Pentraxin family of proteins interact specifically with phosphorylcholine and/or phosphorylethanolamine.

Pentraxins are a family of serum proteins characterized by five identical subunits that are noncovalently linked. The two major types of pentraxins are C-reactive protein (CRP) and serum amyloid P component (SAP). CRP proteins are identified by their calcium-dependent interaction with phosphorylcholine. This study showed that SAP also bound to phosphorylated compounds but had a high specificity for phosphorylethanolamine. Thus, human CRP and SAP show high specificity that is complementary for the related compounds, phosphorylcholine and phosphorylethanolamine, respectively. This relationship suggests a complementary and/or related function for the pentraxins. Pentraxins from other species were also examined. Mouse SAP showed binding interactions and specificity similar to human SAP. Female protein (FP) from hamster and rat CRP showed a hybrid specificity and bound to both phosphorylethanolamine and phosphorylcholine. All of the proteins that bound phosphorylethanolamine also associated with human C4b-binding protein (C4BP). With the exception of human and rat CRP, all the proteins also bound to vesicles containing acidic phospholipids. All of these binding interactions were calcium-dependent and mutually exclusive, suggesting that they involved the same site on the protein. These findings suggest possible ways to examine the function of the pentraxins.     

Serum amyloid P component and C-reactive protein mediate phagocytosis through murine Fc gamma Rs

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are acute-phase serum proteins in mice and humans, respectively. Although SAP binds to DNA and chromatin and affects clearance of these autoantigens, no specific receptor for SAP has been identified. CRP is an opsonin, and we have shown that it binds to FcgammaR. Mice deficient in FcgammaR were used to assess the role of these receptors in phagocytosis by pentraxins using zymosan as a ligand. Phagocytosis of zymosan by bone marrow macrophages (BMM) was enhanced by opsonization with SAP or CRP. BMM from mice deficient in all three FcgammaR or in gamma-chain ingested unopsonized zymosan, but phagocytosis of SAP- or CRP-opsonized zymosan was not enhanced. SAP binding to BMM from gamma-chain-deficient mice was also greatly reduced, indicating little or no binding of SAP to FcgammaRII. SAP and CRP opsonized zymosan for phagocytosis by BMM from mice deficient in FcgammaRII or FcgammaRIII. SAP, but not CRP, opsonized zymosan for uptake by neutrophils that express only low levels of FcgammaRI. Together these results indicate that FcgammaRI and FcgammaRIII are receptors for SAP in the mouse. Opsonization of zymosan by CRP is mediated through FcgammaRI. Pentraxins are major proteins of the innate immune system and arose earlier in evolution than Igs. The use of FcgammaR by the pentraxins links innate and adaptive immunity and may have important consequences for processing, presentation, and clearance of the self-Ags to which these proteins bind.     

Competitive binding of pentraxins and IgM to newly exposed epitopes on late apoptotic cells

A random distribution of phospholipids among the inner and outer leaflet of the cell membrane occurs during apoptosis and is known as membrane flip-flop. Flip-flopped cells have binding sites for various plasma proteins, such as IgM and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP). In this study, we investigated whether pentraxins and IgM antibodies recognize the same binding sites on apoptotic cells, and whether phospholipids constitute these binding sites. Except for SAP which also bound to early apoptotic cells, pentraxins and IgM preferentially bound to late apoptotic cells. Competition experiments with different phosphatemonoesters revealed that CRP and SAP as well as part of the IgM bound to the phospholipids head groups, SAP mainly to phosphorylethanolamine, CRP to phosphorylcholine and phosphorylethanolamine and to a lesser extent to phosphorylserine, and IgM to phosphorylcholine and phosphorylserine. These results were confirmed in experiments in which proteins were adsorbed from plasma with artificial phospholipids particles. IgM and the pentraxins variably competed for the same binding sites on late apoptotic cells, SAP having the highest and CRP the lowest apparent affinity. We conclude that CRP, SAP, and part of the IgM bind to the phospholipid head groups exposed on apoptotic cells. This shared specificity as well as their shared capability to activate complement, suggest that IgM and the pentraxins CRP and SAP exert similar functions in the removal of apoptotic cells.     

