www.ebi.ac. GTTCTGCA-39). Quantitative real-time PCR was performed using the Sybr uk/embl/), Ensembl (www.ensembl.org), DNA Databank of Japan (www. Green PCR Master Mix (Applied Biosystems) in a 7900HT fast real-time PCR ddbj.nig.ac.jp/), and University of California Santa Cruz Genome Bio- system (Applied Biosystems). Data were analyzed with the DDCT method informatics (http://genome.ucsc.edu/) database using the sequence retrieval (Applied Biosystems, Real-Time PCR Applications Guide). A standard curve system and/or basic local alignment search tool (BLAST) (29). Multiple for each reference gene was generated using serial dilutions of a reference sequence alignments were carried out using clustal w (30). sample (tissue cDNA from three control mice). mRNA levels were determined Phylogenetic trees were constructed on the basis of amino acid difference from the appropriate standard curve. Data were normalized by b-actin ex- (p-distance) by the neighbor-joining (NJ) method (complete deletion) using pression. Analysis of all samples was performed in triplicate. Molecular Evolutionary Genetics Analysis version 3.1 and 4 (31). Re- Statistical analyses were carried out with GraphPad Prism software Downloaded from liability of the tree was assessed by bootstrapping, using 1000 bootstrap (version 4; GraphPad, San Diego, CA). Differences were evaluated with replications. Information on the organization of PTX3 and PTX4 genes as Dunnett’s multiple comparison test (one-way ANOVA analysis). well as their chromosomal location was retrieved from the Ensembl (www. PTX4 cloning, expression in , and purification. ensembl.org/) and NCBI (http://ncbi.nlm.nih.gov) databases. E. coli Murine cDNA form bone marrow and spleen and putative exons from genomic DNA were Signal peptide predictions were carried out using SignalP 3.0 (32). sequenced by Primm (Milan, Italy). Calculation of pairwise amino acid identities was carried out using the SIM Alignment tool (33). Full-length PTX4 cDNAwas amplified from total cDNA of mouse thymus andhuman smallintestineusingPhusion High-Fidelity DNApolymerase (New The NetNGlyc 1.0 software (www.cbs.dtu.dk/services/NetNGlyc/) was 9 http://www.jimmunol.org/ used to determine PTX4 potential glycosylation sites. England Biolabs, Beverly, MA) with specific primers (mPTX4 sense: 5 - GAATTCATGAGGTGCTTGAAGAAGAAGAC-39, antisense: 59-CTCGAG The search of conserved domains was performed with reversed position- TTATGGACACTGCTCCAGGCAGG-39;andhPTX4sense:59-GAATTC- specific (RPS)-BLAST at NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/ ATGGGTTGCTCGTGGAGG-39, antisense: 59-CTCGAGTCAGGGACAG- wrpsb.cgi) using the full-length PTX4 protein sequence as query. CGTTCCAG-39) containing EcoRI and XhoI restriction sites, respectively, The SCWRL3.0 program was used for prediction of protein side-chain and cloned into the pGEX-4T-1 expression vector (Amersham Biosciences). conformations (34). E. coli BL21 (DE3) cells were transformed with the recombinant The species analyzed were: Homo sapiens (human); Pan troglodytes plasmids. Expression of the fusion protein was induced with 1 mM iso- (chimpanzee); Macaca mulatta (Rhesus macaque); Mus musculus (mouse); propyl-b-D-thiogalactopyranoside at 20˚C overnight. PTX4-GST fusion Rattus norvegicus (rat); Cavia porcellus (guinea pig); Mesocricetus aur- proteins were extracted and purified by GSTrap-FF affinity chromatogra- atus (golden hamster); Cricetulus migratorius (grey hamster); Canis fa-

