Eur. J. Immunol. 2007. 37: 3477–3488
Innate immunity
A female-specific pentraxin, CrOctin, bridges pattern
recognition receptors to bacterial phosphoethanolamine
Yue Li1, P. Miang Lon Ng1, Bow Ho*2 and Jeak Ling Ding*1
1
2
Department of Biological Sciences, National University of Singapore, Singapore
Department of Microbiology, National University of Singapore, Singapore
Pathogen recognition and binding are crucial functions of innate immunity. It has been
observed that the short pentraxin superfamily including C-reactive protein (CRP) and
serum amyloid P component are pathogen pattern recognition receptors (PRR) in the
plasma. We isolated and characterized a novel and distinctive pentraxin from the
plasma of horseshoe crab, Carcinoscorpius rotundicauda, henceforth named CrOctin,
which binds to bacteria via phosphoethanolamine (PE), a chemical component present
on lipid A and core polysaccharide moieties of bacterial lipopolysaccharide (LPS).
Infection enhances the formation of the PRR interactome constituting CrOctin, CRP and
galactose-binding protein. In particular, infection increases the affinity of CRP to
CrOctin by 1000-fold. Furthermore, we observed that by binding to PE, CrOctin acts as a
linker that bridges the PRR interactome to the inner core of LPS. On the other hand,
under normal physiological conditions, binding of CrOctin to PE appears to obscure
other PRR from interacting directly with PE. Interestingly, the cluster of “CrOctininteractive PRR” is sex specific. We report, for the first time, the change in PRR protein
profiles with a distinctive gender difference during Pseudomonas infection.
Received 17/1/07
Revised 10/8/07
Accepted 9/10/07
[DOI 10.1002/eji.200737078]
Key words:
CrOctin Innate
immunity
Phosphoethanolamine PRR interactome
Sex specificity
Supporting information for this article is available at
http://www.wiley-vch.de/contents/jc-2040/2007/37078_s-pdf
Introduction
One of the critical roles of the innate immune system is
the recognition of diverse pathogens by pattern
recognition receptors (PRR) [1]. The pentraxin super-
* These two authors share the senior authorship of this paper.
Correspondence: Dr. Jeak Ling Ding, Department of Biological
Sciences, National University of Singapore, 14 Science Drive 4,
Singapore 117543
Fax: +65-67792486
e-mail: dbsdjl@nus.edu.sg
Abbreviations: CRP: C-reactive protein GBP: galactose-binding protein hpi: hours post infection LpSAP: Limulus
polyphemus SAP-like pentraxin MS: mass spectrometry PAMP: pathogen-associated molecular pattern PC: phosphocholine PE: phosphoethanolamine pmf: peptide
mass fingerprint POPE: palmitoyl-oleoylphosphatidylethanolamine PRR: pattern recognition
receptor RpL3: ribosomal protein subunit L3 SAP: serum
amyloid P TBS: Tris-buffered saline TSB: tryptic soy broth
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family such as the short pentraxins, C-reactive protein
(CRP) and serum amyloid P (SAP) component, have
been amongst the first PRR to be identified [2, 3]. CRP
and SAP share high homologies in their sequence and
structure [4, 5] and are widespread in occurrence, from
invertebrates to human. They act as acute phase proteins
in different species [6, 7].
CRP and SAP have been shown to bind various
ligands derived from the host and/or pathogens [8], of
which phosphoethanolamine (PE) appears to be a ligand
for both CRP and SAP. PE is a chemical moiety of the
lipopolysaccharide (LPS) on the surface of gramnegative bacteria. PE reduces the net negative charge
of the microbe and thus fortuitously enables its evasion
from attack by cationic antimicrobial peptides [9].
However, as both CRP and SAP have affinity for PE
[10–13], it is possible that the host uses such short
pentraxins to act as PRR to seek out PE on the pathogen
surface in order to surmount the microbial camouflage.
But PE is also a variable moiety of phospholipids found
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in the eukaryotic cell membrane [14]. This appears to
pose a dilemma on self-nonself recognition, although
the exact mechanism for this differentiation remains an
enigma. However, a plausible explanation may be that
the PE groups of LPS are displayed on the outer surface
of a pathogen [15], in an appropriate configuration for
PRR recognition and binding, unlike the pattern and
location of the PE moieties on the inner surface of
normal host cells [16]. Here, we seek to isolate and
characterize the proteins from the plasma of an
estuarine horseshoe crab (Carcinoscorpius rotundicauda) that are able to recognize bacteria via PE. The
horseshoe crab family has been reported to harbor both
SAP-like proteins and CRP, the latter of which is
particularly abundant [17, 18]. As an invertebrate with
no adaptive immunity, the horseshoe crab relies solely
on its innate immune system to tolerate microbiologically challenging habitats and overcome systemic
infection by bacterial pathogens [17] at dosages that
would be lethal to mice [19, 20]. Our recent study has
shown that CRP collaborates with other plasma proteins
to boost the innate immune response [21].
