Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.     

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.     

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.     

Serum amyloid P component is the Shiga toxin 2-neutralizing factor in human blood

It has been suggested that some factor present in human plasma binds to Shiga toxin 2 (Stx2) and neutralizes it in vitro (Bitzan, M., Klemt, M., Steffens, R., and Muller-Wiefel, D. E. (1993) Infection 21, 140-145). This factor does not exist in other species (Caprioli, A., Luzzi, I., Seganti, L., Marchetti, M., Karmali, M., Clarke, I., and Boyd, B. (1994) Recent Adv. VTEC Infect. 353-356). Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDS-polyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.     

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.     

Calumenin interacts with serum amyloid P component

We recently reported the identification of human calumenin, a novel Ca(2+) binding, transformation-sensitive and secreted protein [Vorum et al. (1998) Biochim. Biophys. Acta 1386, 121-131; Vorum et al. (1999) Exp. Cell Res. 248, 473-481] belonging to the family of multiple EF-hand proteins of the secretory pathway that include reticulocalbin, ERC-55, Cab45 and crocalbin. In order to further investigate the extracellular functions of calumenin we immobilized the recombinant protein to a column. After application of a placental tissue extract we were able to elute one protein that interacts with calumenin in the presence of Ca(2+). Amino acid sequencing identified this protein as serum amyloid P component (SAP). Furthermore, we verified and characterized the calumenin-SAP interaction by the surface plasmon resonance technique. The findings indicate that calumenin may participate in the immunological defense system and could be involved in the pathological process of amyloidosis that leads to formation of amyloid deposits seen in different types of tissues.     

Serum amyloid P component is the major calcium-dependent specific DNA binding protein of the serum

Serum amyloid P component (SAP), a member of the highly conserved pentraxin family of plasma proteins, was found to be the only protein in whole normal or acute phase serum which underwent specific calcium-dependent binding to either single or double-stranded DNA immobilised on gel. Isolated purified SAP also bound to long chromatin, to H1-stripped chromatin and to native DNA in solution at physiological ionic strength. Pure SAP which had been immobilised on gel, specifically bound nucleosome core particles from solution. These observations strongly suggest that SAP may bind to extracellular chromatin and DNA in vivo and that this may be its physiological role.     

Determination of amyloid P component in blood by an immunoenzyme method

An ELISA procedure was developed for measuring serum amyloid P-component (SAP). The assay is based on our finding of binding AP to polystyrene microtiter plates. The amount of SAP is determined by concurrent ELISA, wells being sequentially incubated with rabbit anti-AP antiserum and goat anti-rabbit antiserum, conjugated with peroxidase. The limit of sensitivity of the assay is 0.5 micrograms/ml. When applied to the screening of patients plasma, elevation of SAP concentration in patients with rheumatoid arthritis and amyloidosis were found.     

Influenza virus infection is not affected by serum amyloid P component

BACKGROUND: Binding of serum amyloid P component (SAP) to its ligands, including bacteria, chromatin and amyloid fibrils, protects them from degradation, is anti-opsonic and anti-immunogenic. SAP thereby enhances the virulence of pathogenic bacteria to which it binds. However SAP also contributes to host resistance against bacteria to which it does not bind. Human SAP has been reported to bind to the influenza virus and inhibit viral invasion of cells in tissue culture. We therefore investigated a possible role of SAP in either host resistance or viral virulence during influenza infection in vivo. MATERIALS AND METHODS: The clinical course of mouse adapted influenza virus infection, the host antibody response, and viral replication, were compared in wild type mice, mice with targeted deletion of the SAP gene, and mice transgenic for human SAP. The effects of reconstitution of SAP deficient mice with pure human SAP, and of a drug that specifically blocks SAP binding in vivo, were also studied. Binding of mouse and human SAP to immobilized influenza virus was compared. RESULTS: The presence, absence, or availability for binding of SAP in vivo had no significant or consistent effect on the course or outcome of influenza infection, or on either viral replication or the anti-viral antibody response. Mouse SAP bound much less avidly than human SAP to influenza virus. CONCLUSIONS: In marked contrast to the dramatic effects of SAP deficiency on host resistance to different bacterial infections, mouse SAP apparently plays no significant role during infection of mice with influenza virus. Human SAP binds much more avidly than mouse SAP to the virus, but also had no effect on any of the parameters measured and is therefore unlikely to be involved in human influenza infection.     

Acute phase induction of mouse serum amyloid P component. Correlation with other parameters of inflammation

Hepatic mRNA levels of the mouse major acute phase proteins serum amyloid P component (SAP) and serum amyloid A component (SAA) were monitored at timed intervals after i.p. injection of thioglycollate or s.c. injection of azocasein. Both mRNA increased dramatically in response to either inflammatory stimulus. The increase in SAA mRNA levels accompanied an abrupt change in mRNA size from 650 to 750 bases. Peak SAA mRNA concentrations were observed 18 h after either stimulus; by 72 h concentrations had returned to preinflammatory levels. Peak SAP mRNA concentrations were observed 8 h after thioglycollate and 12 to 18 h after azocasein injection; by 36 h concentrations were close to preinflammatory levels. All mRNA species studied (SAP, SAA and the complement components C3, C5 and factor B) were induced more rapidly by the thioglycollate stimulus and reached higher peak concentrations. SAP mRNA levels were correlated with other parameters of inflammation: infiltration of peritoneal exudate cells (PEC) into the peritoneum after thioglycollate injection, and serum concentrations of SAP after azocasein injection. Serum SAP concentrations rose 20-fold in response to the latter stimulus by 36 h, i.e., 18 to 24 h after the peak SAP mRNA levels. The highest numbers of PEC were present 24 h after the thioglycollate stimulus, i.e. 16 h after the maximum SAP mRNA concentration, indicating the continuation of an active local inflammation many hours after one aspect of the systemic response has ceased.     