phy, according to the manufacturer’s protocol (Amersham Biosciences). by guest on September 24, 2021 miliaris (dog); Bos taurus (cow); Sus scrofa (pig); Oryctolagus cuniculus Purified proteins were analyzed by 10% SDS-PAGE under reducing con- (rabbit); Monodelphis domestica (opossum); Gallus gallus (chicken); ditions and analyzed by Western blotting using anti-GST polyclonal Ab Xenopus laevis and tropicalis (African clawed toad); Danio rerio (zebra- (Amersham Biosciences). fish); Takifugu rubripes (pufferfish); L. polyphemus (horseshoe crab); Anopheles gambiae and Anopheles aegypti (mosquito); Drosophila mela- In-gel digestion, MALDI-TOF/TOF mass spectrometry analysis, and nogaster (fruit fly); and Ciona intestinalis and savignyi (sea squirt). protein identification. The murine purified protein was run in a 10% SDS-PAGE and identified according to standard protocols following in gel Human tissues and cells tryptic digestion. Briefly, Coomassie blue-stained gel bands were manually excised from gel, destained overnight with 40% ethanol in 25 mM am- Human normal tissue and bone marrow total RNA was purchased from monium bicarbonate, and washed with increasing concentrations of ace- Applied Biosystems (FirstChoice Total RNA; Foster City, CA). Monocytes, lymphocytes, NK cells, and polymorphonuclear cells were tonitrile in distilled water. Gel slices were incubated with 10 mM isolated from fresh buffy coats of healthy donors (Centro Trasfusionale dithiothreitol in 100 mM ammonium bicarbonate at 56˚C for 30 min to Ospedale Niguarda, Milan, Italy) using Ficoll (Biochrom, Berlin, Germany) reduce disulfide bridges. Thiol groups were alkylated upon reaction with and Percoll (Amersham Biosciences, Uppsala, Sweden) as described (35). 55 mM iodoacetamide in 100 mM ammonium bicarbonate at room tem- Monocytes were incubated with the b form of pro-IL-1 (100 ng/ml; perature in the dark for 20 min. Tryptic digestion was carried out overnight m Dompe`, L’Aquila, Italy) for 4 or 24 h and T lymphocytes with 100 U/ml with 10 ng/ l sequencing modified bovine trypsin (Roche, Basel, Swit- PHA for 48 hours. B cells were prepared from tonsils as described (36). zerland) at 37˚C in 5% acetonitrile in 25 mM ammonium bicarbonate. The HUVECs were obtained as described (6) and stimulated with the b form of reaction was stopped by adding trifluoroacetic acid (0.1% final). pro-IL-1 (20 ng/ml) or LPS (100 ng/ml) for 4 h. Cells were plated at 106 A total of 0.6 ml tryptic digest was loaded on an Opti-Tof 384 Well cell/ml, 3 ml/well. Two to three donors were tested for each condition. Insert (Applied Biosystems) and air-dried; before mass spectrometric analysis, 0.6 ml matrix a-cyano-4-hydroxycinnamic acid was added, and Murine tissues and cells the sample was air-dried. The remaining tryptic digest was desalted, C57BL/6 mice (Charles River Laboratories, Calco, Italy) were used for ptx4 concentrated with C18 ZipTip pipette tips (Millipore, Bedford, MA) and expression studies. When indicated, mice were injected i.p. with 30 mg/kg cocrystallized on the insert with the matrix before mass spectrometric LPS (Escherichia coli O55:B5; Sigma-Aldrich, St. Louis, MO) and sac- analysis. The stock solution of matrix was prepared as saturated solution in rificed after 6 or 24 h. 50% acetonitrile containing 0.1% trifluoroacetic acid, and diluted 1:1 with Mouse peritoneal macrophages and bone marrow-derived dendritic cells 50% acetonitrile containing 0.1% trifluoroacetic acid before mixing with were generated and treated as described (37). Leukocytes and the stromal the sample. Peptide mass fingerprinting and mass spectrometry (MS)/MS compartment of the thymus and spleen were separated by passing the analysis was done on a 4800 MALDI-TOF/TOF mass spectrometer (Ap- tissue through a cell strainer (Falcon, BD Biosciences, San Jose, CA). plied Biosystems). The mass spectra were internally calibrated with trypsin Procedures involving animals and their care conformed to institutional autolysis fragments. The five most abundant precursor ions out of the guidelines in compliance with national (4D.L. N.116, G.U., supplement 40, exclusion mass list (ions from human keratin and trypsin) were selected for 18-2-1992) and international law and policies (EEC Council Directive 86/ MS/MS analysis. The combined MS and MS/MS data were submitted by 609, OJ L 358,1,12-12-1987; National Institutes of Health Guide for the the GPS Explorer version 3.6 software (Applied Biosystems) to the Care and Use of Laboratory Animals, U.S. National Research Council MASCOT database search engine (version 2.1; Matrix Science, Boston, 1996). All efforts were made to minimize the number of animals used and MA) and searched with the following parameters: Swissprot 55.2x data- their suffering. base over all Mus musculus protein sequences deposited, no fixed The Journal of Immunology 3