We report the isolation of a new member of the
pentraxin family, CrOctin, which specifically binds PE.
From the plasma of Pseudomonas aeruginosa-infected
C. rotundicauda, several pentraxins and other lectins
were copurified with CrOctin. An infection-triggered
interaction was demonstrated amongst these PRR.
Through its binding to PE, CrOctin functions as a linker
PRR interacting with other proteins to mediate the
formation of a PRR interactome. Notably, infection
causes a 1000-fold increase in the binding affinity of CRP
to the CrOctin anchored on PE. Furthermore, we
demonstrate that the transcription of CrOctins and
CRP is synchronous and that, interestingly, CrOctins
display gender bias in their gene expression in relation to
the innate immune response against P. aeruginosa
infection.
Eur. J. Immunol. 2007. 37: 3477–3488
proteins (p28 and p29) by mass spectrometry (MS) gave
a peptide mass fingerprint (pmf) that was distinctive
(Fig. 1B) from the known C. rotundicauda CRP (EMBL
Accession Number Q2TS31–39) and dissimilar to other
known proteins of C. rotundicauda.
Isolation of the novel PE-binding PRR by
PE-Sepharose
To isolate the novel PRR that bind bacteria via PE, we
incubated plasma with PE-Sepharose. The PE-associated
proteins were sequentially eluted with sialic acid,
phosphocholine (PC) and EDTA to resolve different
PE-binding proteins. Previous reports have shown that
differential elution of PE-binding proteins by sialic acid,
PC and EDTA yielded limulin (also known as CRP2), CRP
(also known as CRP1) and SAP-like pentraxins,
respectively [18]. Indeed immunodetection of the PCeluted proteins by anti-CRP antibody showed a strong
signal (Fig. 1C, lane PC) confirming it to be CRP [17],
while the sialic acid and EDTA-eluted proteins showed
weak cross-reactions (Fig. 1C, lanes SA and EDTA),
suggesting that these proteins share homology with CRP.
MS analysis showed that the EDTA-eluted fraction
contains p28 and p29, which were also found amongst
the proteins bound to the bacteria (Fig. 1A). Although
also eluted by EDTA, like the Limulus polyphemus SAPlike pentraxin (LpSAP) [18], p28 and p29 (Fig. 1C, lane
EDTA) showed pmf (Fig. 1D) that are distinct from the
predicted pmf of LpSAP. This suggests that p28 and p29
bear characteristics of SAP-like protein although they do
not share high sequence homology. Thus, p28 and p29
may belong to a novel group of SAP-like pentraxins that
specifically bind bacteria via the PE moieties. p28 and
p29 seem to oligomerize under non-reducing condition
(Supporting Information Fig. 1). Since the limulus SAP
forms an octamer in the plasma, we henceforth named
p28 and p29 Carcinoscorpius rotundicauda Octins (p29
as CrOctin-1 and p28 as CrOctin-2).
Results
CrOctin is a novel SAP-like pentraxin
PE is a moiety of LPS targeted by PRR during
bacterial recognition
MS-derived peptide sequences of CrOctin were matched
to four partial cDNA clones previously isolated from a
hepatopancreas cDNA library. The full-length cDNA
were isolated by 50 and 30 RACE. Two major groups of
CrOctin (CrOctins 1 and 2) containing eight possible
sub-isoforms (CrOctin1.1, 1.2, GenBank Accession
DQ092708-DQ092709; CrOctin2.1a, b, c, d and
CrOctin2.2a, b, GenBank Accession DQ648075DQ648080) were characterized (Fig. 2A). However,
we cannot preclude the possibility that PCR mutations
had contributed to the sub-isoforms within the two
groups. Nevertheless, analysis of the two major groups
showed that CrOctin contains a characteristic sequence,
To investigate the plasma proteins that may target
bacteria via the PE moieties of LPS, we incubated
C. rotundicauda plasma with S. enterica Minnesota and
used PE as an eluant. Since CRP is a major immune
response protein in the horseshoe crab plasma and since
it may have affinity to PE [11], we used an anti-CRP
antibody to investigate whether CRP was eluted by PE
from the bacteria. Western blot analysis weakly detected
a protein of about 28 kDa (designated p28; Fig. 1A) that
did not appear in the control. Further analysis of the new
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Eur. J. Immunol. 2007. 37: 3477–3488
Innate immunity
Figure 1. Isolation of new pentraxin-like plasma proteins that bind bacteria via PE. (A) S. enterica Minnesota was incubated with
C. rotundicauda plasma or with buffer as control, before elution using 1 mM PE. Western blot of the eluted proteins using anti-CRP
antibody showed strict specificity for CRP and not GBP. A weaker signal was observed with PE-eluted p28 compared to the strong
signal with the CRP control. Both p28 and p29 were excised from the gel for MS analysis since a similar doublet was also observed in
PE-Sepharose purification with EDTA as eluant (see C). (B) The pmf profiles of p28 and p29 eluted by PE are compared with the pmf
profile of purified CRP (some CrCRP peaks are labeled in white boxes). p28 and p29 share similarity, but are different from CrCRP.
