Abstract
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.
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References
Pepys MB, Baltz ML. (1983) Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv. Immunol. 34: 141–212.
Pepys MB, Dash AC, Fletcher TC, Richardson N, Munn EA, Feinstein A. (1978) Analogues in other mammals and in fish of human plasma proteins C-reactive protein and amyloid P component. Nature 273: 168–170.
Baltz ML, de Beer FC, Feinstein A, et al. (1982) Phylogenetic aspects of C-reactive protein and related proteins. Ann. N.Y. Acad. Sci. 389: 49–75.
Woo P, Korenberg JR, Whitehead AS. (1985) Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component. J. Biol. Chem. 260: 13384–13388.
Mantzouranis EC, Dowton SB, Whitehead AS, Edge MD, Bruns GAP, Colten HR. (1985) Human serum amyloid P component. cDNA isolation, complete sequence of pre-serum amyloid P component, and localization of the gene to chromosome 1. J. Biol. Chem. 260: 7752–7756.
Emsley J, White HE, O’Hara BP, et al. (1994) Structure of pentameric human serum amyloid P component. Nature 367: 338–345.
Srinivasan N, White HE, Emsley J, Wood SP, Pepys MB, Blundell TL. (1994) Comparative analyses of pentraxins: implications for protomer assembly and ligand binding. Structure 2: 1017–1027.
Shrive AK, Cheetham GMT, Holden D, et al. (1996) Three-dimensional structure of human C-reactive protein. Nature Struct. Biology 3: 346–354.
Srinivasan N, Rufino SD, Pepys MB, Wood SP, Blundell TL. (1996) A superfamily of proteins with the lectin fold. Chemtracts-Biochem. Mol. Biol. 6: 149–164.
Thompson D, Pepys MB, Wood SP. (1999) The physiological structure of human C-reactive protein and its complex with phosphocholine. Structure 7: 169–177.
Pinteric L, Assimeh SN, Kells DIC, Painter RH. (1976) The ultrastructure of Clt, a subcomponent of the first component of complement: an EM and ultracentrifuge study. J. Immunol. 117: 79–83.
Pepys MB, Dash AC, Munn EA, et al. (1977) Isolation of amyloid P component (protein AP) from normal serum as a calcium-dependent binding protein. Lancet i: 1029–1031.
Pinteric L, Painter RH. (1979) Electron microscopy of serum amyloid protein in the presence of calcium: alternative forms of assembly of pentagonal molecules in two-dimensional lattices. Can. J. Biochem. 57: 727–736.
Perkins SJ, Pepys MB. (1986) X-ray and neutron scattering studies on CRP and SAP. In: Peeters J (ed). Protides of the Biological Fluids, Vol. 34. Pergamon Press, Oxford, pp. 323–326.
Ashton AW, Boehm MK, Gallimore JR, Pepys MB, Perkins SJ. (1997) Pentameric and decameric structures in solution of serum amyloid P component by X-ray and neutron scattering and molecular modelling analyses. J. Mol. Biol. 272: 408–422.
Sørensen IJ, Andersen O, Holm Nielsen E, Svehag S-E. (1995) Native human serum amyloid P component is a single pentamer. Scand. J. Immunol. 41: 263–267.
Pepys MB. (1994) Amyloidosis. In: Frank MM, Austen KF, Claman HN, Unanue ER (eds). Samter’s Immunologic Diseases, Fifth Ed. Little, Brown and Company, Boston, pp. 637–655.
Pepys MB, Booth DR, Hutchinson WL, Gallimore JR, Collins PM, Hohenester E. (1997) Amyloid P component. A critical review. Amyloid: Int. J. Exp. Clin. Invest. 4: 274–295.
Tennent GA, Lovat LB, Pepys MB. (1995) Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer’s disease and systemic amyloidosis. Proc. Natl. Acad. Sci. USA 92: 4299–4303.
Botto M, Hawkins PN, Bickerstaff MCM, et al. (1997) Amyloid deposition is delayed in mice with targeted deletion of the serum amyloid P component gene. Nature Med. 3: 855–859.
Pepys MB, Butler PJG. (1987) Serum amyloid P component is the major calcium-dependent specific DNA binding protein of the serum. Biochem. Biophys. Res. Commun. 148: 308–313.
Breathnach SM, Kofler H, Sepp N, et al. (1989) Serum amyloid P component binds to cell nuclei in vitro and to in vivo deposits of extracellular chromatin in systemic lupus erythematosus. J. Exp. Med. 170: 1433–1438.
Butler PJG, Tennent GA, Pepys MB. (1990) Pentraxin-chromatin interactions. Serum amyloid P component specifically displaces H1-type histones and solubilizes native long chromatin. J. Exp. Med. 172: 13–18.
Hintner H, Booker J, Ashworth J, Auböck J, Pepys MB, Breathnach SM. (1988) Amyloid P component binds to keratin bodies in human skin and to isolated keratin filament aggregates in vitro. J. Invest. Dermatol. 91: 22–28.
Hind CRK, Collins PM, Baltz ML, Pepys MB. (1985) Human serum amyloid P component, a circulating lectin with specificity for the cyclic 4,6-pyruvate acetal of galactose. Biochem. J. 225: 107–111.
Pepys MB. (1999) The Lumleian Lecture. C-reactive protein and amyloidosis: from proteins to drugs? In: Williams G (ed). Horizons in Medicine, Vol. 10. Royal College of Physicians, London, pp. 397–414.
