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.     

Comparison between intact and desialylated human serum amyloid P component by laser photo CIDNP (chemically induced dynamic nuclear polarization) technique: an indication for a conformational impact of sialic acid

The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is generated by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption- or emission-signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these amino acid residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one Trp/His-characteristic signal is decreased by 0.1 ppm. One Trp/His-signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The Tyr signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N-glycan of each monomer apparently affects surface presentation of distinct CIDNP-reactive amino acids of SAP [1]. A conformational change of the protein part of SAP in relation with a different orientation of the desialylated oligosaccharide chain in comparison to the complete one is a possible explanation of our CIDNP results. This revised version was published online in August 2006 with corrections to the Cover Date.     

Brain Serum Amyloid P Levels are Reduced in Individuals that Lack Dementia While Having Alzheimer’s Disease Neuropathology

The neuropathological signs of Alzheimer’s disease (AD) include beta amyloid plaques and neurofibrillary tangles. There is a significant population of individuals that have these key hallmarks but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). The protective mechanism allowing these individuals to escape dementia is unknown. Serum amyloid P (SAP) is a serum protein associated with wound repair that is elevated in the brains of Alzheimer’s patients and binds to amyloid plaques. Using immunoblotting and immunohistochemistry, we evaluated SAP levels in postmortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels compared to normal controls, while NDAN samples had no significant difference in SAP levels compared to normal controls. Our results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques and tangles.     

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.     

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.     

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.     

Pathogenesis, diagnosis and treatment of systemic amyloidosis

Amyloidosis is a disorder of protein folding in which normally soluble proteins are deposited as abnormal, insoluble fibrils that disrupt tissue structure and cause disease. Although about 20 different unrelated proteins can form amyloid fibrils in vivo, all such fibrils share a common cross-beta core structure. Some natural wild-type proteins are inherently amyloidogenic, form fibrils and cause amyloidosis in old age or if present for long periods at abnormally high concentration. Other amyloidogenic proteins are acquired or inherited variants, containing amino-acid substitutions that render them unstable so that they populate partly unfolded states under physiological conditions, and these intermediates then aggregate in the stable amyloid fold. In addition to the fibrils, amyloid deposits always contain the non-fibrillar pentraxin plasma protein, serum amyloid P component (SAP), because it undergoes specific calcium-dependent binding to amyloid fibrils. SAP contributes to amyloidogenesis, probably by stabilizing amyloid fibrils and retarding their clearance. Radiolabelled SAP is an extremely useful, safe, specific, non-invasive, quantitative tracer for scintigraphic imaging of systemic amyloid deposits. Its use has demonstrated that elimination of the supply of amyloid fibril precursor proteins leads to regression of amyloid deposits with clinical benefit. Current treatment of amyloidosis comprises careful maintenance of impaired organ function, replacement of end-stage organ failure by dialysis or transplantation, and vigorous efforts to control underlying conditions responsible for production of fibril precursors. New approaches under development include drugs for stabilization of the native fold of precursor proteins, inhibition of fibrillogenesis, reversion of the amyloid to the native fold, and dissociation of SAP to accelerate amyloid fibril clearance in vivo.     

Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer''s disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer''s disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.     

Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications

Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in atherosclerotic lesions. We investigated the extent of the colocalization of SAP with apolipoprotein A-I (apoA-I), apoB, apoC-II, and apoE in human coronary arteries and explored potential roles for SAP in these regions, specifically the effect of SAP on the rate of formation and macrophage recognition of amyloid fibrils composed of apoC-II. Analysis of 42 human arterial sections by immunohistochemistry and double label fluorescence microscopy demonstrated that SAP and apoA-I, apoB, apoC-II, and apoE were increased significantly in atherosclerotic lesions compared with nonatherosclerotic segments. SAP colocalized with all four apolipoproteins to a similar extent, whereas plaque macrophages were found to correlate most strongly with apoC-II and apoB. In vitro studies showed that SAP accelerated the formation of amyloid fibrils by purified apoC-II. Furthermore, SAP strongly inhibited the phagocytosis of apoC-II amyloid fibrils by primary macrophages and macrophage cell lines and blocked the resultant production of reactive oxygen species. The ability of SAP to accelerate apoC-II amyloid fibril formation and inhibit macrophage recognition of apoC-II fibrils suggests that SAP may modulate the inflammatory response to amyloid fibrils in atherosclerosis.     

Oxidation of low-density lipoproteins induces amyloid-like structures that are recognized by macrophages

The macrophage scavenger receptor CD36 plays a key role in the initiation of atherosclerosis through its ability to bind to and internalize oxidized low-density lipoproteins (oxLDL). Prompted by recent findings that the CD36 receptor also recognizes amyloid fibrils formed by beta-amyloid and apolipoprotein C-II, we investigated whether the oxidation of low-density lipoproteins (LDL) generates characteristic amyloid-like structures and whether these structures serve as CD36 ligands. Our studies demonstrate that LDL oxidized by copper ions, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), or ozone react with the diagnostic amyloid dyes thioflavin T and Congo Red and bind to serum amyloid P component (SAP), a universal constituent of physiological amyloid deposits. X-ray powder diffraction patterns for native LDL show a diffuse powder diffraction ring with maximum intensity corresponding to an atomic spacing of approximately 4.7 A, consistent with the spacing between beta-strands in a beta-sheet. Ozone treatment of LDL generates an additional diffuse powder diffraction ring with maximum intensity indicating a spacing of approximately 9.8 A. This distance is consistent with the presence of cross-beta-structure, a defining characteristic of amyloid. Evidence that these cross-beta-amyloid structures in oxLDL are recognized by macrophages is provided by the observation that SAP strongly inhibits the association and internalization of (125)I-labeled copper-oxidized LDL by peritoneal macrophages. The ability of SAP to bind to amyloid-like structures in oxLDL and prevent lipid uptake by macrophages highlights the potential importance of these structures and suggests an important preventative role for SAP in foam cell formation and early-stage atherosclerosis.     