The peaks flagged in black boxes show peptide fragments common between p28 and p29 of (B) and (D). (C) Naive plasma from
C. rotundicauda was incubated with PE-Sepharose. The bound proteins were eluted sequentially with 5 mM sialic acid (SA), 10 mM
PC and 10 mM EDTA. The immunoblot shows that the anti-CRP antibody can detect the proteins from all three elutions. (D) MS
analysis of the p28 + p29 doublet shown in (C) from EDTA elution shows pmf(s) of a new SAP-like protein, henceforth referred to as
CrOctin-1 and -2. The peaks corresponding to predictions based on the sequence in Fig. 2A are flagged in black boxes.
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HLCYVWTN, similar to the classical pentraxin motif,
HxCxS(T)WxS, [8] except for two positions (Fig. 2B,
arrows). The Limulus SAP-like pentraxin [4] containing
a HVCHVWSG motif also differs at the same two amino
acids. This indicates that CrOctins appear related to the
SAP based on this characteristic motif, but are distinctive
members of the pentraxin family, and that the most
conserved motif in pentraxins is HxCxxW. According to
the size and the amino acid sequence homology,
CrOctins are classified under the short pentraxin family.
To determine if the full-length CrOctin sequence shows
closer homology to CRP or SAP, an alignment of the
CrOctin with other known horseshoe crab pentraxins
and vertebrate pentraxins was performed. The homo-
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logy tree (Fig. 2C) indicates that CrOctin does not
cluster with either the horseshoe crab CRP or SAP-like
proteins; hence, we deemed CrOctin to be a new
member of the pentraxin family.
Infection triggers CrOctin to interact with PRR
Chiou et al. [22] suggested that PRR such as galactosebinding protein (GBP) interact with other PRR like LPSbinding protein and protein A-binding protein during
their recognition of LPS and protein A. Since CrOctin
also binds gram-negative bacteria, we used the yeast
two-hybrid assay to investigate whether protein-protein
interaction occurs between CrOctin and other PRR.
Figure 2. Isolation of CrOctin clones. (A) Eight CrOctin clones belonging to the pentraxin family were isolated from the
C. rotundicauda hepatopancreas cDNA library. Two sub-isoforms of CrOctin-1 (CrOctin1.1 and 1.2) and six sub-isoforms of
CrOctin-2 (CrOctin2.1a, 2.1b, 2.1c, 2.1d, 2.2a, 2.2b) were characterized. The molecular masses of mature CrOctin-1 and -2 are
estimated to be 29.3 and 28.3 kDa, respectively. (B) Alignment of CrOctin and LpSAP against other pentraxins revealed the region
homologous to the known pentraxin motif, indicating that the three amino acids H, C and W (*) are highly conserved. Arrows
indicate the two amino acids unique to CrOctin LpSAP. The potential N-glycosylation site in the CrOctin sequence is
unglycosylated (see Supporting Information Fig. 2). (C) The phylogenetic analysis of CrOctin and other pentraxin family members
was performed by multiple sequence alignment using CLUSTAL X by Gonnet series protein weight matrix. The unrooted
phylogenetic tree was constructed by the neighbor-joining method based on the alignments. The confidence scores (%) of a
bootstrap test of 1000 replicates are indicated for major branching nodes. The yellow, green and pink areas annotate mammalian
CRP, mammalian SAP and horseshoe crab CRP, respectively. The scale bar corresponds to 0.1 estimated amino acid substitutions
per site. The GenBank accession numbers are listed in Table 1.
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Innate immunity
Table 1. The members of the pentraxin family used for
phylogenetic analysis
Clone name
Source
EMBL
Accession No.
