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.     

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.     

Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component

The normal physiological roles of the phylogenetically conserved human plasma proteins C-reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities are in clinical development as both proteins have significant pathophysiological effects, SAP in promoting amyloidosis and CRP in exacerbating ischemic injury. Both proteins bind to phosphoethanolamine and we show here that, under physiological conditions, phosphoethanolamine is bound with higher affinity by human SAP than by human CRP. An explanation is provided by X-ray crystal structures that show SAP residue Tyr74 allowing additional hydrophobic protein-ligand interactions compared with the equivalent Thr76 of CRP. Docking simulations show many more low energy positions for phosphoethanolamine bound by CRP than by SAP and are consistent with the crystallographic and functional binding results. These fundamental observations on structure-activity relationships will aid the design of improved pentraxin targeting drugs.     

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.     

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.     

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.     

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.     

Selective inhibition of platelet activation by the amyloid P-component of serum

One component of amyloid, protein AP, has a characteristic pentameric structure and is identical with a 9.5s serum alpha 1-globulin designated serum amyloid P-component or SAP. Another pentameric molecule, the acute-phase reactant C-reactive protein (CRP), shares major amino acid sequence homology with SAP although, in man, SAP is not an acute-phase reactant. Recently, we demonstrated that heat-aggregated CRP (H-CRP), like heat-aggregated IgG, activates platelets to reactions of aggregation, secretion, and generation of thromboxane A2. We report here that physiologic concentrations of SAP inhibit platelet aggregation stimulated by H-CRP. SAP must be present before platelet challenge with H-CRP to be effective. Native (unaggregated) CRP does not inhibit platelet activation induced by H-CRP, and the platelet inhibitory effect of SAP is restricted because platelet responses to each heat-aggregated IgG, acid-soluble collagen, DNA, ADP, and thrombin remain unaltered in the presence of SAP. Thus, human SAP seems to selectively modulate platelet reactivity to modified CRP, and as such to down-regulate at least one aspect of the biologic capacity of its acute-phase homologue.     

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.     

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.     

Purification of human serum amyloid P component (SAP) by calcium affinity chromatography

Serum amyloid P component (SAP) has been purified from human serum by means of immobilized metal ion affinity chromatography (IMAC). It was selectively concentrated on carboxymethylated aspartic acid agarose (CM-Asp-agarose) loaded with calcium and, employing very mild conditions, purified to electrophoretical and immunological homogeneity in a single step amounting to about 1900-fold purification. As a purification method our procedure thus compares well with bio-specific affinity chromatography.     

Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis

Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (FcgammaR). We now report that SAP also binds to FcgammaR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcgammaRI, FcgammaRIIa, and FcgammaRIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to FcgammaR. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through FcgammaR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcgammaRIIIb also decreased phagocytosis of SAP-opsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through FcgammaR bearing cells.     

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.     

Isolation and characterization of the complete complementary and genomic DNA sequences of human serum amyloid P component

Complementary and genomic DNA clones corresponding to the human serum amyloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal peptide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATATAAA sequence 29-bp upstream from the cap site, but there is no CAAT box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were bounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.     

不同年龄急性冠脉综合征患者血脂代谢临床特点对比分析

目的:对比研究不同年龄急性冠脉综合征患者急性期血脂代谢特点及相关因素变化,探讨血脂代谢在不同年龄急性冠脉综合征发生过程中的作用及临床意义。方法:222例急性冠脉综合征(ACS)患者及119例稳定型心绞痛(SAP)患者共341例按照年龄分为3组,<60岁为非老年组共68人,其中ACS组33人,SAP组35人;60-74岁为普通老年组共67人,其中ACS组34人,SAP组33人;≥75岁为高龄老年组共206人,其中ACS组155人,SAP组51人。详细记录其基线资料并测定其血脂各项指标(包括TC、TG、LDL-C、HDL-C)及CRP。结果:与SAP患者比较,非老年人组及普通老年人组ACS患者LDL-C、CRP均显著升高;高龄老年人ACS患者LDL-C水平无显著升高,但HDL-C显著降低,CRP显著升高,差异均具有统计学意义(P<0.05),LDL-C虽无显著升高,但未达指南要求水平。各组之间TC、TG之间差异无统计学意义(P>0.05)。结论:脂代谢异常在不同年龄ACS患者的发生过程中均有重要作用,老年ACS患者中,除了应关注脂代谢紊乱外,还应该关注高血压、糖尿病等危险因素。     

Correlation between the glucose level and the development of acute pancreatitis

Objective

To investigate the correlation between the level of glucose in serum and the development of acute pancreatitis (AP).

Human serum amyloid P component is a single uncomplexed pentamer in whole serum

BACKGROUND: Serum amyloid P component (SAP) is a universal constituent of amyloid deposits and contributes to their pathogenesis. SAP also has important normal functions in the handling of chromatin in vivo and resistance to bacterial infection. The atomic resolution crystal structure of SAP is known, but its physiological oligomeric assembly remains controversial. In the absence of calcium, isolated human SAP forms stable decamers composed of two cyclic disk-like pentamers interacting face to face. However, in the presence of its specific low molecular weight ligands and calcium, SAP forms stable pentamers. In the presence of calcium, but without any ligand, isolated human SAP aggressively autoaggregates and precipitates, imposing severe constraints on methods for molecular mass determination. MATERIALS AND METHODS: Gel filtration chromatography and density gradient ultracentrifugation were used to compare SAP with the closely related molecule, C-reactive protein (CRP; which is known to be a single pentamer) and the effect of human serum albumin on SAP autoaggregation was investigated. RESULTS: In most physiological buffers and with the necessary absence of calcium, SAP, whether isolated or from whole serum samples, eluted from gel filtration columns clearly ahead of CRP. This is consistent with the existence of a monodisperse population of SAP decamers, as previously reported. However, in Tris/phosphate buffer, SAP was pentameric, suggesting that decamerization involved ionic interactions. On density gradients formed in undiluted normal human serum, SAP sedimented as single pentamers not complexed with any macromolecular ligand, regardless of the presence or absence of calcium. The calcium-dependent autoaggregation of isolated SAP was completely inhibited by physiological concentrations of albumin and the SAP remained pentameric. CONCLUSIONS: Human SAP exists within serum as single uncomplexed pentamers in the presence or absence of calcium. This oligomeric assembly, thus, does not require a calcium-dependent small molecule interaction. The usual >2000-fold molar excess of albumin over SAP in plasma is apparently sufficient to keep SAP in its physiological conformation.     

河蚌C-反应蛋白的性质研究

采用亲和层析方法从河蚌体液中可以一步获得纯化的C-反应蛋白(CRP)。本文对河蚌CRP一些性质做了进一步的研究。该蛋白是一个糖蛋白;但对鸡血红细胞并无凝集作用。通过免疫电泳法测得在正常河蚌体液中CRP的含量约为8.3mg/l。经荧光光谱研究发现,该蛋白结合Ca~(2+)和磷酸胆碱(PC)后有构象变化。     

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.