1. No category

From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1996 87: 1862-1872
Cloning of mouse ptx3, a new member of the pentraxin gene family
expressed at extrahepatic sites
M Introna, VV Alles, M Castellano, G Picardi, L De Gioia, B Bottazzai, G Peri, F Breviario, M
Salmona, L De Gregorio, TA Dragani, N Srinivasan, TL Blundell, TA Hamilton and A Mantovani
Updated information and services can be found at:
http://www.bloodjournal.org/content/87/5/1862.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
Cloning of Mouse ptx3, a New Member of the Pentraxin Gene Family
Expressed at Extrahepatic Sites
By Martino Introna, Victor Vidal Alles, Marina Castellano, Giuseppina Picardi, Luca De Gioia, Barbara Bottazzi,
Giuseppe Peri, Ferruccio Breviario, Mario Salmona, Laura De Gregorio, Tommaso A. Dragani,
Narayanaswamy Srinivasan, Tom L. Blundell, Thomas A. Hamilton, and Alberto Mantovani
Pentraxins, which include C reactive protein (CRP) and serum amyloid P component (SAP), are prototypic acute phase
reactants that serve as indicators of inflammatory reactions.
Here we report genomic and cDNA cloning of mouse ptx3
(mptx3). a member of the pentraxin gene family and characterize its extrahepatic expression in vitro and in vivo. mptx3
is organized into three exons on chromosome 3: the first (43
aa) and second exon (175 aa) code for the signal peptide
and for a protein portion with no high similarity to known
sequences, the third (203 aa) for a domain related to classical
pentraxins, which contains the “pentraxin family signature.”
Analysis of the N terminal portion predicts a predominantly
LY helical structure, while the pentraxin domain of ptx3 is
accommodated comfortably in the tertiary structure fold of
SAP. Normal and transformed fibroblasts, undifferentiated
and differentiated myoblasts, normal endothelial cells, and
mononuclear phagocytes express mptx3 mRNA and release
the protein in vitro on exposure to interleukin-lp (IL-1p)and
tumor necrosis factor (TNF)a. mptx3 was induced by bacterial lipopolysaccharide in vivo in a variety of organs and,
most strongly, in the vascular endothelium of skeletal muscle and heart. Thus, mptx3 shows a distinct pattern of in
vivo expression indicative of a significant role in cardiovascular and inflammatory pathology.
0 1996 by The American Society of Hematology.
S
for ptx3), its genomic organization (three exons rather than
two as for CRP and SAP), chromosomal localization (3
rather than l), and, more importantly, in vitro expression.
hptx3 is induced by lipopolysaccharide (LPS), IL-I, and
TNFa, but not IL-6, in a variety of cell types, including
HUVEC, fibroblasts, hepatocytes, and monocytes, and, unlike SAP and CRP, is not restricted to hepatocytes.‘.’ Here
we report the genomic and cDNA cloning of mouse ptx3
(mptx3) and characterize its expression and production.
mptx3 is very similar to its human homologue (hptx3). During an acute phase response induced by LPS, mptx3 is expressed in a variety of organs, most prominently in the heart
and skeletal muscle, unlike SAP, which is produced only in
the liver. Thus, ptx3 shows a distinct pattern of in vivo
expression indicative of significance in cardiovascular and
inflammatory pathology.
INCE THE ORIGINAL introduction of the term “acute
phase” by Avery et a1 (for an historical overview, see
ref I), it has become clear that many physiological and biochemical alterations that occur during an inflammatory response are the result of the interactions between cytokines
and different responsive cell types.*,’ For example, the elevation of the serum levels of several acute phase reactants,
including the pentraxins C reactive protein (CRP) and serum
amyloid P component (SAP),4 is the result of the exposure
of liver cells to cytokines, mainly interleukin-6 (IL-6).5
To characterize the molecular response of human umbilical vein endothelial cells (HUVEC) to IL-1, we isolated
several genes by differential screening of a cDNA library.6
Among these, we identified a new member of the pentraxin
gene family and called it p t ~ 3 The
. ~ same gene was also
cloned in tumor necrosis factor (TNF)-induced fibroblasts
and called TSG 14.’
Human ptx3 (hptx3) differs from classical pentraxins in
its size (approximately 23 kD for SAP and CRP and 43 kD
From the Istituto di Ricerche Farmacologiche “Mario Negri, ”
Milano; the Istituto di Patologia Generale, III Cattedra, Universita
di Catania, Catania; the Divisione di Oncologia Sperimentale A,
Istituto Nazionale Tumori, Milano; Section of General Pathology
and Immunology, University of Brescia, Brescia, Italy; ICRF Unit
of Structural Molecular Biology, Department of Crystallography,
Birkbeck College, University of London, London, UK; and Research
Institute, NNI, The Cleveland Clinic Foundation, Cleveland, OH.
Submitted June 20, 1995; accepted October 13, 199.5.
Supported in part by the Associazione Italiana Ricerca sul Cancro
(AIRC)by Telethon (Project No. 371) by the Istituto Superiore Sanita
(progetto AIDS).
Address reprint requests to Martino Introna, MD, Head, Laboratory of Molecular Immunohematology, Department of Immunology,
Istituto di Ricerche Fannacologiche “Mario Negri, ” via Eritrea,
62 201.57 Milano, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-497I/96/8705-0020$3.00/0
1862
MATERIALS AND METHODS
Cells and stimuli. The following cell lines were used as described: the UV-2237M-Cl3 (hereafter referred to as C13)’”and the
mFS6-M4 (hereafter referred to as M4)” fibrosarcoma cell lines and
the fibroblastic NIH-3T3 cell line from ATCC, Rockville, MD; the
myoblastic C2C12 cell line,’* kindly provided by Dr M. Boucht
(Istituto di Istologia, Universita “La Sapienza,” Rome, Italy); the
polyoma middle T (PmT) transformed endothelioma cell lines
bEND, tEND, SEND,” H5V, B9V, ElOV,I4 PY4.1,” FB-B, FB-H,“
the normal endothelial cell lines MAE, MHE, MBE.” and the LMec and B-Mec endothelial cell lines (a kind gift of Dr G. Grau,
Geneva, Switzerland). Thioglycollate (TG) elicited peritoneal macrophages were prepared as previously described.” The COS clones
containing sense and antisense human ptx3 cDNA in the pSGS expression vector were grown and characterized as previously described.’ The fibrosarcoma cell line 838719 was cultured in minimal
essential medium (Seromed, Berlin, Germany) and 10% fetal calf
serum (FCS). All culture reagents contained less than 0.125 ng/mL
LPS, as determined with the Limulus Amoebocyte Lysate assay
(Microbiological Associates, Walkersville, MD). Cells were stimulated with IL-I p (3 to 20 ng/mL) (courtesy of Dr A. Tagliabue,
Dompi?, L’ Aquila, Italy), 500 U/mL TNFa (BASF/Knoll, Ludwighafen, Germany), or 20 ng/mL LPS ( E coli 0S5:B5, Difco, Detroit,
MI).
Genomic cloning. The genomic library was a kind gift of Dr E.
Garattini (Istituto di Ricerche Farmacologiche “Mario Negri,” MiBlood, Vol 87, No 5 (March I ) , 1996: pp 1862-1872
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
CLONING OF LONG PENTRAXIN PTX3 IN MOUSE
Ian, Italy). It consisted of mouse 129/Sv spleen DNA partially digested with Sau 3a and cloned in the Xho I site, after modification,
of the A FIX I1 vector (Stratagene, La Jolla, CA). A total of 4 X
lo5 plaques were screened with the 3zP-labelledhuman ptx3E fulllength cDNA as probe, obtained as described6 according to standard
procedures.
Twenty-one plaques showing a strong hybridization signal were
picked and rescreened four times to obtain single clones and later
on were found to contain one single genomic clone of approximately
24 kb insert length.