CrCRP1
Cr: Carcinoscorpius
rotundicada
Q2TS37
CrCRP2
LpCRP1.1
Q2TS36
Lp: Limulus polyphemus
P06205
LpCRP1.4
P06206
LpSAP
Q8WQK3
TtCRP
Tt: Tachypleus tridentatus
Q9U8Y8
XePTX
Xe: Xenopus laevis
P49263
XeCRP
pigCRP
Q07203
Sus scrofa
pigSAP
ratCRP
O19062
O19063
Rattus norvegicus
ratSAP
P48199
P23680
troutPTX
Oncorhynchus mykiss
P79899
mouseCRP
Mus musculus
P14847
mouseSAP
S-hamsterCRP
P12246
Mesocricetus auratus
S-hamsterSAP
HumanCRP
P49262
P07629
Homo sapiens
HumanSAP
by infection which may be necessary to mediate
interaction between CrOctin and CRP, we further
examined the interaction between CrOctin and CRP
using the GST-pulldown assay. Fig. 3B shows that only
the 3-h post infection (hpi) CRP was pulled down by the
CrOctin1.1 isoform, while more of the 3-hpi CRP was
bound to CrOctin2.1d, thus corroborating the possibility
that infection mediates CrOctin-CRP interaction.
P02741
P02743
chickenCRP
Gallus gallus
Q2EJU6
GuineaCRP
Cavia porcellus
P49254
GuineaSAP
P49255
A-hamsterSAP (FP) Cricetulus migratorius
P15697
Fig. 3A shows that CrOctin interacts with GBP and
hemocyanin (HMC) but not with CRP-1 or CRP-2. Since
the yeast two-hybrid assay lacks the conditions triggered
Infection altered the binding kinetics between
CrOctin and CRP
To quantify the effects of infection on the interaction
between CrOctin and CRP, palmitoyl-oleoyl-phosphatidylethanolamine (POPE) was immobilized on an HPA
BIAcore chip with hydrophobic surface, whereby the PE
motif was available for interaction. We found that
CrOctin was bound avidly to PE at a KD of 6.79 10–9 M
(Fig. 4A). Next, we compared the binding affinity
between naive and infected CRP and CrOctin, the latter
of which was pre-anchored onto PE immobilized on a
chip (an attempt to mimic the biological surface of the
gram-negative bacteria). Naive CRP was found to bind
the CrOctin-PE-chip at a KD of 1.35 10–6 M (Fig. 4B),
which is *10 times lower in affinity than the direct
binding of naive CRP to PE (KD of 2.01 10–7 M)
(Fig. 4C). In contrast, infected CRP bound to the
CrOctin-PE-chip at a dramatically increased affinity of
8.49 10–9 M (Fig. 4D), *1000 times higher than
naive CRP. This increase in affinity was not due to CRP
binding directly to PE since direct binding between
infected CRP and PE occurred at a KD of only
3.88 10–7 M (Fig. 4E), which is comparable to that
of the naive CRP and PE. Taken together, we can possibly
draw the conclusion that infection causes a conformational change to CRP, increasing its affinity for its
protein partner, CrOctin, which is pre-anchored to the
PE component on the LPS molecule. Thus, PE is
preferentially bound by CrOctin (KD of 10–9 M) rather
than by CRP (KD of 10–7 M), and infection then causes
Figure 3. Protein-protein interaction between CrOctin and PRR. (A) Cotransformation of the plasmids of full-length CrOctin2.1d
and other PRR, namely GBP, CRP-1, CRP-2 and hemocyanin (HMCII C-terminal domain) into yeast to show interaction with CrOctin
in vivo. The intensity of the interaction was scored based on the positive control, GAL4. (B) GST-pulldown assay to confirm the
interaction between CrOctin isoforms and purified CRP. GST-CrOctin1.1 and GST-CrOctin2.1d immobilized on glutathioneSepharose were incubated with CRP purified from naive and 3-hpi plasma. Lanes "+" were with 17.5 lg CRP; lanes "–" were with
Buffer A only. Then the beads were washed and incubated with thrombin. The samples are loaded onto SDS-PAGE gels and
detected by anti-CRP antibody.
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Figure 4. Real-time biointeraction analysis
between CrOctin and CRP. POPE was immobilized on an HPA sensor chip in a BIAcore
2000 system. The PE moiety of the immobilized POPE interacts with various purified
proteins flown over the chip surface. The
real-time biointeraction kinetics were determined by injecting: (A) 100 and 200 nM
purified CrOctin; (B) 200 nM CrOctin followed by 50 lL of 2000 and 3000 nM naive
CRP; (C) 500 and 1000 nM naive CRP;
(D) 200 nM CrOctin followed by 2000 and
3000 nM of 3-hpi CRP; (E) 500 and 2000 nM of
infected CRP. This experiment was repeated
with hSAP and hCRP where (F) 5, 25 and
50 nM hSAP; (G) 50, 250 and 500 nM hCRP
were injected over the PE-chip. (H) Of 50 nM
hSAP, 50 lL was injected followed by 50 lL of
50, 250 and 500 nM hCRP. All the surface
plasmon resonance curves in RU were
normalized against Buffer A (50 mM Tris,
150 mM NaCl, 10 mM CaCl2, pH 7.4).
