Pentraxin

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Pentraxin And Its Possible Receptors
Structure_Function Relationship Bidya Sagar,
THI
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pentraxins A superfamily of conserved
Proteins that are characterized by a cyclic
multimeric structure. Family of oligomeric
plasma proteins all of which bind Ca ions
3
They are arranged in a pentagonal or rarely
hexagonal cyclic symmetry
Pentraxin family signature HxCxS/TWxS x -
amino acid
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Short Pentraxins
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Classical Short Pentraxins 25-kDa Within
Vertebrates, there exists two main branches of
the Pentraxin Family CRP-like proteins and
Serum Amyloid P-like proteins (SAP).
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The proteins that bind phosphocholine (in
bacterial and fungal polysaccharides and of most
biological cell membranes) are CRP- like
proteins CRP also binds certain nuclear
constituents, which do not contain PCh, such as
small ribonucleoprotein particles .
7
And the proteins that bind carbohydrate moieties
are SAP-like proteins
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Human Acute-Phase Proteins
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Location The CRP gene has been mapped to
human chromosome 1, between 1q21 and 1q23. It
contains 2263 nucleotides and has a single intron
Synthesis in the liver is responsible for blood
CRP but extrahepatic expression has also been
reported
10
Transcription Both in vitro and in vivo results
have established that interleukin-6 (IL-6) is the
principal inducer of the CRP gene, while IL-1,
glucocorticoids and certain other factors, act
synergistically with IL-6 to enhance its effect
11
Structure
The structure of CRP has been determined by
X-ray crystallography at 3 Å resolution
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Pentameric structure of CRP viewed down the
5-fold symmetry axis. The effector face of the
molecule is on the top, while the calcium- and
PCh-binding sites are on the opposite
recognition' face
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Structure of the human CRP protomer
The five identical 24-kDa protomers consist of
206 amino acids
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The phosphocholine-binding site
The binding of phosphocholine to C-reactive
protein. (a) Difference Fourier map at 2.5 Å,
showing the positions of the two calcium ions
(orange) and a molecule of phosphocholine. (b)
GRASP representation of CRP illustrating the
positions of the five bound molecules of
phosphocholine (orange and black).


Thompson
et al., 1999
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(a) CRP pentamer illustrating the positions of
the clefts present on the A surface (b) CRP
protomer illustrating the charge distribution
in the cleft. Blue, positive charge red,
negative.
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The existence of this pocket encourages design
of branched PC analogues with bulky substituents
at the 2 position that could be bound with
higher affinity than PC. These would be
powerful reagents to investigate CRP function as
well as having the potential as drugs to block
possible harmful effects of CRP in vivo.
These results are in general agreement with
earlier molecular modelling studies
GRASP representation of the phosphocholine-binding
site showing the vacant pocket adjacent to the
ligand.
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CRP consists of five noncovalently
associated protomers arranged symmetrically
around a central pore. The overall dimensions
of the CRP pentamer are about 102 Å outside
diameter with a central pore diameter of 30 Å and
a protomer diameter of 36 Å. The protomer
consists of 206 amino acids folded into two
antiparallel sheets with a flattened jellyroll
topology
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Long Pentraxins
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long pentraxins have an unrelated, long
amino-terminal domain coupled to the
carboxy -terminal pentraxin domain differ, with
respect to short pentraxins in their gene
organization, chromosomal localization,
cellular source, and in their stimuli-inducing
and ligand-recognition ability.
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The subfamily of long pentraxins
Apexin/p50 425 aa long protein was found to be
localized to the anterior pole of guinea-pig
sperm within the acrosomal compartment and thus
named apexin or p50
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The subfamily of long pentraxins
Apexin/p50 425 aa long protein was found to be
localized to the anterior pole of guinea-pig
sperm within the acrosomal compartment and thus
named apexin or p50 XL-PXN1 416 aa with Mr
50kDa. It is one of the most likely to have
divergent amino-terminal domain from the other
long pentraxins
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The subfamily of long pentraxins
Apexin/p50 425 aa long protein was found to be
localized to the anterior pole of guinea-pig
sperm within the acrosomal compartment and thus
named apexin or p50 XL-PXN1 416 aa with Mr
50kDa. It is one of the most likely to have
divergent amino-terminal domain from the other
long pentraxins Rat neuronal pentraxin I
(NPI) 47 kDa secretory glycoprotein,
expression is restricted to neurons of the
cerebellum, hippocampus and cerebral cortex
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The subfamily of long pentraxins
Apexin/p50 425 aa long protein was found to be
localized to the anterior pole of guinea-pig
sperm within the acrosomal compartment and thus
named apexin or p50 XL-PXN1 416 aa with Mr
50kDa. It is one of the most likely to have
divergent amino-terminal domain from the other
long pentraxins Rat neuronal pentraxin I
(NPI) 47 kDa secretory glycoprotein,
expression is restricted to neurons of the
cerebellum, hippocampus and cerebral
cortex Human neuronal pentraxins II (NPTX2) The
human counter part
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The subfamily of long pentraxins TSG-14
Among 8 distinct TNF-stimulated genes (TSG)
identified, TSG-14 was the most frequently
encountered sequence in the cDNA library
Implies high abundance of its mRNA in TNF
stimulated fibroblasts

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ptx3
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• Pentraxin-3 (PTX3) is a newly discovered
  marker of the acute phase response from Pentraxin
  family.

