Download Entry PTX4 Evolution of the Pentraxin Family

Evolution of the Pentraxin Family: The New
Entry PTX4
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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
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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.
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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
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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
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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
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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.
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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
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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
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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.
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Pentraxins 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 (2). The
present study was designed as a search for pentraxin domaincontaining sequences in different databases to understand the relationship among the known short and long pentraxins and their
evolution. 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;
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The short pentraxins CRP and SAP originated diverging from the
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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
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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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sequences. Moreover, the analysis performed on the two domains
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suggests that local genome rearrangement occurred in these long
pentraxin loci during mammalian evolution, but the conservation of
flanking genes indicates some maintenance of synteny. Moreover,
the maintenance of synteny in PTX4 adds confidence to prediction
of orthology among these species.
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THE NEW PENTRAXIN PTX4