Abstract
The secondary structures of human C-reactive protein (CRP) and serum amyloid P component (SAP) in D2O-based solutions in the presence or absence of calcium, magnesium, and phosphorylcholine have been investigated using Fourier transform infrared spectroscopy. Quantitative analysis provided estimations of about 50% beta-sheet, 12% alpha-helix, 24% beta-turn, and 14% unordered structure for CRP and about 54% beta-sheet, 12% alpha-helix, 25% beta-turn, and 9% unordered structure for SAP. With both proteins significant calcium-dependent changes were observed in conformation-sensitive amide I regions assigned to each type of structure. The CRP spectrum was also affected by magnesium, but the changes differed from those induced by calcium. The SAP spectrum was not affected by magnesium. Phosphorylcholine in the presence of calcium also affected the spectrum of CRP but not the spectrum of SAP. Our present study provides the first direct comparison of the secondary structures of the pentraxins human CRP and SAP and hamster female protein (Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S., and Coe, J. E. (1992) Biochemistry 32, 9364-9370). These findings suggest that the three pentraxins have similar secondary structure compositions and calcium-dependent conformational changes, but differ significantly in their responses to phosphorylcholine and magnesium. Such properties are expected to be relevant to the incompletely understood roles of these highly conserved proteins including binding to nuclear proteins, complement activation, and association with amyloids.
Highlights
(CRP) and serum amyloid P component ( S A P ) in D,O- CRP and S A P share 50 and 72% sequence homology with hambased solutions in the presenceor absence of calcium, ster FP, respectively (7).The evolutionary conservation of penmagnesium, and phosphorylcholine have been investi- traxin genes and ubiquitous presence of pentraxins in vertegated using Fourier transform infrared spectroscopy. bratesuggest a n essential function for these proteins
CRP andFP have a primary bindingspecificity for phosphorylcholine (PC) which is absentfrom SAP
Our present High aftinity chromatin bindingactivity was observed in study provides the first direct comparison of the seScAoPn(d1-8).SAP and FP have been identified as peripheral comary structures of the pentraxins human CRPand S A P ponents of a variety of amyloids (19-21), and it appears that andhamsterfemaleprotein(Dong, A., Caughey, B., the levels of FP expression can have a profound influence on Caughey, W
Summary
Based solution.The spectra of human CRP and SAP were measured afier 24-h hydrogen-deuterium exchange at room temperature. The spectral contributions of the buffer and atmospheric water vapor have been subtracted from the original spectra as described under “Materials and Methods.”Human CRP and SAP were at concentrationsof 5 mg/ml in 20 m~ Tris, 100m~ NaCl (pD8).The spectrum of hamster FP is from Dong et al (24). The amide I’ and 11’ bands of proteins in D,O-based solution correspond to the amide I and I1 bands of proteins in HzObased solution. Isolated and purified from human pleural fluid and plasmapheresis samples by affinity chromatography, gel filtration, and ion exchange chromatographyas described previously(15).Human SAP was isolated and purified from human pleural fluid and plasmapheresis samples as Spectra Calc Software (Galactic Industries Corp.) on a 386-based personal computer.
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