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.     

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.     

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.     

The primary structure of human tissue amyloid P component from a patient with primary idiopathic amyloidosis

The amino acid sequence of human tissue amyloid P component (AP) extracted by a modified method from the spleen of a patient with primary idiopathic amyloidosis was determined. AP is a glycoprotein composed of a pair of noncovalently bound pentameric discs with a subunit size of 23-25 kDa. Each subunit consists of 204 residues, a single disulfide bridge linking Cys 36 to Cys 95, and a carbohydrate moiety attached to Asn 32. The precursor of AP is the serum amyloid protein (SAP). The primary structure of AP presented here differs from the amino acid sequence of SAP previously reported, but is identical to the amino acid sequence of mature SAP deduced from the nucleotide sequence of complementary DNA clones. It shares 52% homology with the amended sequence of human C-reactive protein, an acute phase protein, and 68% homology with the Syrian hamster "female protein," another acute phase protein whose response is modulated by sex steroids. AP/SAP, C-reactive protein, and female protein belong to a family of plasma proteins called pentraxins and their considerable sequence homology is probably the result of gene duplication. Neither the physiological function of AP nor its possible pathological role in amyloidosis are yet known.     

Liver-specific and high-level expression of human serum amyloid P component gene in transgenic mice

To analyze the regulation of human serum amyloid P component (SAP) gene expression, we have produced seven transgenic mice. The 3.3 kb human SAP genes containing about 0.8 kb of 5' and 1.5 kb of 3' flanking region were injected into fertilized eggs of C57BL/6 mice. In five of the seven transgenic mice, human SAP was detected in the sera and serum concentrations were higher than that of human serum in three lines. The human SAP gene was expressed only in the liver. Amounts of human mRNA in the liver and serum concentrations of human SAP were roughly proportional to the copy number of the integrated gene. Human SAP production lowered the serum levels of mouse endogenous SAP. With the intraperitoneal administration of lipopolysaccharide, the mRNA levels in the liver and serum levels of mouse SAP increased several-fold in both the control and transgenic mice. On the other hand, neither the mRNA nor the serum levels of human SAP increased significantly.     

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.     

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.     

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.     

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.     

Armenian hamster female protein (serum amyloid P component). Comparison with the sex-regulated homolog in Syrian hamster

Complementary DNA clones for Armenian hamster female protein (FP) were isolated and the complete nucleotide sequence and derived amino acid sequence were determined and compared with relevant data for the closely related Syrian hamster. Although biosynthesis of preSAP is directed by a 1.0-kb mRNA in both genera and the molecular mass of the primary translation product of FP is identical, the FP gene structure and regulation of expression of FP are different in Syrian and Armenian hamsters. Whereas the direction of alteration in FP mRNA levels is divergent in Syrian hamsters during an acute phase reaction, hepatic FP mRNA levels increase in both male and female Armenian hamsters during inflammation. Regulation of expression of Armenian and Syrian hamster FP genes occurs at a pretranslational level.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.     

Effect of hamster pregnancy on female protein, a homolog of serum amyloid P component.

Pentraxins such as human serum amyloid P component (SAP) and C reactive protein (CRP) represent an ancient family of proteins that are ubiquitous in nature and have evolved with little change in structure or regulation. The pentraxin in the Syrian hamster (Mesocricetus auratus) is unique because it is preferentially expressed in the female at high constitutive levels and accordingly called female protein (FP) or FP(SAP) due to its close homology with human SAP. The high levels of FP in female serum (100-fold greater than male serum) suggested its role in hamster pregnancy, one of the shortest of any eutherian mammal. We determined the serum FP concentration in pregnant Syrian hamsters and found a marked decrease (>80%) at term with the nadir at parturition with subsequent increase. A similar downregulation of FP was found in the normal female Syrian hamster after injury (acute phase response), so in both cases the assumed beneficial effects were achieved with less, rather than more pentraxin, a paradoxical pentraxin response. The fall in serum FP concentration could represent a response to protect the fetus from the high and potentially toxic level of FP normally found in the female, that is harmful because of its association with amyloidosis. An FP that is 97.5% identical to Syrian hamster FP is found in the Turkish hamster (Mesocricetus brandti), although serum levels in females are much lower, and amyloid is very rare. During pregnancy/parturition of Turkish hamsters, the serum level of FP remained remarkably constant. In a more distantly related hamster, the Armenian hamster (Cricetulus migratorius), serum FP actually increased during pregnancy and at parturition in a manner similar to that found in the Armenian hamster during an acute phase response. The heterogeneity of FP kinetics during pregnancy in these three species of hamster indicates pleomorphic gene structure for regulation of their similar FPs, and suggests that this protein may have a different function in the pregnancy of each species.     