Table I. Accession number of pentraxins selected in this study retrieved Table I. (Continued) from NCBI or Ensembl databases

Gene Species Database Identification Gene Species Database Identification G. gallus NM_001017413 CRP Homo sapiens M11880 X. tropicalis ENSXETG00000012244 Pan troglodytes ENSPTRT00000002803 T. rubripes ENSTRUG00000012858 Macaca mulatta ENSMMUT00000012470 PTX4 H. sapiens NM_001013658 Oryctolagus M13497 XM_372607 cuniculus M. musculus ENSMUSG00000044172 Cavia porcellus S60422 R. norvegicus XM_220237 Mesocricetus auratu S56005 M. domestica ENSMODG00000016175 Monodelphis ENSMODT00000012826 C. familiaris ENSCAFG00000019558 domestica B. taurus ENSBTAG00000005148 Mus musculus X17496 D. rerio ENSDARG00000038072 Rattus norvegicus M83176 X. tropicalis ENSXETG00000009128 Canis familiaris ENSCAFT00000018706 b6 Drosophila Y17570 Bos taurus ENSBTAT00000018469 melanogaster Xenopus laevis L08166 AGAP005038- Anopheles gambiae XM_558416 Xenopus tropicalis ENSXETT00000027727 PA CRP1(1.4) Limulus polyphemus M14024 CRP3(3.3) L. polyphemus M14025 The pentraxins used to generate the phylogenetic tree are in boldface. CRP4(1.1) L. polyphemus M14026 Downloaded from FP Cricetulus M31610 migratorius modifications, as possible modifications carboamidomethylation of cyste- SAP (APCS) H. sapiens D00097 ine and oxidation of methionine, 1 missed trypsin cleavage, and a mass P. troglodytes ENSPTRT00000002799 tolerance of 60.1 D for the peptide mass values and of 60.3 D for the MS/ M. mulatta ENSMMUT00000009360 MS fragment ion mass values. A protein was regarded as identified if M. domestica ENSMODT00000012843 MASCOT protein score, based on combined MS and MS/MS data, was M. auratus L22024 above the 5% significance threshold for the database (score .51) (38). http://www.jimmunol.org/ M. musculus M23552 R. norvegicus X55761 C. porcellus S60421 Sus scrofa NM_213887 Results B. taurus ENSBTAT00000026133 Phylogenetic analysis of the pentraxin family Gallus gallus ENSGALG00000022137 To understand the relationship among the known short and long X. tropicalis ENSXETT00000052578 L. polyphemus AY066022 pentraxins and their evolution, we performed a phylogenetic Mptx R. norvegicus NM_001037642 analysis looking for conserved sequences in mammals, lower NP1 (NPTX1) H. sapiens NM_002522 vertebrates, arthropods, and nematodes. All available orthologous P. troglodytes ENSPTRT00000017905 sequences of known short and long pentraxins were retrieved from by guest on September 24, 2021 M. mulatta ENSMMUT00000001825 M. domestica ENSMODT00000002976 various sequence databases by extensive systematic BLAST M. domestica ENSMODT00000002979 searches (Table I). M. musculus NM_008730 Orthologous molecules have been found so far for the short R. norvegicus U18772 pentraxin CRP and SAP, the long pentraxin PTX3, and NP1, NP2, C. familiaris ENSCAFG00000005689 and NPR in human, mouse, rat, opossum, chicken, but also in lower B. taurus ENSBTAT00000011769 X. tropicalis ENSXETT00000042466 vertebrates, such as zebrafish, pufferfish, and frog (Xenopus) (En- Danio rerio ENSDART00000066418 sembl and NCBI database). In the rat, a short pentraxin, called Mptx Takifugu rubripes NEWSINFRUT00000135826 (NM_001037642), has been described; Mptx is a colon pentraxin NP2 H. sapiens U29195 for which the expression is downregulated by dietary heme (39). P. troglodytes ENSPTRT00000035972 M. mulatta ENSMMUT00000016767 According to our analysis, Mptx is different from other short pen- M. musculus AF318618 traxins and has putative orthologs in the mouse (NM_025470) and