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CRP to be recruited at 1000 times higher affinity to the
immobilized CrOctin.
In humans, it is reported that SAP rather than CRP
binds preferentially to PE [8]. Here, we compared the
real-time biointeraction of (i) human (h)SAP to PE and
(ii) hCRP to PE (Fig. 4, G). We found that the binding
characteristics of hSAP and hCRP to the PE moiety are
similar to those of CrOctin and CRP, respectively. We
therefore tested the potential interaction between hCRP
and hSAP by injecting hCRP over a hSAP-PE-chip.
Fig. 4H shows that hCRP can bind immobilized hSAP
with a KD of 3.27 10–6 M. Whether the interaction
between hCRP and hSAP would show a similar level of
increase in affinity during infection as observed in
Carcinoscorpius remains to be further investigated.
Innate immunity
Infection mediates gender-specific transcription
of CrOctin and CRP
From the PE-Sepharose pulldown profile, we observed
differences in the levels of CrOctin and CRP in the
plasma of male and female animals. After elution of the
bound PRR from the male plasma (either naive or
infected) with PC followed by sialic acid, further elution
with EDTA did not dissociate any more CrOctin, but the
female plasma showed strong and persistent levels of the
CrOctin over the course of infection (Fig. 5A). It was
therefore imperative to examine the transcription of
CrOctin and CRP in the males and females during
infection. Between the two dominant immune responsive tissues, hemocyte and hepatopancreas, CrOctin was
Figure 5. Infection mediates gender- and tissue-specific transcription of CrOctins. (A) Naive, 1-hpi and 6-hpi plasma samples from
male and female animals were incubated with PE-Sepharose. After sequential elution with sialic acid and EDTA, the proteins were
resolved by SDS-PAGE and visualized by silver staining. Only the female plasma contained CrOctins, which was slightly enhanced
by P. aeruginosa infection, and was the only PRR eluted finally by EDTA. (B) The gene-specific primers of CrOctin-1 and CrOctin-2
were used for RT-PCR of mRNA templates from hemocytes and hepatopancreas of female animals infected at different time points.
RpL3 was used as loading control. (C) The real-time PCR analysis of the transcriptional levels of CrOctins and CRP in naive and
1–72-hpi hepatopancreas of both males and females. All data are taken from the mean of three individual experiments and
normalized against naive CRP-2. CrOctin1 mRNA was undetectable in the male samples.
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mainly synthesized by the latter (Fig. 5B), which is
functionally equivalent to the mammalian liver [23]. By
real-time PCR analysis, the CRP transcript levels in the
male and female hepatopancreas were determined
(Fig. 5C). The CrOctin-1 mRNA was undetectable in
the males while CrOctin-2 mRNA was expressed at
*1000-fold lower levels than that of CRP. In contrast, in
the females, CrOctin-1 was highly expressed from 1 hpi
onwards and CrOctin-2 transcription underwent a
surge, even superceding the level of the CRP transcripts
by 10–100-fold. In marked contrast, in the female, the
CRP and CrOctins share synchronous expression profiles
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exhibiting biphasic transcriptional activities with earlier
peaks at 1 and 24 hpi. Taken together, our finding
suggests that the gender difference can substantially
influence the activity of pentraxins during infection.
Discussion
During infection, the plasma PRR bind to the surface of
the invading pathogens. A PRR may operate individually
or as a complex to target the pathogen-associated
molecular patterns (PAMP). We postulate that the PRR
Figure 6. A hypothetical model for pathogen recognition assembly via interaction between CrOctin and PE displayed on the surface
of a gram-negative bacterium. Infection triggers CrOctin to anchor on the PE groups on the inner core and lipid A of LPS. Various
combinations of PRR interactomes may bind to the invading bacteria via CrOctin's contact with the bacterial PE moieties.