Proteomic characterization of novel serum amyloid P component variants from human plasma and urine

Serum amyloid P component (SAP) is a human plasma protein that has been widely studied for its influence on amyloid plaque formation and stabilization. SAP was characterized directly from human plasma and urine samples via novel affinity mass spectrometry-based proteomic technology that is able to readily discriminate between mass-altered protein variants. These analyses were able to identify several variants of SAP that have not been previously reported. These variants include microheterogeneity of the glycan structure, from the loss of one or both terminal sialic acid residues, as well as the loss of the C-terminal valine residue. Moreover, the analysis of urine allowed for the consistent identification of serum amyloid P component as a normal constituent of the urine proteome.     

Heparin influence on the complex of serum amyloid P component and complement C4b-binding protein

Serum amyloid P component (SAP) forms a calcium-dependent complex with C4b-binding protein (C4BP) in human serum. this study demonstrated that heparin interacted with SAP in a calcium-dependent manner and prevented formation of the SAP.C4BP complex. Furthermore, the SAP-heparin interaction interfered with SAP binding to membranes. Therefore, all three of these interactions involved similar sites on SAP, or each interaction sterically obstructed the other binding sites. In addition to heparin, SAP bound to heparan sulfate and chondroitin sulfate. In each case, a distinct multimeric species was generated. Gel filtration and sucrose density gradient ultracentrifugation suggested that heparin and heparan sulfate produced a dimer of SAP. The dimer appeared to be the most stable structure since it was not dissociated by excess heparin. While low molecular weight heparin interacted with SAP and inhibited SAP association with membranes, the SAP dimer was not detected in sucrose density gradient ultracentrifugation studies. Polybrene prevented the interaction between SAP and heparin in both a purified system and in human serum that was enriched in SAP and heparin. In contrast, Polybrene did not seem to alter the SAP.C4BP complex. While the function of the SAP.C4BP complex is unknown, it may be important for regulation of complement and/or transport of SAP to sites in the body. Dissociation of the SAP.C4BP complex by sulfated polysaccharides such as heparin may be a physiological response that could be important during tissue damage or complement activation.     

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.     

Ligand-assisted aggregation of proteins. Dimerization of serum amyloid P component by bivalent ligands

A comprehensive series of solution and crystallographic studies reveal how simple, achiral, bivalent ligands of the cyclic pyruvate of glycerol promote face-to-face complex formation of the pentraxin, serum amyloid P component (SAP) into decamers. SAP, a protein of the human innate immune system, is universally present in amyloids, including cerebral amyloid deposits found in the brain of Alzheimer disease patients. Removal of SAP through a specific aggregation mechanism mediated by multivalent ligands appears to provide therapeutic benefit in the progression of this disease. Crystallographic studies reveal that in our novel series of ligands only the methyl and carboxylate moieties of the pyruvate ketal directly interact with the protein, but the geometric constraints imposed by the tether dictate which of two chair conformations are adopted by the pyruvate dioxane ring. Solution studies, as interpreted through a simple thermodynamic model, account for the distribution of pentameric and decameric bound states at different ligand concentrations and indicate that differences in the flexibility of the tether determine the geometry and stability of the specific aggregates formed between SAP and two different bivalent ligands. The factors affecting the design of ligands promoting face-to-face protein dimerization as well as potential biological implications are discussed.     

New insights into the role of serum amyloid P component, a novel lipopolysaccharide-binding protein

Serum amyloid P component (SAP) is a highly preserved plasma protein named for its ubiquitous presence in amyloid deposits. Although SAP is described to bind many ligands, no clear biological function has been ascribed to it as yet. This review summarizes the current knowledge about the protein SAP, its ligands and functional properties. Finally, the author focuses on the recent finding of the binding of SAP to lipopolysaccharide (LPS) and Gram-negative bacteria and the possible functional consequences of these interactions.     

Chromatin-independent binding of serum amyloid P component to apoptotic cells.

Human serum amyloid P component (SAP) is a glycoprotein structurally belonging to the pentraxin family of proteins, which has a characteristic pentameric organization. Mice with a targeted deletion of the SAP gene develop antinuclear Abs, which was interpreted as evidence for a role of SAP in controlling the degradation of chromatin. However, in vitro SAP also can bind to phosphatidylethanolamine, a phospholipid which in normal cells is located mainly in the inner leaflet of the cell membrane, to be translocated to the outer leaflet of the cell membrane during a membrane flip-flop. We hypothesized that SAP, because of its specificity for phosphatidylethanolamine, may bind to apoptotic cells independent of its nuclear binding. Calcium-dependent binding of SAP to early, nonpermeable apoptotic Jurkat, SKW, and Raji cells was indeed observed. Experiments with flip-flopped erythrocytes confirmed that SAP bound to early apoptotic cells via exposed phosphatidylethanolamine. Binding of SAP was stronger to late, permeable apoptotic cells. Experiments with enucleated neutrophils, with DNase/RNase treatment of late apoptotic Jurkat cells, and competition experiments with histones suggested that binding of SAP to late apoptotic cells was largely independent of chromatin. Confocal laser microscopic studies indeed suggested that SAP bound to these apoptotic cells mainly via the blebs. Thus, this study shows that SAP binds to apoptotic cells already at an early stage, which raises the possibility that SAP is involved in dealing with apoptotic cells in vivo.     

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