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The subfamily of long pentraxins An
essentially identical gene, differing by only
one amino acid (L202M), has been independently
cloned from human vascular endothelial cells
in response to interleukin-1 and named
ptx3 TSG-14/PTX3
was the first long pentraxin described and is
the best characterized of the long pentraxins

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Phylogenetic relationship among the pentraxin
family members Evolutionary neighbor-joining tree
relationship of TSG-14 w.r. to pentraxins
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• Human TSG-14 is a single copy gene
• It has three exons and two introns
• Located in chromosome 3 at band q25

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Properties TSG-14 gene and protein Its cDNA
encodes a 381 amino acid protein having a
putative hydrophobic signal sequence of 17 amino
acids The mature protein, lacking the signal
peptide, has a predicted Mr of 42 kDa and pI
4.69 The NH2-terminal half of PTX3 shows no
similarity to any known protein sequence and
initiates with a putative signal peptide
indicating that PTX3 is secreted
31

• TSG-14/PTX3 is produced as a 1020-subunit
  multimer protein
• Its synthesis can strongly induced at the
  transcriptional level by the inflammatory
  cytokines
• tumor-necrosis factor
• interleukin-1
• Bacterial lipopolysaccharide (LPS)
• in fibroblasts, Endothelial cells, Cells of the
  monocyte/macrophage lineage

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• Hansson group recently showed that immuno-
  histochemical staining of advanced
  atherosclerotic plaques revealed strong
  expression of PTX3.
• In contrast, no PTX3 expression was observed
  in non-atherosclerotic internal mammary arteries.
  
• They observed that PTX3 was produced
  principally by macrophages and endothelial cells.
  
• Infrequent expression by smooth muscle cells
  was also
• observed.

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FACS analysis of Ptx3 binding to conidia of A.
fumigatus and C. albicans. Binding was revealed
by biotinylated Ptx3 followed by fluorescein
isothiocyanate (FITC)- labelled streptavidin.
Heat-inactivated (a) or viable (b) conidia were
used with similar results. Galactomannan
(20 µg ml-1) abolished binding (b). d, Specific
binding of Ptx3 to A. fumigatus conidia. Binding
is saturable with a kd of 7.8 10-7 M
(calculated on the basis of the Ptx3 protomer
mass of 45 kDa). MFI, mean fluorescence
intensity. e, f, Binding to bacteria. Dotted
curves show control fluorescence in the absence
of PTX3.
Interaction of Ptx3 with selected pathogens
Nature, 2002
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The finding that Ptx3 facilitated recognition of
conidia by macrophages implied the existence of a
receptor for this molecule. Ptx3 bound to
mononuclear phagocytes and dendritic cells, but
not to T lymphocytes Thus, the long pentraxin
Ptx3 is a secreted pattern-recognition receptor
that has a non- redundant role in resistance to
selected microbial agents, in particular to the
opportunistic fungal pathogen Aspergillus
fumigatus.
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Receptors The presence
of a receptor for CRP was first suggested by
reports of opsonic activity of CRP Work in Du
Clos' laboratory has provided strong evidence
that Fc receptors for IgG are also the receptors
for CRP.
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The functions of FcRs. Pathogens, allergens or
autoantigens are bound by specific antibodies in
a process called opsonisation. These opsonised
antigens are bound by cell-surface FcRs leading
to aggregation of FcR
ITAM Immunoreceptor tyrosine-based
activation motif
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The FcRs for the main serum immunoglobulin IgG
can be subdivided into three classes and
represent type I transmembrane proteins.
Fc?RII, CD32 Fc?RIII, CD16 (Kd10-5-10-7 M)
Fc?RI, CD64 (Kd10-8 M)
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FcRs are in clinical focus since they are
important mediators of the disregulated immune
system. Fc?RI is the high affinity IgE
receptor expressed on mast cells. It is the
primary effector in allergic reactions and
asthma. Fc?Rs play a role in infections,
autoimmune reactions and cancer.
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Fc Receptors

• Diversity of FcRs
• 3 Receptors for IgG
• Fc?RI, Fc?RII,Fc?RIII
• Fc?R -receptor for IgE
• Different cells have
• different FcRs