Bickerstaff MCM, Botto M, Hutchinson WL, et al. (1999) Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity. Nature Med. 5: 694–697.
Baltz ML, de Beer FC, Feinstein A, Pepys MB. (1982) Calcium-dependent aggregation of human serum amyloid P component. Biochim. Biophys. Acta 701: 229–236.
de Beer FC, Baltz M, Holford S, Feinstein A, Pepys MB. (1981) Fibronectin and C4-binding protein are selectively bound by aggregated amyloid P component. J. Exp. Med. 154: 1134–1149.
Schwalbe RA, Dahlbäck B, Nelsestuen GL. (1990) Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes. J. Biol. Chem. 265: 21749–21757.
Schwalbe RA, Dahlbäck B, Nelsestuen GL. (1991) Heparin influence on the complex of serum amyloid P component and complement C4b-binding protein. J. Biol. Chem. 266: 12896–12901.
de Frutos PG, Dahlbäck B. (1994) Interaction between serum amyloid P component and C4b-binding protein associated with inhibition of factor I-mediated C4b degradation. J. Immunol. 152: 2430–2437.
de Frutos PG, Härdig Y, Dahlbäck B. (1995) Serum amyloid P component binding to C4b-binding protein. J. Biol. Chem. 270: 26950–26955.
Hawkins PN, Tennent GA, Woo P, Pepys MB. (1991) Studies in vivo and in vitro of serum amyloid P component in normals and in a patient with AA amyloidosis. Clin. Exp. Immunol. 84: 308–316.
de Beer FC, Pepys MB. (1982) Isolation of human C-reactive protein and serum amyloid P component. J. Immunol. Methods 50: 17–31.
Pepys MB, Dash AC, Fielder AHL, Mirjah DD. (1977) Isolation and study of murine C3. Immunology 33: 491–499.
Reay P. (1982) Use of N-bromosuccinimide for the iodination of proteins for radioimmunoassay. Ann. Clin. Biochem. 19: 129–133.
Hawkins PN, Wootton R, Pepys MB. (1990) Metabolic studies of radioiodinated serum amyloid P component in normal subjects and patients with systemic amyloidosis. J. Clin. Invest. 86: 1862–1869.
Hind CRK, Collins PM, Renn D, et al. (1984) Binding specificity of serum amyloid P component for the pyruvate acetal of galactose. J. Exp. Med. 159: 1058–1069.
Pepys MB, Dash AC, Markham RE, Thomas HC, Williams BD, Petrie A. (1978) Comparative clinical study of protein SAP (amyloid P component) and C-reactive protein in serum. Clin. Exp. Immunol. 32: 119–124.
Nelson SR, Tennent GA, Sethi D, et al. (1991) Serum amyloid P component in chronic renal failure and dialysis. Clin. Chim. Acta 200: 191–200.
Hohenester E, Hutchinson WL, Pepys MB, Wood SP. (1997) Crystal structure of a decameric complex of human serum amyloid P component with bound dAMP. J. Mol. Biol. 269: 570–578.
Pepys MB, Rademacher TW, Amatayakul-Chantler S, et al. (1994) Human serum amyloid P component is an invariant constituent of amyloid deposits and has a uniquely homogeneous glycostructure. Proc. Natl. Acad. Sci. USA 91: 5602–5606.
Vigushin DM, Pepys MB, Hawkins PN. (1993) Metabolic and scintigraphic studies of radioiodinated human C-reactive protein in health and disease. J. Clin. Invest. 91: 1351–1357.
Pepys MB. (1979) Isolation of serum amyloid P component (protein SAP) in the mouse. Immunology 37: 637–641.
Osmand AP, Friedenson B, Gewurz H, Painter RH, Hofmann T, Shelton E. (1977) Characterisation of C-reactive protein and the complement subcomponent Clt as homologous proteins displaying cyclic pentameric symmetry (pentraxins). Proc. Natl. Acad. Sci. USA 74: 739–743.
Roux KH, Kilpatrick JM, Volanakis JE, Kearney JF. (1983) Localization of the phosphocholine-binding sites on C-reactive protein by immuno-electron microscopy. J. Immunol. 131: 2411–2415.
Wood SP, Oliva G, O’Hara BP, et al. (1988) A pentameric form of human serum amyloid P component: crystallization, X-ray diffraction and neutron scattering studies. J. Mol. Biol. 202: 169–173.
Hind CRK, Collins PM, Pepys MB. (1984) Calcium-dependent aggregation of human serum amyloid P component. Inhibition by the cyclic 4,6-pyruvate acetal of galactose. Biochim. Biophys. Acta 802: 148–150.
Booth DR, Gallimore JR, Hutchinson WL, et al. (1999) Analysis of autoaggregation and ligand binding sites of serum amyloid P component by in vitro mutagenesis. In: Kyle RA, Gertz MA (eds). Amyloid and Amyloidosis 1998. Parthenon Publishing, Pearl River, New York, pp. 23–25.
Acknowledgments
This work was supported by Medical Research Council (U.K.) Programme Grant G97900510 to M. B. Pepys and Human Frontier Science Program Fellowship to E. Hohenester.
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Hutchinson, W.L., Hohenester, E. & Pepys, M.B. Human Serum Amyloid P Component is a Single Uncomplexed Pentamer in Whole Serum. Mol Med 6, 482–493 (2000). https://doi.org/10.1007/BF03401789
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DOI: https://doi.org/10.1007/BF03401789