R. norvegicus NM_001034199 human (XM_001131442). Hamster female protein (FP) is a short M. domestica ENSMODT00000038226 pentraxin with close homology to SAP, which is preferentially C. familiaris ENSCAFT00000024339 expressed in females at high constitutive levels and is differentially B. taurus ENSBTAT00000010374 G. gallus ENSGALT00000005641 regulated in different hamster species during pregnancy (40). In X. tropicalis ENSXETT00000051833 arthropods, orthologs of the short pentraxins CRP and SAP and D. rerio ENSDART00000055071 a long pentraxin XL-PXN1 have been found in L. polyphemus.As T. rubripes NEWSINFRUT00000135347 the L. polyphemus genome sequence is still incomplete, the exis- C. porcellus U13234 Apexin D. NPR (NPTX2) H. sapiens NM_014293 tence of other pentraxins cannot be excluded. Among insects, in M. musculus NM_030689 melanogaster and Anopheles spp., we found multidomain proteins R. norvegicus NM_030841 containing a pentraxin domain, which are not related to the verte- X. tropicalis ENSXETT00000015097 brate long pentraxins (Y17570 in D. melanogaster, XM_558415 in D. rerio ENSDART00000078201 A. gambiae and AAEL011440 in A. aegypti). Finally, we did not D. rerio ENSDART00000059181 T. rubripes NEWSINFRUT00000151340 find putative orthologs of short or long pentraxins in C. elegans or in XL-PXN1 X. laevis L19881 the ancient chordate Ciona spp. We found multidomain proteins PTX3 H. sapiens X63613 containing a pentraxin-domain in C. elegans (W02C12.1 in Chro- M. musculus NM_008987 mosome IV, NCBI: AAB37995) and in C. intestinalis (ENSC- R. norvegicus ENSRNOT00000016541 M. domestica ENSMODG00000015690 ING00000010582). (Table continues) Pentraxins coding sequences or amino acid sequences were aligned using the ClustalW algorithm and then uploaded into 4 THE NEW PENTRAXIN PTX4 molecular evolutionary genetics analysis (Molecular Evolutionary The first cluster includes the short pentraxins, CRP and SAP, Genetics Analysis version 3.1) (41). We used different algorithms which originated diverging from the common ancestor of all for the construction of phylogenetic trees: the maximum- pentraxins and can be found in chordates (mammals) as well as in parsimony method and the NJ method. NJ trees were constructed arthropods (Limulus) and X. laevis XL-PXN1, which is a long on the basis of the following distances: the uncorrected proportion pentraxin (Fig. 1). Because Limulus pentraxins evolved earlier in of amino acid difference (p) and the Poisson-corrected proportion the pentraxin evolution, they appear on the branch before mam- of amino acid differences. The results obtained are shown in Fig. 1, malian short pentraxins, forming a separate clade. representing the tree for selected short and long pentraxins. Similar The second group includes all the NPTXs that cluster as results were obtained aligning coding sequences and amino acid a subclass of long pentraxins found in mammals as well as in lower sequences. vertebrates (Fig. 1). According to the length of branches, among The overall topology of the pentraxin family tree consists of five the NPTXs, NPR is the oldest that diverged from a common an- major distinct clusters containing nearly all the vertebrate pen- cestor of NPTXs; subsequently NP2 and finally NP1 appeared. traxins and, in a separate clade, the invertebrate pentraxins. 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 Downloaded from 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 http://www.jimmunol.org/ 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 by guest on September 24, 2021 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. Phylogenetic analysis of the C- and N-terminal domains of long pentraxins 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 se- FIGURE 1. Phylogenetic analysis of short and long pentraxins. Acces- quence of PTX3 shows only 10% identity with the human NP1 sion numbers of all available orthologous sequences of known short pen- N-terminal domain