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FcR Receptors overview

• Diversity of FcRs
• Distribution of FcRs on different cell types
  different functions
• Phagocytosis
• Placental transport
• Antibody dependent cellular cytotoxicity
• Mast cell degranulation
• etc

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Schematic representation of the structures of the
Ig superfamily FcRs
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Structures of ligand-free FcR
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Structure of the Fc RIII complexed with Fc
portion of IgG1
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Fc?RIIIFc interface. (A) Interactions between
Fc?RIII (green) and chain A of Fc (cyan).
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Antibody-Fc?RIII binding and ligand induced
receptor aggregation model
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The effect of Fc R binding on the symmetry axis
of IgG Fc fragment
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Additionally to membrane-bound forms, Soluble
FcRs are present in serum and other body fluids.
(sFc RI, sFc RII, sFc RIII and sFc RI ) They
are produced either by proteolysis, alternative
splicing or by separate genes and a physiological
relevance in immune regulation seems possible
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Potential inhibitors of FcR/Fc fragment binding
and applications
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Potential of FcR-(ant)agonists
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The binding of human Fc?RIII to IgG Fc fragment
in relation to the position of various
antibody-derived peptides that modulate receptor
binding.
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It was initially demonstrated that CRP binds to
the high affinity IgG receptor, Fc RI, on
mononuclear cells transfected with a cDNA
encoding this receptor More recently,
transfection experiments using Fc?RIIa cDNA and
parallel studies on monocytic cell lines
expressing this receptor, demonstrated that it
is the main receptor for CRP Experiments using
FcR -chain-deficient and Fc RII-deficient mice,
indicated that similarly to man, murine Fc RI
and Fc RII are the receptors for CRP
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Precipitation of FcR by CRP
THP-1 cells express both Fc RI and Fc RII
These bands with Mr of 70 and 42 kD corresponded
to the bands precipitated by mAb to CD64 and CD32
Immobilized CRP precipitates Fc?RI and Fc?RII
from radiolabeled, pronase-treated human
monocytic cell line (THP-1) cells
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Crystal structure of the human leukocyte Fc
receptor, Fc?RIIa Ribbon diagram of an Fc?RIIa
dimer viewed down the twofold axis.
Maxwell
et al., Nature St. Biol, 1999
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Molecular surface of the Fc?RIIa dimer
An orientation of IgG and Fc RIIa dimer which
allows complex formation. Fc RIIa is positioned
with Ctermini down. The Fc RIIa dimer is
represented as a molecular surface with the
ligand binding region in red
Ig binding surface and interaction with IgG
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Lack of specific receptors for C-reactive
protein on white blood cells
Hundt et al., Eur.
J. Immunol, 2001 competition assays demonstrated
that binding of biotinylated, partially purified
CRP was only inhibited by partially purified CRP
and IgG, but not by highly purified and
recombinant CRP. Recombinant CRP bound to U937
cells only after contamination with 0.5?g IgG
per 100 ?g CRP before biotinylation.
Therefore, they concluded that CRP itself is
not bound to white blood cells and strongly
suggested a reassessment of previous data.
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C-reactive protein-mediated phagocytosis and
phospholipase D signalling through the
high-affinity receptor for immunoglobulin G
(Fc?RI)
Katherine et al., Immunology,
2002 CRP-mediated phagocytosis may be indirect,
through activation of complement and complement
receptors, or direct, through receptors for the
Fc portion of immunoglobulin G (IgG Fc?Rs) or
even a putative CRP-specific receptor. No
strong evidence has been shown to indicate
which receptors may be responsible for
phagocytosis or signalling responses.
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Using BIAcore technology, they confirmed that
CRP binds directly to the extracellular portion
of Fc?RI with a threefold higher affinity than
IgG (K D  081X10-9M). Binding is Ca2
dependent and is inhibited by IgG1 but not by
phosphorylcholine (PC). CRP opsonization (using
CRP concentrations within the normal human serum
range) of PC-conjugated sheep erythrocytes
increased phagocytosis of these particles by
COS-7 cells transfected with Fc?RI-II chimaera
or Fc?RI/ -chain.
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(No Transcript)
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Interferon-?-treated U937 cells, which signal
through Fc?RI to activate phospholipase D (PLD)
in response to cross-linked IgG, were also
activated by CRP without any requirement for
further cross-linking. These studies indicate
that CRP is capable of binding to and
cross-linking Fc?RI thereby resulting in PLD
activation and increased phagocytosis. Uptake
by Fc?RI has been reported to promote various
acquired immune responses suggesting that CRP
could act in a similar way.
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Speculation During the acute phase response,
high levels of circulating CRP may influence
signaling by IgG complexes through Fc?RII.
Whether CRP will have a or influence ?
What is the direct effect of CRP on receptor
activation and the effect of CRP on
IgG-mediated signaling ? What is the binding
site ?