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.     

Pentraxins and IgA share a binding hot‐spot on FcαRI

The pentraxins, C‐reactive protein (CRP), and serum amyloid P component (SAP) have previously been shown to function as innate opsonins through interactions with Fcγ receptors. The molecular details of these interactions were elucidated by the crystal structure of SAP in complex with FcγRIIA. More recently, pentraxins were shown to bind and activate FcαRI (CD89), the receptor for IgA. Here, we used mutations of the receptor based on a docking model to further examine pentraxin recognition by FcαRI. The solution binding of pentraxins to six FcαRI alanine cluster mutants revealed that mutations Y35A and R82A, on the C‐and F‐strands of the D1 domain, respectively, markedly reduced receptor binding to CRP and SAP. These residues are in the IgA‐binding site of the receptor, and thus, significantly affected receptor binding to IgA. The shared pentraxin and IgA‐binding site on FcαRI is further supported by the results of a solution binding competition assay. In addition to the IgA‐binding site, pentraxins appear to interact with a broader region of the receptor as the mutation in the C′‐strand (R48A/E49A) enhanced pentraxin binding. Unlike Fcγ receptors, the H129A/I130A and R178A mutations on the BC‐ and FG‐loops of D2 domain, respectively, had little effect on FcαRI binding to the pentraxins. In conclusion, our data suggest that the pentraxins recognize a similar site on FcαRI as IgA.     

The interaction of C-reactive protein and serum amyloid P component with nuclear antigens

The pentraxins are a family of proteins characterized by cyclic pentameric structure, calcium-dependent ligand binding and sequence homology. The two main representatives of this family are the serum proteins, C-reactive protein (CRP) and serum amyloid P component (SAP). In man CRP is an acute phase reactant which increases up to 1000 fold during the acute phase response whereas SAP is a constitutive protein expressed at about 30 g/ml. These proteins activate complement through the classical pathway and participate in opsonization of particulate antigens and bacteria. In the past several years it has been determined that both of these pentraxins interact with nuclear antigens including chromatin and small nuclear ribonucleoproteins (snRNPs). Both CRP and SAP have nuclear transport signals which facilitate their entry into the nuclei of intact cells. Furthermore, these pentraxins have been shown to affect the clearance of nuclear antigens in vivo. It is now believed that one of the major functions of the pentraxins could be to interact with the nuclear antigens released from apoptotic or necrotic cells. This interaction could mitigate against deposition of these antigens in tissue and autoimmune reactivity.Abbreviations CRP C-reactive protein - HSA human serum albumin - PC phosphocholine - SAP serum amyloid P component - snRNP small nuclear ribonucleoprotein - SLE systemic lupus erythematosus     

Isolation and characterization of SAP and CRP,two pentraxins from Pangasianodon (Pangasius) hypophthalmus

From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine–Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca²⁺ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 μg SAP and 56 μg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1–2 mM), respectively. Circular dichroism studies revealed that antiparallel β-pleated sheets are dominating the secondary structure. Upon removing the Ca²⁺ ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.     

A protease-sensitive site in the proposed Ca(2+)-binding region of human serum amyloid P component and other pentraxins.

Serum amyloid P component (SAP) is a decamer of 10 identical 25.5-kDa subunits. Limited proteolysis of SAP with alpha-chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of Ca2+ (10 mM). Cleavage with alpha-chymotrypsin prevents the Ca(2+)-dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The alpha-chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C-reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca(2+)-binding site in calmodulin and related Ca(2+)-binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.-Y., 1986, J. Biol. Chem. 261, 10456-10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that are similar to those obtained with alpha-chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS-PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by alpha-chymotrypsin, resulting in the loss of Ca(2+)-binding (as shown by equilibrium dialysis) and Ca(2+)-dependent binding reactivities (Kinoshita, C.M., Ying, S.-C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28, 9840-9848).(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum amyloid P component binds to histones and activates the classical complement pathway.