Consequently, different immune pathways are initiated to kill the bacteria: (A) complement pathways mediated by CRP [35];
(B) direct killing caused by CRP-2 [36]; (C) phagocytosis mediated by CRP [35], or CrOctin itself [37]; (D) prophenoloxidase pathway
mediated by hemocyanin (HMC) [38, 39]. Since PE is located at the proximal end of LPS, the binding between CrOctin-PRR and PE
ensures that the immune reactions occur efficiently, proximal to the pathogen. The CRP-CrOctin complex in the center of the
model is supported by the BIAcore data of Fig. 4. The other two complexes (CrOctin-GBP-CRP, and CrOctin-HMC on the left and
right sides of the model) are implied from Fig. 3A and Supporting Information Fig. 3. These are represented by dashed arrows
indicating the potential to form interactomes, and possibly resulting in the immune defense pathways. GlcN: glucosamine;
P: phosphate; PE: phosphoethanolamine; Ara: arabinose; Gal: galactose; Glc: glucose; Hep: heptose; KDO: keto-deoxyoctunolate;
NAG: N-acetylglucosamine.
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complex constitutes different plasma proteins to form a
PRR interactome (Fig. 6), which bears great significance
to the innate immune response. In this work, we
discovered a novel PRR, CrOctin, which targets bacterial
PE. Furthermore, we provide mechanistic insights into
how CrOctin might act as a bridge to link other PRR to
form a PRR interactome for nonself recognition during
immune defense.
PE is a cell membrane component found on the lipid
bilayer of both the prokaryotes and eukaryotes [16, 24].
To evade antimicrobial peptides, which have a net
positive charge, gram-negative bacteria harbor PE (with
net positive charge) on their core polysaccharide and
modify their lipid A by adding ethanolamine on the LPS
phosphate group. This chemical composition seems to
neutralize the net negative charge of the bacterial
membrane and therefore enables the microbe to avoid
the detrimental effects of cationic antimicrobial peptides
[9]. In contrast, PE is located at the inner leaflet of the
normal eukaryotic cell membrane so that it is remote
from immune surveillance, hence avoiding self recognition. However, apoptotic cells that have undergone
disruption of membrane asymmetry [25] will expose its
PE. Notwithstanding the display of these potential
danger signals, the recognition of PE is immunologically
beneficial for the host. PE-Sepharose provides a specific
“binding surface” for the complex of PRR (CRP, SAP and
CrOctin), thereby simulating the surface of the bacteria.
Christner and Mortensen [26] reported that immobilized hCRP can bind soluble hSAP. Here, we show
that immobilized CrOctin can bind soluble CRP and that
infection enhances binding (Fig. 4). The increase in their
affinity may be a result of the conformational change in
the PRR during infection. Real-time interaction analysis
of CRP showed its strong affinity for CrOctin prebound
to the PE-on-chip. Thus, it is reasonable to envisage that
in vivo CrOctin binds to the PE motif on LPS and
facilitates the stable anchorage of CRP close to the core
region of the invading microbe, where the immune
assault would occur effectively.
The yeast two-hybrid results (Fig. 3A) confirmed that
CrOctin can interact with GBP, which is another
important PRR in the horseshoe crab innate immune
system. Interestingly, CrOctin and GBP were not
copurified from the naive, but from the infected plasma.
It indicates that either (a) some factors are released
during infection to permit the interaction of these PRR
for a downstream response or (b) infection mediated a
conformational change in CrOctin to facilitate its
interaction with other PRR. In addition, the yeast
two-hybrid analysis revealed that CrOctin also interacts
with hemocyanin (Fig. 3A), an initiator of the
prophenoloxidase cascade. All of these interactions
between CrOctin and other PRR suggest that CrOctin
plays a crucial role in protein-protein networking in the
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Innate immunity
innate immune system to bridge various PRR to form a
PRR interactome, which confers greater binding capacity for the PAMP at the initial period of infection.
We also observed that when GlcNAc (a ligand of
GBP) was used to elute the 6-hpi plasma proteins from
the PE-Sepharose beads, both GBP and CRP were
dissociated (Supporting Information Fig. 4). This result
suggests that there is a population of CRP that binds to
the PRR complex via GBP. Fig. 6 illustrates a hypothetical model of the pathogen recognition PRR interactome, in which CrOctin plays a crucial role in
monitoring/sensing the inner biochemical environment,
seeking the PE. When bound onto PE on the surface of
the pathogen, CrOctin probably forms the initial
component of the PRR interactome. Simultaneously,
infection triggers the expression and conformational
change of other PRR such as CRP, GBP and HMC, which
will bind to CrOctins. Meanwhile, CRP and GBP also
bind to each other to stabilize the PRR complex. A
stabilized complex of PRR evokes downstream immune
responses that effectively destroy the pathogen.