Analysis of human blood serum using the off-line coupling of capillary isoelectric focusing to matrix-assisted laser desorption/ionization time of flight mass spectrometry

Off-line coupling of capillary IEF (CIEF) with matrix-assisted laser desorption/ionization mass spectrometry was utilized for the analysis of human blood serum. Serum proteins were initially separated by CIEF, and fractions of the isoelectric separation were eluted sequentially to a MALDI-TOF MS sample target. During pressure elution of the CIEF sample, voltage was maintained across the capillary system utilizing a sheath flow arrangement to minimize band broadening induced by the laminar flow field. Both pI and mass information were obtained from the complex biological sample, similar to traditional 2-DE techniques, and the platform was faster (hours versus days), more automatable, and simpler than 2-DE. The volume of raw sample present in the actual analysis was approximately 100 nL, making this technique well suited for very rare specimens. Additionally, the speed and simplicity of the technology make it an attractive technique for performing initial comparative analyses of complex samples.     

Goodpasture antigen-binding protein/ceramide transporter binds to human serum amyloid P-component and is present in brain amyloid plaques

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease.     

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.     

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.     

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.     

Congo Red Inhibits Proteoglycan and Serum Amyloid P Binding to Amyloid β Fibrils

Abstract: Various data suggest that Alzheimer's disease results from the accumulation of amyloid β (Aβ) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to Aβ fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar Aβ might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to Aβ is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of Aβ. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to Aβ fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar Aβ from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.     

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.     

Regulation of the antibody response by the acute phase reactant: mouse serum amyloid P-component (SAP)

Serum amyloid P-component (SAP) is the major acute phase reactant (APR) of mice. Purified mouse SAP at 0.1 to 10.0 micrograms/ml selectively suppressed the secondary in vitro IgG antibody plaque-forming cell (PFC) response to the T-dependent antigen TNP-KLH but not to the T-independent antigens TNP-LPS and DNP-Lys-Ficoll. The suppression was antigen nonspecific. The mechanism of suppression occurred primarily through the activation of Lyt-1+, I-J+ suppressor-inducer cells, which in turn activated a Lyt-2+ suppressor T-cell population. The activity of preexisting, antigen-specific Lyt-2+ suppressor T cells was not influenced by SAP. The antigen-nonspecific suppressor T cells generated by SAP were sensitive to cyclophosphamide. Removal of SAP from the culture fluid with rabbit anti-Mo SAP antibody or agarose beads abrogated the suppression. Pentraxin proteins closely related to mouse SAP, such as human SAP and hamster female protein (FP), also displayed immunoregulatory activity of the antibody response by the same cellular mechanism. The results suggest that SAP regulates antibody responses by the activation of suppressor-inducer T cells and that the regulation of the antibody response during the acute stage of inflammation may occur via SAP.     

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.     

Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization

In this work, a novel CIEF methodology for the analysis of the glycated hemoglobin, HbA(1c), in dimethylpolysiloxane coated fused-silica capillaries (DB-1, 50 microm I.D., 27 cm, 0.20 microm coating thickness), using a narrow pH ampholyte mixture (4% pH 6-8:pH 3-10, 10:1, v/v) in 0.30% methylcellulose, was developed. In the focusing procedure, a 0.100-mol l(-1) phosphoric acid solution was used as anolyte and a 0.040-mol l(-1) NaOH solution was used as catholyte. During method development, two types of mobilization of the focused hemoglobins were tested: pressure and chemical mobilization. Chemical mobilization performed better, allowing the complete baseline resolution of the hemoglobin of interest, HbA(1c), from its adjacent peak, HbA, in less than 8 min. In the chemical mobilization procedure, the catholyte was replaced by a 0.040-mol l(-1) NaOH solution containing 0.080 mol l(-1) NaCl. The proposed methodology was applied to the analysis of 31 hemolysate samples and validated with respect to the selectivity, inter-assay and intra-assay precision (both migration time and hemoglobin percentage concentration). In addition, HbA(1c) determinations were compared for the CIEF method and a chromatographic standardized procedure using cation-exchanger columns (Variant, Bio-Rad), adopted in a local clinical laboratory, showing excellent correlation (r(2)=0.872, n=31). The slope was found to be statistically equal to one but the intercept differed from zero. Also the Bland-Altman plot indicates bias, implying that the CIEF method yields HbA(1c) concentration higher than the reference method. The separation of the hemoglobins HbA, HbA(2), HbF and HbA(1c) and the variants HbS and HbC was also demonstrated (8 min run). The resolving power of the proposed CIEF method allowed baseline resolution of hemoglobins with a pI difference as small as ca. 0.03, as it is the case for the pairs HbC/HbA(2) and HbA/HbA(1c).