Mapping of mptx3 in the genome. Details on the interspecific
testcross population of 106 male (C3WHe X M.spretus X C57BL/
65, (CfS)B6 mice have already been reported.” A total of 225 genetic markers were mapped in the (C3S)B6 population. The loci were
identified using restriction fragment length polymorphisms (RFLPs),
multilocus and minisatellite probes that detect multiple bands, and by
polymerase chain reaction (PCR) based analysis of simple sequence
length polymorphism (SSLP) loci;’ as described?’
The marker loci D3Mit3 and D3Mit9 were identified by PCR
amplification of genomic DNA from (C3S)B6 population using the
appropriate oligonucleotide primers.” HmgI4-rsS was identified by
hybridization of Southern blot of TaqI digested DNAs from (C3S)B6
population with the probe pM14C.”
PCR was used to search length polymorphisms in the Ptx3 gene
among the three parental strains. A total of 75 ng of genomic DNA
from each parental strain was suspended in PCR mix containing: 1
x PCR buffer, 1.5 mmoVL Mg++, 200 pmollL of each d N ” , 0.5
U of Taq Polymerase (Roche Molecular System, Inc, Branchburg,
NJ), 0.5 pCi (a3*P)dCTP(3,000 Cdmmol) and 2 pmol of specific
primers (5’-TTGTGAAAATCTACTTGAAGCC-3‘ from position
1249 to 1270 and 5’-AAATI’ACACAACTACCTTCTCC-3‘ from
position 1749 to 1770; see Fig 1B) derived from the mptx3 cDNA
sequence to amplify a fragment of 470 bp in the 3’ UTR region of the
Ptx3 gene. The final volume was 12.5 pL. Samples were subjected to
25 cycles consisting of denaturation at 93°C for 25 minutes, primer
annealing at 58°C for 30 minutes, and chain elongation at 72°C for
30 minutes.
A genetic linkage map was constructed by multipoint analysis of
the data using the MAPMAKEREXP pr~gram.’~.’~
Genetic distances were computed using Haldane’s mapping function. Linkage
between markers was considered significant if the LOD (Logarythm
of the Odds) score was more than 3.
cDNA cloning. Total RNA was isolated from C13 cells stimulated for 4 hours with 20 ng/mL IL-lp and 500 U/mL TNFa. Poly
(A+) RNA was further purified by affinity chromatography on oligo
(dT) cellulose. A cDNA library was constructed in the A-ZAP I1
vector (Stratagene, La Jolla, CA) as described.6 A total of 60,000
plaques were transferred onto nitrocellulose membranes in duplicate
by standard procedures and screened as described! Fourteen plaques
were found to hybridize with the 3’ specific probe, five plaques were
found to hybridize with the 5’ specific probe, and only two plaques
were found positive for both probes and were subsequently screened
three times to obtain single clones.
Southern analysis. A total of 10 pg of the monophage recombinant DNA were digested with several restriction enzymes and blotted
onto nitrocellulose membranes. To obtain a probe containing the
first and second exons of human ptx3, the plasmid ptx3E6 was
digested with Sac11 and the 500-bp fragment isolated. To obtain a
probe containing the third exon of human PTX3, we have used the
partial clone ptx3/A described previously! Hybridization with 3zPlabelled probes was performed overnight at 42°C in 50% deionized
formamide, 4 XSSC, 1 X Denhardt’s solution, SO mmoUL phosphate
buffer pH 6.5, 0.1% sodium dodecyl sulfate (SDS), and 100 pg/mL
salmon sperm DNA. Two SacVSacI fragments (no. 2 and no. 5)
were identified as specific for the first and the second exon following
1863
further restriction and subcloning in the pBluescript vector. One
EcoRI fragment was identified as specific for the third exon and
subsequently restricted and subcloned in pBluescript.
RNA extraction and Northern analysis. RNA was extracted and
purified using the guanidine/isothiocyanate (Merck, Darmstadt, Germany) method as described previ0usly.2~Probes labelling and hybridization conditions were as previously de~cribed.’~
PCR ampl$cation. The following oligomers were purchased
from Integrated DNA Technologies, Inc, Coralville, IA: the 5’ forward CTCCAGCAATGCACCTCCC (19mer) and the 3’ reverse
‘ITCTCCAGCATGATGAACAGC (2lmer) corresponding to position 92 to position 110 and from nucleotide 273 to nucleotide 293,
respectively, of mptx3 cDNA sequence. The forward is a sequence
coded by the first exon and the reverse by the second exon.
A total of 1 pg of total RNA was reverse transcribed with random
primers and subsequently amplified for 30 cycles according to standard procedures. The amplified fragment of 200 nucleotides was
resolved in agarose gels in the presence of ethidium bromide.
In vivo expression. C57BY6 mice were injected intravenously
(IV) with 30 pg LPS in 0.2 mL saline. At different times, three
animals per experimental group were killed, their organs pooled in
50 mL conical tubes and lysed in 5 mL of guanidine-isothiocyanate
solution, and mechanically disrupted with a bench top homogenizer.
Total RNA isolation, electrophoresis conditions, and hybridization
were as described above. The @actin probe has been previously
and the rat SAP cDNA probe (prSAP1) was a kind gift
by Dr S. Bruce Dawton, (School of Medicine, Washington University, St Louis, MO).
In situ hybrrdization. In situ hybridization was done using the
method described previously?’ Radiolabeled ’H-cDNA probes were
prepared by in vitro transcription from T3 and T7 RNA polymerase
promoters in pBluescript containing the 1280-bp HindIIVHindIII genomic subclone containing most of the third exon sequence as indicated in Fig 1A.The antisense probe for ptx3 was prepared by linearizing this plasmid with Xba I. For preparation of the sense strand probe,
the plasmid was digested with Bam HI. In vitro transcription and
labeling of RNA probes was performed as described.” Tissue sections
to be hybridized were processed essentially as
Western analysis. The rat anti-hptxf GP-1 (IgG 2a), cross-reactive with mptx3 was used for Western analysis, essentially as described.’
Sequence analysis and structure prediction. The sequences were
screened against the EMBL Data bank” using the FASTA p r ~ g r a m , ~ ’
as implemented on the EMBnet computer resource.32Screening of
the SWISS-Prot protein sequence data bank33and detailed sequence
analysis were performed with the PC/Gene sequence analysis package (IntelliGenetics, Mountain View, CA).
The secondary structure was predicted for the region preceding
the pentraxin domain in mptx3 using a combination of different
approaches encoded in SPETOR (Zhu and Blundell, submitted),
SAPIENS,34PHD,35the LEEDS sequence analysis pa~kage,’~
and
aligned sequences of mptx3 and hptx3. SPETOR incorporates
knowledge about tertiary structural constraints on the secondary
structure f~rmation,~’
while SAPIENS uses knowledge about structural environment-dependent amino-acid substitutions in homologous proteins.38PHD uses the neural network technique. The LEEDS
package incorporates nine different schemes such as Chou-Fasman
and GOR methods. A consensus prediction is obtained using the
criterion that, in a given residue position, at least three of the four
prediction schemes should result in the same prediction. A threedimensional model for the penraxin domain in mptx3 was generated
from the high resolution (2 A) crystal structure of human SAP”
using the rule-based procedure, COMPOSER““.41incorporated in
SYBYL 6.1 (Tripos Inc, St Louis, MO). In this procedure, the aminoacid sequence of the pentraxin domain in mptx3 was aligned with
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1864
INTRONA ET AL
A
B
16
11
151
42
226
61
301
92
316
Ill
451
142
526
167
601
192
676
217
751
242
826
261
901
292
916
317
1051
342
1126
367
1201
381
1216
1351
1426
1501
1576
1651
1126
1801
that of SAP. The conserved regions, which include almost all the
@-strands and an a-helical segment, were modelled on the SAP
structure and the structurally variable regions were modelled using
a database search for fragments in known three-dimensional struct u r e ~The
. ~ ~spatial proximity of two cysteine residues (not conserved
with SAP) in the loop regions was exploited to select an appropriate
fragment that permits formation of an unstrained disulphide. Side
chain conformations were predicted using a set of 1,200 rules based
on the conformers in equivalent residues in SAP or from a rotamer
library, depending on the nature of substitutions and the type of
secondary s t r ~ c t u r e .Energy
~’
minimization of the model was used to
relieve short contacts and rectify inconsistencies in stereochemistry.