sequence; however, the amino acid identity in traxins (CRP, SAP, hamster FP, rat Mptx), long pentraxins (NP1, NP2, the amino-terminal domain among the NPTXs is higher and ranges NPR, apexin, PTX3, PTX4, Xenopus PXN1) and pentraxin domain-con- between 28% and 38%, suggesting the existence of subclasses of taining sequences (Drosophila and Anopheles pentraxins) used to generate molecules among the long pentraxins. The sequence similarity this NJ tree are reported in Table I. The five clusters identified are marked between NP1, but also NP2, and PTX3 at the N-terminal level is with circles. The name of species analyzed is reported as follows: anogam, restricted to the extreme N terminus; this characteristic and the A. gambiae (mosquito); capo, C. porcellus (guinea pig); crimi, C. migratorius (grey hamster); drome; D. melanogaster (fruit fly); gaga, G. longer size of NP1 and NP2 suggest the presence of a third domain gallus (chicken); hosa, H. sapiens (human); lipo, L. polyphemus (horseshoe localized between the N-terminal and the pentraxin domains (8). crab); meau, M. auratus (golden hamster); mumu, M. musculus (mouse); To better understand the evolution and biology of the N- and C- orycu, O. cuniculus (rabbit); rano: R. norvegicus (rat); xela: X. laevis terminal domains of long pentraxins, we performed a second (African clawed toad). analysis using separately the sequences of the two domains of each The Journal of Immunology 5

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; N- terminal domains represented by a dotted circle. Orthologous N-terminal sequences cluster together in Downloaded from 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. http://www.jimmunol.org/

pentraxin. The results shown in Fig. 2 indicate that for NPTXs, the search tool at NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.

C-terminals and the N-terminals of all orthologs form two sepa- cgi) did not reveal significant relationship or similarity among the by guest on September 24, 2021 rate groups. In contrast, the N-terminal domains of PTX3 of each members of the subfamily or with other known proteins. species cluster with the entire molecule because of the low levels Identification of a new pentraxin: PTX4 of sequence identity among the orthologous N-terminals, whereas the C-terminal domains cluster with the orthologs of other species. In an attempt to find new pentraxin domain-containing proteins, and The same clusterization occurs for PTX4 N- and C-terminal do- in particular the murine and human counterpart of guinea pig apexin mains. As expected, short pentraxins cluster together. These re- (accession number U13236, http://ncbi.nlm.nih.gov), we used the sults further support the hypothesis that in long pentraxins, the N- apexin amino acid sequence to search for undefined pentraxins in terminal domain evolved independently of the pentraxin domain. databases. This analysis led to the identification of the murine clone In particular, N-terminal domains of each NPTX are evolutively IMAGE: 1294272 39 in the Expressed Sequence Tags database, close compared to the N-terminal of PTX3 and PTX4, which deposited in the NCBI database under the accession number present sequence divergence among orthologs. AI563675 (http://ncbi.nlm.nih.gov) and defined as “similar to The search of conserveddomains in the N-terminal portion of long SW:APEX_CAVPO P47970 APEXIN PRECURSOR.” This partial pentraxins using reversed position-specific basic local alignment nucleotide sequence was used as bait for searches in murine, rat,

Table II. PTX4 orthologs in vertebrates

