Abstract
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. 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. 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. 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.
Highlights
Serum amyloid P component (SAP) is a normal plasma glycoprotein which, together with the classical acute-phase protein, C-reactive protein (CRP), forms the pentraxin family of calciumdependent ligand binding plasma proteins [1]
Fractionation of whole serum on conventional Sephacryl® S-300 and S-300HR columns (Table 2, Fig. 2) showed the same clear separation of pentraxins seen in Fast protein liquid chromatography (FPLC) on Superose 12HR, with SAP eluting well ahead of CRP
Intact native CRP is known from previous gel filtration, ultracentrifuge, X-ray and neutron solution scattering studies to be a pentamer [14,35,45], and its crystal structure confirms the electron microscopic appearance of cyclic pentameric arrangement of the protomers [2,8,10,46,47]
Summary
Serum amyloid P component (SAP) is a normal plasma glycoprotein which, together with the classical acute-phase protein, C-reactive protein (CRP), forms the pentraxin family of calciumdependent ligand binding plasma proteins [1]. These molecules are phylogenetically conserved, 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. Conclusions: Human SAP exists within serum as single uncomplexed pentamers in the presence or absence of calcium This oligomeric assembly, does not require a calcium-dependent small molecule interaction. 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
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