Two members of the pentraxin family of proteins, C-reactive protein (CRP) and serum amyloid P component (SAP), bind to chromatin and may be involved in the solubilization and clearance of nuclear material. Previous studies demonstrated that CRP binding to chromatin is mediated by histones. SAP differs from CRP in being able to bind to DNA, but SAP binding to histones has not been reported. CRP is an activator of the classical C pathway, and C-dependent cleavage of chromatin in the presence of CRP and serum has been shown. Oligomers of SAP have recently been found to bind to C1q and consume total C and C4, indicating that SAP can activate C as well. The present study examined CRP and SAP binding to histones H1 and H2A and C activation after binding. SAP binding to histones H1 and H2A was observed as well as SAP binding to chromatin. In contrast to CRP, SAP binding to chromatin did not require H1. SAP partially inhibited CRP binding to chromatin and to H1. However, neither pentraxin inhibited binding of the other to H2A. Binding of either CRP or SAP to H2A activated C in SAP-depleted serum leading to the deposition of C4 and C3. C activation required C1q and produced C4d indicating that it occurred through the classical pathway. These findings demonstrate that CRP and SAP share histone as well as chromatin binding, and that both pentraxins can activate the classical C pathway after ligand binding.     

Jeltraxin, a frog egg jelly glycoprotein, has calcium-dependent lectin properties and is related to human serum pentraxins CRP and SAP

The egg jelly that encapsulates amphibian eggs is essential for fertilization, but its molecular composition and roles remain largely unknown. We identified a calcium-dependent lectin from the pentraxin superfamily in the egg jelly coat from the South American burrowing frog, Lepidobatrachus laevis. This lectin, jeltraxin, was related to the host-response acute phase serum proteins C-reactive P component (CRP) and serum amyloid P component (SAP). The amino acid sequence of jeltraxin is 44% identical to that of Xenopus laevis CRP, 31-35% identical to those of mammalian CRP and SAP, and 21-27% identical to those of the large fusion pentraxins. Expression of jeltraxin mRNA was restricted to the oviduct, which distinguishes it as the first serum-related pentraxin not expressed in the liver. Purified jeltraxin was previously shown to exist in an oligomeric complex of approximately 250 kDa comprised of self-associating subunits. We have demonstrated by MALDI-TOF that this configuration is due to a decameric complex of 27.7 kDa subunits. Biotinylated jeltraxin bound to the high-molecular mass components of the egg jelly in a calcium-dependent manner with specificity for beta-galactose residues. On the basis of homology modeling, we predict that jeltraxin will coordinate two calcium ions. The function of jeltraxin will likely be related to its calcium-dependent lectin properties.     

The human C-reactive protein gene (CRP) and serum amyloid P component gene (APCS) are located on the proximal long arm of chromosome 1

The genes encoding two pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are located on the proximal long arm of human chromosome 1. Mapping of the CRP and SAP genes between the centromere and band q32 was achieved by Southern blot analysis of DNA from a panel of human × Chinese hamster somatic cell hybrids carrying defined fragments of human chromosome 1. Both genes were localized more precisely between bands q12 and q23 by in situ hybridization to human metaphase chromosomes.     

The Long Pentraxin PTX3 Promotes Fibrocyte Differentiation

Monocyte-derived, fibroblast-like cells called fibrocytes are associated with fibrotic lesions. The plasma protein serum amyloid P component (SAP; also known as pentraxin-2, PTX2) inhibits fibrocyte differentiation in vitro, and injections of SAP inhibit fibrosis in vivo. SAP is a member of the pentraxin family of proteins that includes C-reactive protein (CRP; PTX1) and pentraxin-3 (PTX3). All three pentraxins are associated with fibrosis, but only SAP and CRP have been studied for their effects on fibrocyte differentiation. We find that compared to SAP and CRP, PTX3 promotes human and murine fibrocyte differentiation. The effect of PTX3 is dependent on FcγRI. In competition studies, the fibrocyte-inhibitory activity of SAP is dominant over PTX3. Binding competition studies indicate that SAP and PTX3 bind human FcγRI at different sites. In murine models of lung fibrosis, PTX3 is present in fibrotic areas, and the PTX3 distribution is associated with collagen deposition. In lung tissue from pulmonary fibrosis patients, PTX3 has a widespread distribution, both in unaffected tissue and in fibrotic lesions, whereas SAP is restricted to areas adjacent to vessels, and absent from fibrotic areas. These data suggest that the relative levels of SAP and PTX3 present at sites of fibrosis may have a significant effect on the ability of monocytes to differentiate into fibrocytes.     