Chromosome/ Database Identification Amino Identitya versus Similaritya versus Identitya versus Similaritya versus Species Scaffold (Protein) Acids Human (1) Human (1) Human (2) Human (2) Homo sapiens 16 ENSP00000293922 (1) 473 100 100 88.5 88.9 XP_372607 (2) 478 88.5 88.9 100 100 Mus musculus 17 ENSMUSP00000055984 482 57.7 67.9 64 75 Sequenced from cDNAb 478 58.9 65.4 69 76.2 Rattus norvegicus 10 ENSRNOP00000022697 478 57.2 68.5 63.3 74.8 Monodelphis 6 ENSMODP00000020216 492 49.0 60.5 53.2 64.7 domestica Canis familiaris 6 ENSCAFP00000028929 478 61.1 69.4 68.8 77.5 Bos taurus 25 XP_602381 468 58.8 67.9 66.2 75.9 Danio rerio 3 ENSDARP00000055499 478 33.1 50.3 35.5 52.9 Xenopus tropicalis Scaffold_702 ENSXETP00000020030 482 35.5 51.0 38.5 55.3 Sequences listed under Database Identification (Protein) were retrieved from Ensembl (www.ensembl.org) and EMBL-European Bioinformatics Institute (www.ebi.ac.uk/). aIdentity and similarity were compared to the two human sequences reported: ENSP00000293922 (1) and XP_372607 (2). bMouse protein predicted according to sequenced cDNA from thymus. 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 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 FIGURE 4. Analysis of PTX4 gene. Comparison of the genomic orga- human, mouse, rat, opossum, frog (X. laevis), and chicken. For nization of the human, mouse, rat, and opossum PTX4 genes. Boxes rep- Downloaded from PTX3, in Xenopus (scaffold S-50), chicken (chromosome 9), and resent exons. Exon sizes are indicated within the boxes; intron sizes are opossum (chromosome 7), the gene order is SHOX2, VEPH1, given underneath the introns. The three nucleotide residues surrounding PTX3, CCNL1 (Fig. 3A). Further chromosomal rearrangements each splice site are shown; coding residues are represented by capitals. The appear in the mouse (chromosome 3 E1) and rat (chromosome actual splice donor and acceptor residues are indicated in boldface. The two 2q31) involving both order and orientation, which are conserved alternative human sequences (1, NM_001013658 and 2, XM_372607) are reported. Accession numbers for PTX4 genes are reported in Tables I and II.

in human (chromosome 3q25.32). For PTX4, in opossum, the gene http://www.jimmunol.org/ order is CLCN7, PTX4, TELO2, IFT140, TMEM204 (chromosome 6), and it is maintained in human (chromosome 16p13.3), codon and thus for the first exon; second and third exons are the whereas the entire chromosomal region changes orientation in rat same in the two sequences (Fig. 4). Identity and similarity between (chromosome 10q12) and mouse (chromosome 17A3.3) (Fig. 3B). PTX4 orthologs in vertebrates and the XM_372607/XP_372607 Analysis of the predicted PTX4 (www.ebi.ac.uk/) sequence is higher compared to NM_001013658/ ENSP00000293922 (www.ensembl.org) (Table II). The comparison of the genomic organization of human, murine, rat, Conservation in evolution among mammalian PTX4s and the and opossum PTX4 revealed a well-conserved gene consisting of presence of a putative signal peptide only in the sequence three exons. The exons are almost of identical length and all introns XM_372607 (data not shown) suggest that the correct human PTX4 by guest on September 24, 2021 9 9 contain well-recognizable 5 donor (gt) and 3 splice acceptor (ag) ortholog is XM_372607. Moreover, we failed to amplify the total sites (Fig. 4). Identity and similarity between human and other cDNA or the first exon using primers designed on the species sequences are reported in Table II. NM_001013658 sequence, whereas we amplified a full-length For human PTX4, two alternative in silico transcripts PTX4 cDNA from human small intestine total cDNA using primers (NM_001013658 [www.ensembl.org] and XM_372607 [EMBL- designed on the XM_372607 sequence. European Bioinformatics Institute; www.ebi.ac.uk/) (Tables I, II), The complete nucleotide sequence of murine ptx4 (EN- which differ in the first exon, are proposed in several databases. In SMUSG00000044172, www.ensembl.org) consists of a 74 bp 59 particular, two alternative possibilities were described for the start 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 FIGURE 3. Comparison of the syntenic blocks around PTX3 (A) and in ENSP00000293922. PTX4 (B) in vertebrates: order, orientation, and chromosome location are The NetNGlyc 1.0 computer analysis of the murine amino acid reported. sequence showed the presence of three potential N-linked The Journal of Immunology 7