Specific recognition of PAMP by CrOctin and a 1000fold increase in the affinity of CRP for CrOctin during
infection demonstrates that this mechanism is able to
prevent the immune system from mistriggering against
the host cells, thus ensuring a tight regulation of selfnonself recognition. Our study shows that, when
CrOctin binds PE, it blocks naive CRP from associating
with CrOctin (Fig. 4B). In vivo this surveillance may
prevent autoimmune responses under normal physiological conditions. In contrast, infected CRP exhibited
1000-fold greater affinity for CrOctin (Fig. 4D), indicating that infection may have indeed altered the CRP
molecule, enhancing it to bind CrOctin, to form the PRR
interactome leading to the antimicrobial effects. The
interaction between hSAP and hCRP also shows that,
like CrOctin, when naive hSAP binds PE, the hCRP is
prevented from binding PE (Fig. 4F–H). Taken together,
these results indicate that the mechanism and function
of interaction between these two short pentraxins are
conserved through evolution and presumably play a
crucial role in the innate immune system.
Our real-time PCR results showed that CrOctin and
CRP share a synchronous transcriptional regulation
profile, particularly in the female (Fig. 5C). It appears
that the female expresses these two pentraxins more
highly and rapidly than the male. Such gender-biased
expression of pentraxin members have been observed in
(i) a “female protein” (an SAP homologue in the Syrian
and Armenian hamsters), controlled by female hormones and inflammation [27, 28]; (ii) hCRP, controlled
by testosterone [29]; and (iii) PTX3 which is essential
for female fertility [30]. Consistently, we observed that
in infection, the CRP mRNA level is higher than the
CrOctin mRNA level in the male C. rotundicauda. Taken
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Yue Li et al.
together, these observations suggest that the functions
of pentraxins in the immune system are compensated for
by the two genders having different preferences for
pentraxin members.
In the human, SAP has been associated with many
diseases such as systemic lupus erythematosus [31, 32]
and amyloidosis [33, 34]. Thus, it seems that the
regulation of SAP expression in vivo should be extremely
timely and precise. This fine balance of expression of
pentraxin members is evidently observed in our
experiment where the rapid and biphasic responses of
CrOctin and CRP isoforms, particularly in the females,
occur during the acute phase of infection and inflammation (Fig. 5C). By regulating the transcription of
CrOctin/SAP, the levels of these PRR may decisively
control the immune response against the pathogen or
against the apoptotic cells and other sex-linked physiological dysfunctions. On the other hand, our observation
of the interaction between CrOctin and other PRR
stimulated at the acute phase of infection provides a new
insight into how the innate immune system differentiates self from nonself. Our work presents a radical
means of analyzing the clinical implications on the
members of the short pentraxin superfamily and
implicates the gender-specific expression and binding
properties of CrOctin and SAP.
Materials and methods
Organisms
Horseshoe crabs (Carcinoscorpius rotundicauda) were collected from the Kranji estuary of Singapore. The animals were
handled according to the guidelines of the National Advisory
Committee for Laboratory Animal Research, Singapore.
Hepatopancreas and plasma from the male and female animals
were examined. Pseudomonas aeruginosa ATCC 27853 was
used for in vivo infection studies. Salmonella enterica
Minnesota R595 ATCC 49284, which displays PE on its outer
membrane, was used for in vitro bacterium affinity chromatography to isolate PRR.
Infection of C. rotundicauda
Overnight cultures of P. aeruginosa in tryptic soy broth (TSB)
were washed and resuspended in saline at 1 107 CFU/mL.
C. rotundicauda (140 10 g) were bled prior to and after
intracardial injection with 1.2 106 CFU P. aeruginosa/100 g
body weight. Plasma was collected into pyrogen-free tubes and
clarified by centrifugation at 150 g for 15 min at 4 C. The
cell-free plasma was centrifuged at 5000 g before aliquoting
and storing at –70 C.
f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2007. 37: 3477–3488
Binding of CrOctin to bacteria
S. enterica Minnesota was cultured overnight in TSB at
230 rpm, 37 C. The culture was inoculated into fresh TSB
for 3.5 h at 37 C, 230 rpm. Bacteria were washed thrice and
resuspended in saline. An aliquot of bacteria equivalent to
OD600 nm of 1.0 U/mL was pelleted and resuspended in 500 lL
of either 50 mM Tris-buffered saline (TBS) pH 7.4 with 5 mM
EDTA or 10% plasma in TBS, and incubated for 3 h at room
temperature. The bacteria were pelleted at 5000 g for 5 min
and washed thrice with TBS. Proteins bound to the bacteria
were eluted using 1 mM PE in TBS. The eluate was collected
for Western blot analysis using anti-CRP antibody (Supplementary Information Fig. 5). An immunopositive protein band
(p28) was subjected to MS analysis (Supplementary Information).