Dietary modulation and structure prediction of rat mucosal pentraxin (Mptx) protein and loss of function in humans

Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive "top" face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents.     

C-reactive protein: How conformational changes influence inflammatory properties

Recent evidence suggests that the prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP belongs to the family of pentraxins and as such consists of five identical non-covalently linked subunits. Contradictory data on the characteristics of CRP as either being pro- or anti-inflammatory may be explained by the existence of two conformations of the protein: the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. In vitro both isoforms exhibit a very distinct inflammatory profile. We recently identified a localized, physiologically relevant pCRP dissociation mechanism by activated platelets and apoptotic cells and showed the deposition of mCRP in inflamed tissue. Here we review the literature on the causal role of p- and mCRP in the light of our findings and critically analyze the current controversies around CRP. The novel understanding of the localized dissociation of circulating pentameric CRP to the distinctively pro-inflammatory monomeric CRP allows for a new view on CRP in inflammatory reactions and further highlights mCRP and the pCRP dissociation process as a potential therapeutic target.     

Pentraxins in innate immunity: lessons from PTX3

The innate immune system constitutes the first line of defence against microorganisms and plays a primordial role in the activation and regulation of adaptive immunity. The innate immune system is composed of a cellular arm and a humoral arm. Components of the humoral arm include members of the complement cascade and soluble pattern recognition molecules (PRMs). These fluid-phase PRMs represent the functional ancestors of antibodies and play a crucial role in the discrimination between self, non-self and modified-self. Moreover, evidence has been presented that these soluble PRMs participate in the regulation of inflammatory responses and interact with the cellular arm of the innate immune system. Pentraxins consist of a set of multimeric soluble proteins and represent the prototypic components of humoral innate immunity. Based on the primary structure of the protomer, pentraxins are divided into two groups: short pentraxins and long pentraxins. The short pentraxins C-reactive protein and serum amyloid P-component are produced by the liver and represent the main acute phase proteins in human and mouse, respectively. The long pentraxin PTX3 is produced by innate immunity cells (e.g. PMN, macrophages, dendritic cells), interacts with several ligands and plays an essential role in innate immunity, tuning inflammation and matrix deposition. PTX3 provides a paradigm for the mode of action of humoral innate immunity.     

Three pentraxins C-reactive protein,serum amyloid p component and pentraxin 3 mediate complement activation using Collectin CL-P1

BackgroundPentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition.MethodsWe used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay.ResultsWe demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation.ConclusionOur current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation.General significanceCL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.     

Amino acid sequence homology between rat and human C-reactive protein.

The rat serum protein that undergoes Ca2+-dependent binding to pneumococcal C-polysaccharide and to phosphocholine residues, and that is evidently a member of the pentraxin family of proteins by virtue of its appearance under the electron microscope, has been variously designated as rat C-reactive protein (CRP) [de Beer, Baltz, Munn, Feinstein, Taylor, Bruton, Clamp & Pepys (1982) Immunology 45, 55-70], 'phosphoryl choline-binding protein' [Nagpurkar & Mookerjea (1981) J. Biol. Chem. 256, 7440-7448] and rat serum amyloid P component (SAP) [Pontet, D'Asnieres, Gache, Escaig & Engler (1981) Biochim. Biophys. Acta 671, 202-210]. The partial amino acid sequence (45 residues) towards the C-terminus of this protein was determined, and it showed 71.7% identity with the known sequence of human CRP but only 54.3% identity with human SAP. Since human CRP and SAP are themselves approximately 50% homologous, the level of identity between the rat protein and human SAP is evidence only of membership of the pentraxin family. In contrast, the much greater resemblance to human CRP confirms that the rat C-polysaccharide-binding/phosphocholine-binding protein is in fact rat CRP.