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 myo- sin-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 Downloaded from 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 http://www.jimmunol.org/ deposited XM_372607 sequence. 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 by guest on September 24, 2021 cells and leukocytes, in particular monocytes, resting and PHA- activated 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. Pre- dicted nucleotide and protein sequence of mouse ptx4 (EN- SMUSG00000044172; 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 confir- matory 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 position- specific basic local alignment search tool at NCBI (www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi) with the murine full-length ptx4 FIGURE 6. Expression of human PTX4 mRNA by real-time PCR in protein sequence as query indicates the presence of the pentraxin normal tissues. 8 THE NEW PENTRAXIN PTX4

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 Downloaded from through a cell strainer. Error bars indicate the standard deviation of three replicate samples. Results are rep- resentative of one out of three independent experi- ments. Asterisks indicate a significant difference. pp , 0.05; ppp , 0.01, one-way ANOVA analysis. http://www.jimmunol.org/ by guest on September 24, 2021 very low, and it was not induced by IL-1 or LPS (endothelium, were analyzed by SDS-PAGE, and a protein with an apparent m.w. monocytes, and neutrophils) or PHA (lymphocytes). of ∼75 kDa was observed (Fig. 8). Given that the mass contri- Murine ptx4 expression was analyzed by real-time PCR in bution from the GST tag, present in both chimeric proteins, is 26 several tissues in normal conditions and posttreatment with LPS kDa, the observed immunoreactive bands at ∼75 kDa are likely to and in leukocytes. As shown in Fig. 7A, ptx4 was expressed at low correspond to the PTX4-GST fusion protein. Moreover, little or no levels in all tissues analyzed and was not induced by LPS. On the signal was detected without isopropyl-b-D-thiogalactopyranoside contrary, ptx4 expression was downmodulated in the liver (p , induction (Fig. 8). Murine ptx4-GST was purified by GSTrap-FF 0.01), lung, heart, and spleen. The only exception is the thymus, affinity chromatography followed by in gel tryptic digestion, 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). Expression of human and murine PTX4 protein FIGURE 8. Expression of recombinant human and murine PTX4. Hu- man and murine PTX4 cDNA were amplified from small intestine and To produce the putative proteins, murine and human PTX4 cDNA thymus, respectively, and cloned into pGEX-4T1 vector and expressed in were amplified from total cDNA of mouse thymus and human E. coli. The bacterial lysates were analyzed by Western blotting using the small intestine and cloned into the pGEX-4T1 vector and ex- polyclonal anti-GST Ab. The observed 75-kDa immunoreactive bands are pressed in E. coli. Human and murine PTX4 have a predicted m.w. likely to correspond to the PTX4-GST fusion proteins (52.3 kDa plus 26 of 52.339 Da and 53.084 Da, respectively. The bacterial lysates kDa for human PTX4 and 53.1 kDa plus 26 kDa for murine ptx4). The Journal of Immunology 9 peptide mass fingerprinting, and MS/MS analysis. MALDI-MS of Analysis of the new entry PTX4, in silico and by transcript purified murine ptx4 resulted in the detection of 18 peptides for expression, shows that the gene is well conserved among mammals. which the molecular masses are reported in the Supplemental For human PTX4, two alternative cDNA sequences that differ in the Table I. Three of these peptides were successfully sequenced by first exon have been published in databases. We failed to amplify MALDI-MS/MS. The 18 peptides identified in our analysis (178 the PTX4 cDNA using primers