RESULTS
Genomic cloning, chromosomal localization, and cDNA
cloning qf mptx3. To clone the genome of mptx3, we
Fig 1. Genomic organization and
cDNA sequence of mptx3. (A) Genomic
clone of mptx3: one A clone was isolated and partially characterized. Thick
lines indicate the subcloned regions of
the gene; no. 2 and no. 5 indicate the
t w o Sad subclones. The dashed lines
indicate the portions of the clones that
were subcloned and partially sequenced. The open bars indicate the introns or the flanking ragions. The
arrows indicate the lengths and positions of the sequences performed. S,
Sad; B, Bam HI; E, EcoRI; H, Hindll; P,
fsd. The genomic clones and the subclones are schematically depicted on
different scales. (B) Complete nucleotide and amino acid sequence of mptx3
cDNA. Nucleotides are numbered from
1 t o 1841 on the left side. The longest
open reading frame and its translated
protein sequence are shown. The amino
acids are numberedfrom 1t o 381 on the
right side. The potential signal peptide
starting with the first methionine is underscored with a thin line. The double
line underscores the eight amino acids
that constitute the ”pentraxin consensus signature.” The polyadenylation
signal is underscored three times. The
triangles indicate the two cysteine residues that are conserved in all members
of the pentraxin family. The positions of
the three exons (ex 1-31 relative t o the
cDNA sequence are indicated with
arrows. These sequence data are now
available under accession no. X83601.
screened approximately 400,000 plaques of a spleen-derived
Sau3a-digested genomic library with the human full-length
cDNA probe.6 Twenty-one plaques were found positive,
which was representative of one recombinant phage, containing an insert of approximately 24 kb. On Sac I digestion,
which cuts at both ends of the insert, five bands were detectable in gel electrophoresis. These were blotted and hybridized with a hptx3 cDNA fragment, corresponding to the first
and most of the second exon.6 Only two bands were found
positive (no. 2 and no. 5 of 6.0 kb and 1.O kb, respectively),
subcloned and partially sequenced as shown in Fig 1A. The
sequence of the Bum-Sac subclone of band no. 2 (Fig 1A)
could be aligned perfectly with the nucleotide sequence of
the hptx3 first exon.h Similarly, the sequence of band no. 5
was shown, by alignment, to include the remaining portion
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
CLONING OF LONGPENTRAXINPTX3
IN
MOUSE
of the first exon, the first intron, the second exon, and the
5' portion of the second intron (Fig IA).
In addition, EcoRI digestion of the phage showed one 4.5
kb fragment whichhybridizedwiththe3'portion
of the
human cDNA.h representative of the 3' half of the third exon
(Fig IA). Two overlapping portions of this fragment (EcoRIPstI and Hind 111-Hind 111) weresubclonedandpartially
sequenced as shown in Fig IA, and found, on alignment
with the human nucleotide sequence, to contain the 3' end
of the second intron and the entire third exon including the
putative polyadenylation signal (data not shown).
Thus, the characterization of the murine genome of ptx3
indicates that the mouse gene is encoded by three exons in
a similar way to that of hptx3. It also shows that the exon/
intron boundaries are exactly at the same positions in the
two genes6 (see below).
To localize the genomic locus for mptx3, we firstsearched
for a lengthpolymorphism in the 3' untranslatedregion
(UTR) of the gene by PCR-based analysis between the M
sprerus and the C3H/He and C57BU6J mice. Oligonucleotides were designed in the 3' UTR (see Materials and Methods and Fig 1B). Such polymorphism was found in that the
M sprerus allele was slightly longer than the expected 470bp seen in the other two strains. The M sprerus and the
C3WHe derived alleles showed the expected 1:1 segregation
pattern. We analyzed the segregation of the M spretus poly-
Fig 2. Chromosomal localization of Ptx3. (A) Autoradiography of a representative example of the
segregation of the
Pbr3 3' UTR polymorphism in the
(C3S)B6 male population. When the individualgenotype is C3B6, both alleles have the same length and
comigrate throughout the as
gela single band. When
the individual genotype isSB6, two distinct bands
can be seen on the autoradiography, each corresponding t o its parental allele. (B) Haplotype data of
completely typed (C3SIB6 mice for the loci flanking
Ptx3 locus. Each column represents the type of chromosome identified in the progeny. The number of
progenyexhibiting each typeofchromosome
is
listed at the bottom.
The open squares represent the
M. spretus allele; the black squares stand for the
C3HIHe allele (top). The genetic linkagemapof
mouse chromosome3 showing the locations of the
Ptx3 locus. The recombination frequencies are expressed as genetic distances in centimorgans (Haldane's function) andare shown t o the left (bottom).
1865
morphic fragment in the (C3S)B6 population (a representative example of the segregation is shown in Fig 2A), and
we assigned the ftx3 locus to murine chromosome 3. Figure
2B shows the linkage map obtained for chromosome 3 with
distance and recombination fractions between couples of adjacent markers expressed as genetic distances in centimorgans. Prx3 locus maps 11.3 CM proximal to D3Mir9 in
a region that is similar to human chromosome 3q (q24-28)"
In agreement with our results, thehuman genomic locus
HPTX3 hadbeenpreviouslylocalizedonhumanchromosome 3q25.h
To obtain the complete cDNA for mptx3, we constructed
a cDNA library from polyA(+) RNA purified from theC13
line stimulated for 4 hours with IL-l and TNF. Only two of
200.000 plaques contained the entire cDNA. The two phages
were subcloned and found to contain an identical 1,841 nt
long cDNA after complete sequencing of the two strands,
as shown in Fig IB. The putative start codon is at position
1 0 0 preceded by 99 nt of S' untranslated sequence. The TAA
stop codon is at position 1243 followed by 598 nt of a 3'
untranslated region containing a canonical polyadenylation
signal, as underlined in Fig I .
Alignment with the human cDNA shows an overall 82%
identity that is maintained along the 1145 ntof the open
reading frame, as well as the S' and 3' untranslated regions
(data not shown). In addition, alignment of the cDNAs con-
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
INTRONA ET AL
1866
firms that the exodintron junctions are conserved between
mice and humans.
The predicted protein sequence of 381 amino acids is
shown in Fig 1B. Although the full-length cDNA starts with
an open reading frame, the most 5' methionine residue is
likely to be the one that is effectively used, as it fits the
Kozak consensus sequence45(Fig l e ) . Furthermore, this methionine is immediately followed by a typical signal peptide
sequence (underlined in Fig 1B) as predicted according to
the method of von Heijr~e.~'
As previously reported for hptx3, mptx3 also shows a
significant alignment between the COOH-terminal portion
of the protein from the cysteine at position 179 to the COOH
terminus and the other sixteen cloned members of the pentraxin gene family (data not shown). In particular, the "pentraxin consensus ~ignature"~'and the two half-cystines at
positions 210 and 271 (which maintain the same positions
in all cloned members of the pentraxin
are indicated in Fig 1B.