PE-conjugated Sepharose
Proteins from naive and infected plasma that interact with PE
were isolated by using PE-Sepharose. The PE-bound proteins
were eluted with sialic acid, PC, GlcNAc and EDTA in various
sequential orders. An aliquot of 500 lL of PE-Sepharose was
incubated with 1 mL plasma and the final volume was made up
to 5.25 mL with Buffer A (50 mM Tris pH 7.4 containing
150 mM NaCl and 10 mM CaCl2). The mixture was incubated
overnight, loaded into a column and washed with Buffer A
until OD280 nm was less than 0.01. Bound proteins were eluted
with 5 mM sialic acid (Sigma) in Buffer A, or 10 mM PC
(Sigma) in Buffer A containing 10 mM EDTA. The PESepharose was prepared by conjugating O-PE (Sigma) onto
ECH Sepharose 4B (Amersham, GE Healthcare) according to
the manufacturer's instructions. The proteins were concentrated and dialyzed against Buffer A using Vivaspin columns.
Cloning the CrOctins
Total RNA was isolated from hepatopancreas using TRIZOL
Reagent (Invitrogen). mRNA was purified using the Oligotex
kit (Qiagen). The cDNA library was synthesized using the
MATCHMAKER Library Construction and Screening Kit for RTPCR (Clontech). Details on cloning the CrOctin cDNA are given
in the Supplementary Information.
Yeast two-hybrid assay
The mature CrOctin was cloned into pGBKT7 and cotransformed with different prey plasmids into AH109 according to
the manual of Clontech. Details on the yeast two-hybrid assay
are given in the Supplementary Information.
Surface plasmon resonance analysis
POPE (Avanti Polar Lipids, Inc.) in chloroform contained in a
glass tube was dried by N2 gas. The pellet was redissolved at
0.06 mg/mL in phosphate-buffered saline pH 7.4 containing
10 mM EDTA. The solution was sonicated at 32 C for 10 min,
vortexed for 10 min and centrifuged at 13 000 g at room
temperature for 2 min. Aliquots of 100 lL POPE solution were
injected onto the HPA sensor chip (BIAcore) in a BIAcore 2000
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Eur. J. Immunol. 2007. 37: 3477–3488
system, with water as running phase at a flow rate of 2 lL/min.
Skimmed milk (50 lL at 100 mg/mL; DIFCO) was injected to
coat the nonspecific binding sites, followed by 20 lL 0.1 M
NaOH to remove the multiple layers of lipids and proteins.
Prior to injection, the protein samples were equilibrated in
Buffer A. Each protein at 50 lL was injected at a flow rate of
30 lL/min and then dissociated for 180 s. The curve was
normalized against Buffer A injected as blank. The bound
protein was completely removed with 50 mM Tris containing
10 mM EDTA and 150 mM NaCl, pH 7.4. The data were
analyzed by BIAevaluation Version 3.2.
Real-time PCR
A reaction mixture of 25 lL containing 12.5 lL 2 SYBR
Green PCR Master Mix (Applied Biosystems), 0.6 lM each of
forward and reverse primers and 0.05 lL of the cDNA was
subjected to the following thermal program for real-time PCR
on the ABI prism 7000 Sequence Detection System (Applied
Biosystem): 1 cycle of 95 C for 10 min, 40 cycles of 95 C for
15 s and 60 C for 1 min. The readings of all the samples were
normalized with ribosomal protein subunit L3 (RpL3) and
compared with the level of naive CRP-2. Three independent
experiments were carried out for each time point post
infection.
Acknowledgements: This study was supported by
grants from the Agency for Science Technology and
Research (A*STAR, R-154–001–286–305) and the Ministry
of
Education,
Singapore,
AcRF
Tier
2
(R154–000–283–112), awarded to J.L.D. and B.H. Y.L. is a
Research Scholar supported by the National University
of Singapore. Note: The nucleotide sequence data
reported are available in GenBank databases under
the accession numbers: CrOctin1.1: DQ092708;
CrOctin1.2:
DQ092709;
CrOctin2.1a:
DQ648078;
CrOctin2.1b:
DQ648077;
CrOctin2.1c:
DQ648076;
CrOctin2.1d:
DQ648075;
CrOctin2.2a:
DQ648080;
CrOctin2.2b: DQ648079. In publications after year
2000, the Japanese horseshoe crab GBP was renamed
Tachypleus Plasma Lectin 1 and 2.
Conflict of interest: The authors declare no financial or
commercial conflicts of interest.
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