designed on the NM_001013658 aa residues) represent 39% of the entire primary structure of the sequence, whereas we amplified the PTX4 cDNA from small in- protein, and the combined MASCOT protein score of MS and MS/ testine using primers designed on the XM_372607 sequence. The MS analysis was 98 [protein scores .51 are significant (p , sequence of the amplified cDNA suggests that, at least in this tissue, 0.05)], making certain the identification of murine ptx4 (EN- the transcribed PTX4 corresponds to XM_372607. Moreover, SMUSP00000055984). identity and similarity between PTX4 orthologs in vertebrates and Using the polyclonal Abs directed against three murine ptx4 this latter sequence (XM_372607/XP_372607) is higher compared peptides, we performed immunohistochemistry and found that ptx4 to NM_001013658/ENSP00000293922. Finally, a predicted signal is indeed present in liver as expected (not shown). peptide for human PTX4 is present in XP_372607 but not in ENSP00000293922. Collectively, these data suggest that the hu- Discussion man PTX4 corresponds to XM_372607/XP_372607. Whether an Pentraxins are a superfamily of multifunctional conserved proteins, alternatively spliced form corresponding to NM_001013658 exists some of which are components of the humoral arm of innate in particular conditions has to be determined. immunity and behave as functional ancestors of Abs (2). The Finally, PTX4 has a unique pattern of mRNA expression. In present study was designed as a search for pentraxin domain- particular, the results suggest that expression of PTX4 is distinct Downloaded from containing sequences in different databases to understand the re- from that of other members of the family. For instance, unlike lationship among the known short and long pentraxins and their NPTXs, PTX4 expression is low in the brain. Unlike CRP and SAP, evolution. The results discussed in this paper indicate that based in spite of expression in the liver, it does not behave as an acute on phylogenetic analysis, the pentraxin superfamily consists of phase gene (1). The high expression in the stroma of thymus and five distinct clusters: short pentraxins, which can be found in spleen is unique among pentraxins. Thus, PTX4 is a new unique

chordate and arthropods and originated diverging from the com- member of the pentraxin superfamily, conserved in evolution. http://www.jimmunol.org/ mon ancestor of all pentraxins; NPTXs, a subgroup of long pen- Further studies are needed to define its function. traxins, with NPR being the first to diverge from a common ancestor; the prototypic long pentraxin PTX3, which originated Acknowledgments very early at the divergence of vertebrates; Drosophila B6, a long We thank Alfredo Cagnotto, Istituto Mario Negri, for the generation of pentraxin localized near PTX3 and PTX4 in the phylogenetic tree; peptides. 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 Disclosures common ancestor of all pentraxins. Both can be found in chordates The authors have no financial conflicts of interest. by guest on September 24, 2021 as well as in arthropods, suggesting that the duplication event that gave rise to these highly homologous proteins possibly occurred References very early in the evolution. However, this phylogenetic analysis 1. Pepys, M. B., and G. M. Hirschfield. 2003. C-reactive protein: a critical update. supports previous studies that proposed that the duplication of CRP J. Clin. Invest. 111: 1805–1812. 2. Garlanda, C., B. Bottazzi, A. Bastone, and A. Mantovani. 2005. Pentraxins at the or SAP, followed by sequence divergence and evolution of function, crossroads between innate immunity, inflammation, matrix deposition, and fe- occurred independently along the chordates and arthropods rather male fertility. Annu. Rev. Immunol. 23: 337–366. than in a common ancestor (42). In fact, the arthropod (Limulus) 3. Liu, T. Y., F. A. Robey, and C. M. Wang. 1982. 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