The alignment of the predicted protein sequence of mptx3
with hptx3 shows that 312 of 381 amino acids are identical
(82%), and 35 1 are substituted by similar residues (92%) in
the two proteins (data not shown). Sequence and structure
analyses of the N-terminal portion and of the C-terminal
pentraxin-like domain, coded by the third exon of the gene,
were then conducted.
The N-terminu L portion: Sequence ana Lysis and structural
predictions. A search in the data bank of protein sequences
for homologous or analogous sequences resulted in no significant hit for the full-length of 178 residues in the portion
preceding the pentraxin domain. However, three sequences
(apart from hptx3) analogous to a part of this portion were
identified: the TNF a inducible human b94 protein,'" secretin
from Sus Scrofa, Bos Taurus, and Cavia Porcellu?" and
human transforming protein HST/KS3 (HST, human stomach tumor; KS3, Kaposi's Sarcoma 3)'' An alignment of
the sequences of mptx3, hptx3, and potential analogues is
shown in Fig 3. Many aligned positions after position 70 are
similar. Sequence identity between mptx3 or hptx3 with
these analogues, varies from 22.9% (between hptx3 and b94)
and 33.3% (between mptx3 and hbg4).
Crystal or nuclear magnetic resonance (NMR) structures
of b94 protein and human transforming protein are not yet
known. Secretin belongs to the glucagon family and the
secondary structure, assigned by NMR,'*,'' is a-helical between positions 116 and 122 and between 128 and 137 (Fig
3). The intermittent linker region takes up a helical turn
conformation resulting in a overall longer helix-like structure. The NMR structures of similar proteins, glucagons4 and
growth hormone releasing factor,55 and the crystal structure
of glucagon5' have highly helical structures.
Comparison of secretin with either mptx3 or hptx3 does
not involve any insertion or deletion in these regions, and it
is likely that the equivalent region of mptx3 and hptx3 adopts
a helical conformation.
Secondary structure prediction (Fig 3) indicates a largely
a-helical conformation, including the signal peptide region
between 1 and 17. The region from 109 to 135 (Fig 3) is
also predicted as helical, supporting the prediction based on
a
(D
b94
hbg4
w*
hpt.3
mptx3
MRLLAILfCALWSAVLAENSDDYDLhYVNLDNElDNGLBPTEDPTPCACG
MRLPAILLCALWSAVVAETSDDYELMVNLDNEIDNGLBPTEDPTPCDCR
=a"OmOmaa""ae
c+
m
bQ4
hbg4
.Kr
hptx3
mptr3
mmamm*ameaaee
~ ~ ~ o a ~ o 1 a 0 1 0 1
a
11"
b84
hk4
wcr
hptr.3
mptr3
1w
PPTVEELKAALERGQLEAAR-PLLALERELA
AAVALLPAVLLALLAPWAG-RCGAA
LLLLLPPLLLLACCAA
QERSEWDKLFIMLENSQMRERMLLQATDDVLRGELQRLREELGRLAESLA
QEASEWDKLflMLENSQMREGMLLQATDDVLRGELQRLRAELGRLAGGMA
m,2eamamaaam=maama,,
IM
120
*IO
IS0
A A A A A G G V - - 5 E E E L V R R Q S K V E A L Y E L L R D -- 9 V L G V L R R P L E A P P E R L
APTAPNGT--LEAELERK---WESLVA------LSLARLPVAAQPKEAAV
R P A P P R A P R R S D G T P T S E - -- L S R L R D S A R L Q R L L Q C L V C K R S Q Q D P E N N
RPCAPGAP- -AEAKLTSA---LDELLQATRDAGRRLARHEGAEAQRPEEA
RPCAAGGP--ADARLVRA---LEPLLQESRDASLRLARLEDAEARRPEAT
~
(Imam011101
~
~
~
1m
11"
1110
~
~
~
lvcl
hptx3
GRALAAVLEELRQTRADLBAVQG-------WA*RSWLG
mptx.3
VPGLGAVLEELRRTRADLSAVQS--------WVARBWLPAG
hbg4
~
o
o
=
a
~
~
~
a
n
~ O Qom a a~e
o
~
~
~
~
19"
RQALAVVAEQEREDRQAAAAGPGT5GLAATR.--PRRWLQ
QSGAGDYLLGIKRLRRLYCNVGIGF----~----ELQALPDG
TAWKSGEDRLCQLWSEMPALQAWMPMKPPVDQAWSPWLPPG
h04
o
~
o
~
~
Fig 3. Secondary structure predictionof the region preceding pentraxin domain and alignment of analogous sequences. b94, hbg4,
secr. hptx3, and mptx3 refer to b94 protein, HST/KSB, secretin, human and mouse ptx3, respectively. The consensus secondary structure prediction derived from four diffarent prediction schemes is
shown at the bottom, a predicted a-helices.
the alignment with secretin. Segments that link contiguous
helices are likely to be short, except the one around 40.
wherein many polar residues and prolines are present.
The pentruxin-Like domain: Sequence analysis und structurd prediction. Figure 4 shows the alignment of the pentraxin domain of mptx3 and hptx3 with three classical pentraxins, human SAP, human CRP, and hamster SAP and Fig
5 shows a model based on the crystal structure of human
SAP.39Conservation of the pentraxin signature sequence occurs between 93 and 100 (SAP numbering). The calcium
binding sites, hydrophobic pocket residues, and the residues
involved in the interprotomer recognition in the pentamer
are also indicated.
Comparisons of sequences of mptx3 and hptx3 with SAP,
CRP, hamster SAP, and Limulus CRP" show that they are
almost equally related to ptx3 variants, with Limulus CRP
being the member nearest to ptx3 (Fig 4 and data not shown).
In mptx3 almost all the &strands and the a-helical segments
of SAP are conserved, but the helix in mptx3 is likely to be
slightly shorter because of the presence of two contiguous
glycyl residues near the C-terminus.
The only disulphide bond in SAP is formed between halfcystines at positions 36 and 95. These Cys residues are conserved in mptx3 (as in all known members of the family)
and result in a disulphide bond. The Cys residues at positions
5 and 177 of mptx3 (SAP numbering) are positioned close
enough to form a good disulphide bond.58 This disulphide
is conserved in hptx3. Figure 5 shows the fold of mptx3
with the two disulphides.
~
~
~
~
~
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1867
CLONING OF LONG PENTRAXIN PTX3 IN MOUSE
10
a0
10
40
b5PP
+PPbbb
PPbbPPbPb
P P
P
eo
*
*lo
*
A
110
+
+RPP+PPBP
33s
a----a.x--
PP
4
PP
P
CVGGGFDESLAPSGR1TGPNI~RVLSEEEI~ASGGVBSCEIRGNVVG~
c
A
Y)
110
,*I
- L I ~ ~ i a r i ~ ~ ~ . 9 a ~ ~ i ~ ~ ~ a . . ~ p p ~ ~ ~ i
4
P
CETAIIPPYBSXXIPGSVEPVRPYXLESPSTCIWVKATDVL--N
CETAILFPYBSKXIPGSVEPVRPYRLESFSACIWXATDVL- - N
QTDMSRXAPVPPKESDTSYVSLKAPLTKPLKAFTVCLI3FYT-ELSSTR
T D L T G X V P V P P R q S E T D Y V K L l P R L D K P L Q N P T V C F R A Y S - D L S .- R
10
LM
B 1.0
*D
A
bl~LlfkVP.Ppiii.lb~ViLi~pl~kpl~~P~L~F€AyR-dl‘--i
M
CVGGGlDETLAFSGRLTGPNl~SVLS~EBlRETGGAESCElBGNlVG~
-PGCNFEGSQSLVGDIGNVNUWDIVLSPOIIWTIYL-OcP
-YGGCFDNYQSFVGEICDLNMWDSVLTPEEIKSVYQ-GVP-LEPNILDW
m
U0
*
*.CD.PO.