Molecular chaperone properties of serum amyloid P component

The selective binding of serum amyloid P component (SAP) to proteins in the pathological amyloid cross-beta fold suggests a possible chaperone role. Here we show that human SAP enhances the refolding yield of denatured lactate dehydrogenase and protects against enzyme inactivation during agitation of dilute solutions. These effects are independent of calcium ions and are not inhibited by compounds that block the amyloid recognition site on the B face of SAP, implicating the A face and/or the edges of the SAP pentamer. We discuss the possibility that the chaperone property of SAP, or its failure, may contribute to the pathogenesis of amyloidosis.     

Calcium-mediated hemagglutination by serum amyloid P component and the inhibition by specific glycosaminoglycans

Human serum amyloid P component (SAP) was found to agglutinate erythrocytes in the presence of calcium ion. The hemagglutination was strongly inhibited by hyaluronic acid as well as by heparan sulfate and dermatan sulfate, but not by chondroitin 4-sulfate and keratan sulfate. A specific binding of SAP to hyaluronic acid, heparan sulfate, and dermatan sulfate was also confirmed by the fact that these glycosaminoglycans blocked the binding of SAP to agarose, a specific ligand of SAP.     

Structure of the mouse serum amyloid P component gene

A genomic DNA clone corresponding to the mouse serum amyloid P component (SAP) has been isolated and characterized for the first time. The numbers of exons, the relative sites of intron/exon junctions, and the size of the coding region for mature SAP protein are all in complete agreement with those of the human SAP gene. In the 5'-flanking region of the mouse SAP gene, there is a small DNA segment (43-base pairs) which is highly homologous with the corresponding region of the human SAP gene. However, most parts of the 5'-flanking regions are not conserved between the mouse and human SAP genes, and several phorbol ester-responsive element-like sequences are present only in the mouse SAP gene.     

Biosynthesis and secretion of amyloid P component in rat liver

Amyloid P component was isolated from rat liver and serum; its properties, biosynthesis, and glycosylation in the liver were investigated. The molecular weights of intracellular and serum amyloid P component were estimated to be 28,000 and 30,500, respectively. The two forms were immunologically identical, and kinetic study revealed a clear precursor-product relationship between them. The total mRNA was prepared from rat liver with or without turpentine treatment, and the RNA content of amyloid P component was estimated by the incorporation of [35S]methionine into the in vitro translation products. The turpentine treatment induced a marked increase in the level of translatable mRNA of the amyloid P component (approximately 46 fold), while the serum level of the protein elevated only moderately (approximately 1.7 fold). Most of the intracellular amyloid P component was sensitive to endo H. Various subcellular fractions were prepared from rat livers previously labeled in vivo with [35S]methionine. The protein prepared from the rough and smooth microsomes and heavy Golgi fraction were all sensitive to endo H, whereas that from the light Golgi fraction was a mixture of forms sensitive to and resistant to endo H. This result suggests that the processing of the mannosyl oligosaccharide chains and the subsequent addition of terminal sugars to convert the liver amyloid P component (28,000 daltons) to serum counterpart (30,500 daltons) were performed in the trans-Golgi region just before secretion of the amyloid P component.     

Serum amyloid P aids complement-mediated immunity to Streptococcus pneumoniae

The physiological functions of the acute phase protein serum amyloid P (SAP) component are not well defined, although they are likely to be important, as no natural state of SAP deficiency has been reported. We have investigated the role of SAP for innate immunity to the important human pathogen Streptococcus pneumoniae. Using flow cytometry assays, we show that SAP binds to S. pneumoniae, increases classical pathway-dependent deposition of complement on the bacteria, and improves the efficiency of phagocytosis. As a consequence, in mouse models of infection, mice genetically engineered to be SAP-deficient had an impaired early inflammatory response to S. pneumoniae pneumonia and were unable to control bacterial replication, leading to the rapid development of fatal infection. Complement deposition, phagocytosis, and control of S. pneumoniae pneumonia were all improved by complementation with human SAP. These results demonstrate a novel and physiologically significant role for SAP for complement-mediated immunity against an important bacterial pathogen, and provide further evidence for the importance of the classical complement pathway for innate immunity.     

The distribution of amyloid P component in normal human skin

The distribution of amyloid P component in normal human adult skin was studied using fluorescent immunohistochemical techniques on frozen sections. Amyloid P component is associated with elastic fibres of all sizes, and is present in the basement membrane of sweat gland ducts. It is not demonstrable in the basement membrane at the epidermo-dermal junction or in the secretory portion of the sweat glands. In the latter site there is however a spiral, fibrillar, elastic plexus closely related to the basement membrane.     

The distribution of amyloid P component in normal human cervix

The distribution of amyloid P component in normal human adult cervix was studied using fluorescent immunohistochemical techniques on frozen sections. Amyloid P component is associated with elastic fibres which are particularly concentrated in a sub-epithelial plexus in the ectocervix. This plexus does not extend into the endocervix but terminates at, or just caudal to, the squamocolumnar junction. Amyloid P component was not demonstrated in any of the epithelial basement membranes.