a L~
n y~i i~i g~~ §
V iI
i k:p l~r i~r ~ I : ~ - ) . P .,SLPL~T~~~~~SLLV~~~~.~-~Y~LII-Q~
P P P P P P P B ~ R B + P P P P P P b B 4 PRBBBR 4+81988BR
C I
k L
h
P P
PBPBI
P P
KTILPSYGTXm(PYEI~LYLSS~SLVLVVGG-KENKLAADTVVSL~~GRW
KTILPSYGTXRNPYEIQLYLSYQSIV~VVGG-EENKLVAEAMVSL-.GRW
GYSIPSYATKR~DNEILIPWKDI-GY~PTV-GGSEILIEVFEVTV~APPESLPSYNTEYGENELLIYKE~IG-EYELYI-GNQGTKVRGVEEPA~SF-
tm
110 I
,lo
,a0
PPPP
P P P
PP
VTElQAEGG----AQYVS
VTEIQPBGG----AQYVS
ALKYEVQGIVPTKPQLW
ALNYEMNGYAVIRPRCVALSSYNKIS
*
rE I C V S E 5 II iQi A i F i I no- t p L r k k a 1 I 9 f i f V i 1P I: I V L C g k q i L PPBBPBPR
+PPPPBP4+ PP
88 4 4
4 PRRR4
P
P P
P PPPV
c ‘ C
SELCGTWSB~CSYSLWANGELVATTVElUgSHSVPECCLLqlOPEGGLL~lG~EKNGC
TELCGTWWSEEGLTSLWVNGELAATTVBYATGEIVPEGGILQIG~EKNGC
VBICTSW8SASCIVEPWVDG-KPRVRKSLKKGYTVGAEASIILGqEQDS-
UPPER CASE
Ian?
iWic
bnn
VEPCTSWESSSGIAEPWVNG-KPWVKKGLNKGYTVKNKPSIILGQEQDN-
tilde
bold
&
CedilL
Fig 4. Alignment of the pentraxin domain of ptx3. Alignment of the sequences of the pentraxin domains of mouse and human ptx3 Imptx3
and hptx31 with human SAP 12rap). human CRP (crp) and hamster SAP (hsap). The structural environments at the residue positions in SAP
have been derived from the crystal structure of S A P and are represented using the program JOY.” SAP numbering is shown at the top.
Calcium binding sites are indicated by c; hydrophobic pocket residues are indicated by h; interprotomer recognition involved residues are
indicated by p.
Two further Cys residues in mptx3 are adjacent at positions 139A and 139B (SAP numbering) and are situated in
an exposed loop (Fig 5). It is probable that they are involved
in disulphide formation. Formation of a disulphide between
two Cys residues adjacent in sequence is extremely rare, as
it requires a strained cis peptide bond and significant deviation from planarity of the
When it occurs, it is
usually associated with the function of the protein as in
acetylcholine receptof ’ and in methanol dehydrogenase.62
In ptx3, it is more likely that the Cys residues are involved
in interdomain or in intermolecular cross-links. For example,
there are four Cys residues at positions 9, 47, 49, and 103
in the helix-rich portion preceding the pentraxin domain (Fig
3). One of them (at 9) is situated in the signal peptide region.
The remaining three Cys residues are predicted to be exposed
either in a loop region or at the helix termini, and they
may form disulphides with the adjacent Cys residues of the
pentraxin domain. The most likely candidates are Cys47 and
Cys49 (Fig 3).
The calcium-binding sites in SAP are the backbone carbonyl at 136 and side chains at Asp58, Asn59, Glu136,
Asp138 and Gln148 (SAP numbering) (Fig 4). These side
chains are replaced in mptx3 by Pro58, Tyr59, Glu136,
Asn138, and Leu148 precluding calcium binding. It is not
yet known if ptx3 binds to metal ions. The hydrophobic
pocket residues in SAP, Leu62, Tyr64, and Tyr74 are replaced by Gln62, Tyr64, and Gly74 in mptx3; hence, the
mode of recognition of ligands by ptx3 would be different
from classic pentraxins.
Some of the residues at the interface of two protomers
in pentameric SAP (Fig 4) are conserved or conservatively
variant, such as Val 4 Phe at position 10 and Pro Pro at
position 12. But, many residues are completely different; for
example, Val Thr in 115, Lys + Thr in 116, Lys Val
in 117, and Gly Glu in 118. Insertion or deletion occurs
in some of the interprotomer regions such as around position
40 and position 196. These observations imply that ptx3 is
unlikely to form a pentamer.
In vitro expression of murine ptx3. Two fibrosarcoma
cell lines (C13 and M4) and the normal fibroblastic 3T3 cell
line responded to 6 hours exposure to IL-l/TNF with an
increase in mptx3 message, as shown in Fig 6A (and data
not shown). Neither undifferentiatedproliferating myoblastic
C2C12 progenitors nor the in vitro derived terminally differentiated myotubes showed message under baseline conditions, as for the cells studied at different times during the
differentiation, but a strong signal for mptx3 was induced on
exposure to IL-1, in both undifferentiated and differentiated
cells, which peaks after 6 hours exposure (Fig 6A and data
not shown).
No message could be seen in a panel of nine PmT transformed murine endothelioma cell lines either in the absence
or in the presence of activating cytokines (data not shown).
When five normal endothelial cell lines were studied, a clear
+
-+
+
+
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1868
INTRONA ET AL
Fig 5. Three-dimensional representation of the fold of the pentraxin domain in mouse ptx3. The secondary structural regions are
shown. The positions of the two predicted disulphides and other
Cys residues are marked. This figure has been generated using the
software SETOR.”
A
U
U
E
?
E
2
$
5I 2; iI ;d
7:;;
uu
+
7
L-Mec
E
2
$
f
+
7
U
9-Mec
E
E
D
+
m
Z
E
Medium
IL-1
‘id 2d 3d 4d‘V
7
2
Endothelial cells
B
b
5
I
LPS
‘lh 2h 4h 10h 20h‘
q-9
U
undlfferenhated differenrtated
c2c12
Fibroblasts
SAP
E
uu
normal transformed
3T3
M4
PTX3
U
iiii I
E ?
induction of ptx3 mRNA was observed on IL-I and TNF
exposure only in the lung-derived L-Mec line (Fig 6A). In
anology with what is observed in the human,’ mptx3 mRNA
is not present in unstimulated macrophages, but is rapidly
(2 hours) and transiently induced on LPS exposure (Fig 6A).
Thus, mptx3 is inducible by inflammatory signals such as
LPS, IL-I, and TNF in a broad variety of cell lineages with
similar rapid and transient kinetics.
To investigate the production of mptx3, we took advantage
of a rabbit antiserum and of a monoclonal antibody (MoAb)
(GPI) raised against the recombinant human protein, crossreactive with mptx3 (data not shown). By Western analysis,
we detected mptx3 in the supernatants of resting and stimulated (IL-I and TNF) M4 cells. A clear band of the expected
MW of 42 kD was visible already in the untreated cells, but
its intensity clearly augments ( I O times at the densitometry
scanning) on cytokine stimulation (data not shown).
Expression in vivo. C57BI/6 mice were injected IV with
30 k g LPS, killed 4 hours after the inoculum, and their
organs extracted. A strong message for mptx3 is evident in
the heart and in the limb muscle (Fig 6B). A much weaker
signal is also visible in the lung, ovary, and thymus. On
the contrary, spleen, skin, kidney, intestinal wall and, most
notably, liver are all negative even after prolonged exposure
Of the blots (Fig 6B and data not shown)’ As can be Seen in
Fig 6B, a strong signal for SAP, the prototypic acute phase
reactant in the mouse, is detectable only in the liver, as
expected.6’.ffl By reverse transcriptase (RT) PCR, ptx3 induction was detectable in the liver (data not shown).
c 2 c12
Muscle cells
Peritoneal
macrophages
Fig 6.
ptx3 gene expression
in vitro and in vivo. (A) The fibrosarcoma cell line M4,the normal 3T3 fibroblast line, and the
endothelial cell lines L-Mec and
6-Mec were exposed t o 10 n g l
mL IL-1p and 500 UlmLTNFafor
6 hours. The undifferentiated
proliferating myoblastic C2C12
cells were stimulated in the absence and in the presence of 3
nglmL IL-lp for 6 hours. Upon
serum change, the cells were left
t o differentiate for several days,
and the terminally differentiated
myotubes were exposed t o 3 n g l
mL IL-1p for 6 hours. TG-elicited
peritoneal macrophages were
incubated for the indicated
times with 20 nglmL LPS. (6)
C57BV6 were injected IV with 30
p g LPS and after 4 hours total
RNA was isolated from several
organs. The sign - indicates the
organs isolated from control, saline-treated mice; the sign indicates the organs isolated from
the LPS-treated animals. The
same blot was hybridized first
with mptx3 cDNA and subsequently with the rat SAP cDNA.
The figure shows the overnight
exposure of the blot.
+
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1869
1
Fig 7. In situ hybridization in heart and skeletal muscle. C57B1/6 mice were injectedwith 30 p g of LPS in 100 pL PBS 4 hours before killing.
Heart and skeletal muscle were obtained, rinsed briefly in phosphate-buffered saline (PBS) and fixed in buffered formalin. Sections were
prepared and used for in situ hybridization with a 'H-labeled antisense riboprobe for the third exon of mptxt as described in Materials and
Methods. Slides were exposed for 3 weeks. (AI skeletal muscle, bright field; (6)skeletal muscle, dark field; IC) heart, bright field; ID) heart,
dark field. Magnification x 400. Similar results were obtained in t w o separate experiments.
When male and female mice of similar age were compared, no significant difference could be found (data not
shown), thus ruling out possible gender effects as described
for hamster SAP.6s
The expression of mptx3 was also studied at later times
after LPS injection, but no significant signal could be found
(data not shown), thus confirming in vivo the rapid and
transient nature of ptx3 expression.
In situ hybridization. In sections hybridized with a
sense-oriented strand (negative probe controls) and in sections of tissue from animals injected with saline alone (treatment controls), only minimal grain frequency was observed,
and this was distributed evenly over the entire section (data
not shown). Tissues obtained from animals exposed to LPS
for 4 hours and hybridized with radiolabelled, antisenseoriented riboprobe encoding mptx3 exhibited a distinct
pattem. In both skeletal muscle and heart tissue, a highly
restricted subset of cells exhibited a strongly positive hybridization signal (Fig 7). The positive cells were distributed
evenly throughout the tissue. The muscle fibers themselves
were uniformly negative. The positive cell types generally
exhibited an elongated morphology, and in some cases, were
interconnected to one another in long arrays that appeared
to correspond to the microvasculature (Fig 7). These results
strongly suggest that the predominant cell type responsible
for ptx3 expression in response to LPS in vivo are vascular
endothelial cells lining the capillaries, arterioles, and/or venules within striated muscles. Several tissues, which were
negative for mptx3 expression when examined by northem
hybridization, were also examined using the in situ hybridization technology (data not shown). These included liver
and kidney. The liver tissue was essentially negative, while
occasional positive cells were observed in the kidney. Positive cells in the kidney appeared to be within the extensive
microvasculature of this organ. The frequency of positive
cells in these organs was very low as compared with that
seen in either the heart or skeletal muscle samples. Thus it
appears that LPS can induce mptx3 mRNA expression in
vivo from selected populations of microvascular endothelial
cells.
DISCUSSION
Here we describe the isolation of the genomic clone and
full-length cDNA of mptx3 and characterize its in vitro and
in vivo expression. mptx3 is organized in three exons: the
first codes for 43 aa including the signal peptide, the second
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1870
for a 135 aa domain with no high similarity to known sequences (except for hptx3), and the third for a 203 amino
acid domain with sequence similarity to pentraxins. The pentraxin-like domain maintains the two Cys residues at positions 179 and 271 and a canonical “pentraxin consensus
signature” (H-x-C-X-S/T-W-X-S).~~
Ptx3 is localized on mouse chromosome 3, in a region
that is homologous to the human chromosome 3q (q24-28),
in agreement with the documented localization of HPTX3
in region 3 q25.6
Overall, mptx3 is remarkably similar to hptx3 in terms of
gene organization, localization, and sequence.6 Specifically,
sequence identity is 82% between the human and mouse
gene, which reaches 92% when conservative substitutions
are considered. The high degree of sequence similarity between human and mouse ptx3 is reminiscent of the “classical” pentraxins that have been highly conserved in evoluti~n.~
No homologous or analogous sequences have been found
for the complete N-terminal portion, while three sequences
were identified that show some analogy to a portion of this
domain; they include TNF-inducible human b94 protein, secretin, and human transforming protein HSTKS3. The Nterminal portion is predicted to be predominantly a-helical.
It is of interest that secretin, a member of the glucagon
family whose secondary structure has been determined in
part by NMR, is also predominantly a - h e l i ~ a l . ~ * . ~ ~
The crystal structure of the human SAP pentame8’ defines
a tertiary fold of antiparallel p-strand jelly roll topology
reminiscent of legume lectins, animal S-type lectins, and
some bacterial enzymes.57The sequence of the pentraxin
domain of ptx3 is accommodated well on the tertiary fold
of SAP with almost all of the P-strands and the a-helical
segment conserved. An a-helix segment between 166 and
174 (SAP numbering, see Fig 4) buries the residues in a pstrand formed by the pentraxin signature sequence, H-X-CX-S/T-W-X-S, between 93 and 100 (SAP numbering); this
motif is conserved in PTX3.
Two calcium ions bound to SAP, on an exposed P sheet
face, bind directly to methyl 4,6-0-( 1 -carboxyethylidene)P-D-galactopyranoside (MOPDG) or phosphoethanolamine
(PE). The binding of MOPDG is facilitated by a hydrophobic
pocket formed by the residues Leu62, Tyr64, and Tyr74.
Our analysis has shown differences in the calcium binding
sites, hydrophobic pocket residues (therefore ligand binding
sites), and in residues involved in interprotomer recognition,
indicating a different binding role. Consistent with this prediction, we found that the ligand binding properties of ptx3
differ from those of CRP and SAP and include complement
components (Bottazzi et al, unpublished data). Preliminary
evidence also suggests that ptx3 forms covalently linked
multimers (Bottazzi et al, unpublished data).
A variety of mouse cell types, including normal and transformed fibroblasts, some endothelial cells, undifferentiated
and differentiated myocytes, and mononuclear phagocytes,
expressed mptx3 mRNA in response to the inflammatory
cytokines IL-1 and TNF. Transcript expression was associated with release in the supernatants of a mature protein of
approximately 42 kD.
INTRONA ET AL
Injection of LPS, used as a prototypic inflammatory signal
and inducer of the acute phase response, caused rapid expression of mptx3 in a variety of organs. Using a polyclonal
antiserum directed against recombinant TSG- 14 protein, Lee
et a19 recently found induction by LPS in serum of immunoreactive TSG-lWptx3. Unlike SAP, the main acute phase
response pentraxin in the mouse: confined to the liver,
mptx3 was most abundantly expressed in the heart and in
skeletal muscle. In situ hybridization analysis suggests that,
under these conditions, while muscular cells are negative,
endothelial cells are a major cell type expressing mptx3.
High levels of mptx3 expression in the heart were also detected in preliminary experiments in a model of myocardial
infarction (data not shown). Thus, mptx3 shows a pattern of
in vivo expression distinct from that of classical pentraxins,
with prominent involvement of vascular endothelium in organs such as the heart and skeletal muscle. The role and
significance of ptx3 in cardiovascular and inflammatory pathology deserve further study.
Recently other genes with a carboxy terminal pentraxinlike domain were cloned, including guinea pig ape~in,~‘.‘~
Xenopous XL-PXN1 and two neuronal pentraxins, one
from rat69and one from man.’” In all cases, the molecules
show N-terminal domains of length comparable to ptx3, but
very poor alignment among them~elves.~.~’
Ptx3, therefore,
represents the first isolated member of a family of “long
pentraxins” (when compared with the classical CRP and
SAP) in which a pentraxin module is coupled to distinct and
unrelated N-terminal domains. Unlike classical pentraxins,
long pentraxins can be expressed at extrahepatic sites.
ACKNOWLEDGMENT
We thank the members of pentraxin group, expecially Dr Steve
Wood and Prof Mark Pepys for discussions.
REFERENCES
1. Mc Carty M: Historical perspective on C-reactive-protein. Ann
N Y Acad Sci 389:1, 1982
2. Ciliberto G: Transcriptional regulation of acute phase response
genes with emphasis on the human C-reactive protein gene, in Pepys
M (ed): Acute Phase Proteins and the Acute Phase Response. Heidelberg, Germany, Springer Verlag, 1989, p 29
3. Steel DM, Whitehead AS: The major acute phase reactants:
C-reactive protein, serum amyloid P component and serum amyloid
A protein. Immunol Today 1531, 1994
4. Pepys MB, Baltz ML: Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxis) and serum
amyloid A protein. Adv Immunol 34:141, 1983
5. Baumann H, Gauldie J: The acute phase response. Immunol
Today 15~74,1994
6. Breviario F, d’Aniello EM, Golay J, Peri G, Bottazzi B, Bairoch A, Saccone S, Marzella R, Predazzi V, Rocchi M, Della Valle
G, Dejana E, Mantovani A, Introna M: Interleulun-I-inducible genes
in endothelial cells. Cloning of a new gene related to C-reactive
protein and serum amyloid P component. J Biol Chem 267:22190,
1992
7. Lee GW, Lee TH, Vilcek J: TSG-14, a tumor necrosis factorand IL-I-inducible protein, is a novel member of the pentaxin family
of acute phase proteins. J Immunol 150:1804, 1993
8. Vidal Alles V, Bottazzi B, Peri G, Golay J, Introna M, Mantovani A: Inducible expression of PTX3, a new member of the pen-
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
CLONING OF LONG PENTRAXIN PTX3 IN MOUSE
traxin family, in human mononuclear phagocytes. Blood 84:3483,
1994
9. Lee GW, Goodman AR, Lee TH, Vilcek J: Relationship of
TSG-14 protein to the pentraxin family of major acute phase proteins. J Immunol 153:3700, 1994
10. Giavazzi R, Miller L, Hart IR: Metastatic behavior of an
adriamycin-resistant murine tumor. Cancer Res 435081, 1983
1 1. Giavazzi R, Alessandri G, Spreafico F, Garattini S, Mantovani
A: Metastasizing capacity of tumor cells from spontaneous metastases of transplanted murine tumours. Br J Cancer 42:462, 1980
12. Blau HM, Chiu CP, Webster C: Cytoplasmic activation of
human nuclear genes in stable heterocaryons. Cell 32:1171, 1983
13. Williams RL, Risau W, Zerwes HG, Drexler H, Aguzzi A,
Wagner EF: Endothelioma cells expressing the polyoma middle T
oncogene induce hemangiomas by host cell recruitmeat. Cell
57:1053, 1989
14. Garlanda C, Parravicini C, Sironi M, De Rossi M, Wainstok
de Calmanovici R, Carozzi F, Bussolino F, Colotta F, Mantovani
A, Vecchi A: Progressive growth in immunodeficient mice and host
cell recruitment by mouse endothelial cells transformed by polyoma
middle-sized T antigen: Implications for the pathogenesis of opportunistic vascular tumors. Proc Natl Acad Sci U S A 91:7291, 1994
15. Dubois NA, Kolpack LC, Wang R, Azizkhan RG, Bautch
VL: Isolation and characterization of an established endothelial cell
line from transgenic mouse hemangiomas. Exp Cell Res 196:302,
1991
16. Bocchietto E, Guglielmetti A, Silvagno F, Taraboletti G,
Pescarmona GP, Mantovani A, Bussolino F: Proliferative and migratory responses of murine microvascular endothelial cells to granulocyte-colony-stimulating factor. J Cell Physiol 155:89, 1993
17. Modzelewski RA, Davies P, Watkins SC, Auerbach R, Chang
MJ, Johnson CS: Isolation and identification of fresh tumor-derived
endothelial cells from a murine RIF-I fibrosarcoma. Cancer Res
54:336, 1994
18. Introna M, Bast RCJ, Tannenbaum CS, Hamilton TA, Adams
DO: The effect of LPS on expression of the early “competence”
genes JE and KC in murine peritoneal macrophages. J Immunol
138:3891, 1987
19. Bottazzi B, Polentamtti N, Acero R, Balsari A, Boraschi D,
Ghezzi P, Salmona M, Mantovani A: Regulation of the macrophage
content of neoplasms by chemoattractants. Science 220:210, 1983
20. Manenti G, Binelli G, Gariboldi M, Canzian F, De Gregorio
L, Falvella FS, Dragani TA, Pierotti MA: Multiple loci affect genetic
predisposition to hepatocarcinogenesis in mice. Genomics 23: 118,
1994
21. Dietrich W, Katz H, Lincoln SE, Shin HS, Friedman J, Dracopoli NC, Lander ES: A genetic map of the mouse suitable for typing
intraspecific crosses. Genetics 131:423, 1992
22. Johnson KR, Cook SA, Bustin M, Davisson MT: Genetic
mapping of the murine gene and 14 related sequences encoding
chromosomal protein HMG-14. Mamm Genome 3:625, 1992
23. Lincoln SE, Daly M, Lander ES: Constructing genetic maps
with MAPMAKEREXP 3.0. Whitehead Institute Technical Report
3rd edition, 1992
24. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ,
Lincoln SE, Newburg L: MAPMAKER: an interactive computer
package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174, 1987
25. Introna M, Hamilton TA, Kaufman RE, Adams DO, Bast
RCJ: Treatment of murine peritoneal macrophages with bacterial
lipopolysaccharide alters expression of c-fos and c-myc oncogenes.
J Immunol 137:2711, 1986
26. Golay J, Capucci A, Arsura M, Castellano M, Rizzo V, Introna M: Expression of c-myb and B-myb, but not A-myb, correlates
with proliferation in human hematopoietic cells. Blood 77: 149, 1991
1871
27. Angerer LM, Stoler MH, Angerer RC: In situ hybridization
with RNA probes: An annotated recipe, in Valentino KL (ed): In
situ Hybridization, New York, NY, Oxford, 1987, p 42
28. Narumi S, Wyner LM, Stoler MH, Tannenbaum CS, Hamilton
TA: Tissue-specific expression of murine IP-IO mRNA following
systemic treatment with interferon-gamma. J Leukoc Biol 52:27,
1992
29. Ransohoff RM, Hamilton TA, Tani M, Stoler MH, Shick HE,
Major JA, Estes ML, Thomas DM, Tuohy VK: Astrocyte Expression
of Messenger RNA Encoding Cytokines IP-10 and JEIMCP-I in
Experimental Autoimmune Encephalomyelitis. FASEB J 7592,
1993
30. Stoher PJ, Cameron GN: The EMBL data library. Nucleic
Acids Res 19:2227, 1991
31. Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85:2444, 1988
32. Robertson MJ, Soiffer RI,Wolf SF, Manley TJ, Donahue C,
Young D, Hemnann SH, Ritz J: Response of human natural killer
(NK) cells to NK cell stimulatory factor (NKSF): Cytolytic activity
and proliferation of NK cells are differentially regulated by NKSF.
J Exp Med 175:779, 1992
33. Bairoch A, Boeckmann B: The SWISS-PROT protein sequence data bank. Nucleic Acids Res 19:2247, 1991
34. Wako H, Blundell TL: Use of amino acid environment-dependent substitution tables and conformational propensities in structure
prediction from aligned sequences of homologous proteins. 11. Secondary structures. J Mol Biol 238:693, 1994
35. Rost B, Sander C: Prediction of protein secondary structure
at better than 70% accuracy. J Mol Biol 232584, 1993
36. Eliopoulos E, Geddes AJ, Brett M, Pappin DJC, Findlay JBC:
A structural model for the chromophore-binding domain of ovine
rhodopsin. Int J Biol Macromol 4:263, 1982
37. Blundell TL, Zhu ZY: The alpha-helix as seen from the protein tertiary structure: A 3-D structural classification. Biophys Chem,
55:167, 1995
38. Overington J, Johnson MS, Sali A, Blundell TL: Tertiary
structural constraints on protein evolutionary diversity: Templates,
key residues and structure prediction. Proc R SOCLond B Biol Sci
241:132, 1990
39. Emsley J, White HE, O’Hara BP, Oliva G, Srinivasan N,
Tickle IJ, Blundell TL, Pepys MB, Wood SP: Structure of pentameric
human serum amyloid P component. Nature 367:338, 1994
40. Blundell TL, Sibanda BL, Sternberg MJE, Thomton JM:
Knowledge-based prediction of protein structures and the design of
novel molecules. Nature 326:347, 1987
41. Blundell T, Carney D, Gardner S, Hayes F, Howlin B, Hubbard T, Overington J, Singh DA, Sibanda BL, Sutcliffe M: 18th Sir
Hans Krebs lecture. Knowledge-based protein modelling and design.
Eur J Biochem 172513, 1988
42. Topham CM, McLeod A, Eisenmenger F, Overington JP,
Johnson MS, Blundell TL: Fragment ranking in modelling of protein
structure. Conformationally constrained environmental amino acid
substitution tables. J Mol Biol 229:194, 1993
43. Sutcliffe MJ, Hayes FR, Blundell TL: Knowledge based modelling of homologous proteins, Part 11: Rules for the conformations
of substituted sidechains. Protein Eng 1:385, 1987
44. Prins JB, Meisler MH, Seldin M F Mouse chromosome 3.
Mamm Genome 5:S40, 1994
45. Kozak M: The scanning model for translation: An update. J
Cell Biol 108:229, 1989
46. von Heijne G: A new method for predicting signal sequence
cleavage sites. Nucleic Acids Res 14:4683, 1986
47. Bairoch A: PROSITE: A dictionary of sites and patterns in
proteins. Nucleic Acids Res 19:2241, 1991 (suppl)
48. Nguyen NY, Suzuki A, Boykins RA, Liu TY: The amino
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1872
acid sequence of Limulus C-reactive protein. Evidence of polymorphism. J Biol Chem 261:10456, 1986
49. Sarma V, Wolf FW,Marks RM, Shows TB, Dixit VM: Cloning of a novel tumor necrosis factor-alpha-inducible primary response gene that is differentially expressed in development and capillary tube-like formation in vitro. J Immunol 148:3302, 1992
50. Kopin AS, Wheeler MB, Leiter AB: Secretin: Structure of
the precursor and tissue distribution of the mRNA. Proc Natl Acad
Sci U S A 87:2299, 1990
51. Delli Bovi P, Curatola AM, Kem FG, Greco A, Ittmann M,
Basilic0 C: An oncogene isolated by transfection of Kaposi’s sarcoma DNA encodes a growth factor that is a member of the FGF
family. Cell 50:729, 1987
52. Clore GM, Nilges M, Brunger A, Gronenbom AM: Determination of the backbone conformation of secretin by restrained molecular dynamics on the basis of interproton distance data. Eur J Biochem 171:479, 1988
53. Gronenbom AM, Bovermann G, Clore GM: A IH-NMR
study of the solution conformation of secretin. Resonance assignment and secondary structure. FEBS Lett 215:88, 1987
54. Braun W, Wider G, Lee KH, Wuthrich K: Conformation of
glucagon in a lipid-water interphase by 1H nuclear magnetic resonance. J Mol Biol 169:921, 1983
55. Clore GM, Martin SR, Gronenbom AM: Solution structure
of human growth hormone releasing factor. Combined use of circular
dichroism and nuclear magnetic resonance spectroscopy. J Mol Biol
191:553, 1986
56. Sasaki K, Dockerill S, Adamiak TA, Tickle IJ, Blundell TL:
X-ray analysis of glucagon and its relationship to receptor binding.
Nature 257:751, 1975
57. Srinivasan N, White HE, Emsley J, Wood SP, Pepys MB,
Blundell TL: Comparative analyses of pentraxins-implications for
protomer assembly and ligand-binding. Structure 2: 1017, 1994
58. Sowdhamini R, Srinivasan N, Shoichet B, Santi DV, Ramakrishnan C, Balaram P: Stereochemical modeling of disulfide
bridges. Criteria for introduction into proteins by site-directed mutagenesis. Protein Eng 3:95, 1989
INTRONA ET AL
59. Ramachandran GN, Sasisekaran V: Conformation of polypeptides and proteins. Adv Prot Chem 23:283, 1968
60. Chandrasekaran R, Balasubramanian R: Stereochemical studies of cyclic peptides. IV energy calculations of cyclic disulphide
cysteinyl-cysteine. Biochim Biophys Acta 188:I , I969
61. Mosckovitz R, Gershoni JM: Three possible disulfides in the
acetylcholine receptor alpha-subunit. J Biol Chem 263: 1017, 1988
62. Blake CCF, Ghosh M, Harlos K, Avezoux A, Anthony C: The
active-site of methanol dehydrogenase contains a disulfide bridge
between adjacent cysteine residues. Nature Str Biol 1: 102, 1994
63. Kushner I, Feldman G: Control of the acute phase response.
Demonstration of C-reactiveprotein synthesis and secretion by hepatocytes during acute inflammation in the rabbit. J Exp Med 148:466,
I978
64. Zahedi K, Whitehead AS: Acute phase induction of mouse
serum amyloid P component. Correlation with other parameters of
inflammation. J Immunol 143:2880, 1989
65. Rudnick CM, Dowton SB: Serum amyloid P (female protein)
of the Syrian hamster. Gene structure and expression. J Biol Chem
268:21760, 1993
66. Reid MS, Blobel CP: Apexin, an acrosomal pentaxin. J Biol
Chem 269:32615, 1994
67. Noland TD, Friday BB, Maulit MT, Gerton GL: The sperm
acrosomal matrix contains a novel member of the pentaxin family
of calcium-dependent binding proteins. J Biol Chem 269:32607,
1994
68. Seery LT, Schoenberg DR, Barbaux S, Sharp PM, Whitehead
AS: Identification of a novel member of the pentraxin family in
Xenopus laevis. Proc R SOCLond B Biol Sci 253:263, 1993
69. Schlimgen AK, Helms JA, Vogel H, Perin MS: Neuronal
pentraxin, a secreted protein with homology to acute phase proteins
of the immune system. Neuron 14:519, 1995
70. Hsu YC, Perin MS: Human neuronal pentraxin I1 (NPTX2):
Conservation, genomic structure, and chromosomal localization. Genomics 28:220, 1995
7 1 . Evans SV: SETOR: hardware-lighted three-dimensional solid
model representation of macromolecules. J Mol Graph 1 I :134, 1993