Cryo-Electron Microscopy and Biochemical Analysis Offer Insights Into the Effects of Acidic pH, Such as Occur During Acidosis, on the Complement Binding Properties of C-Reactive Protein.

The pentraxin family of proteins includes C-reactive protein (CRP), a canonical marker for the acute phase inflammatory response. As compared to normal physiological conditions in human serum, under conditions associated with damage and inflammation, such as acidosis and the oxidative burst, CRP exhibits modulated biochemical properties that may have a structural basis. Here, we explore how pH and ligand binding affect the structure and biochemical properties of CRP. Cryo-electron microscopy was used to solve structures of CRP at pH 7.5 or pH 5 and in the presence or absence of the ligand phosphocholine (PCh), which yielded 7 new high-resolution structures of CRP, including pentameric and decameric complexes. Structures previously derived from crystallography were imperfect pentagons, as shown by the variable angles between each subunit, whereas pentameric CRP derived from cryoEM was found to have C5 symmetry, with subunits forming a regular pentagon with equal angles. This discrepancy indicates flexibility at the interfaces of monomers that may relate to activation of the complement system by the C1 complex. CRP also appears to readily decamerise in solution into dimers of pentamers, which obscures the postulated binding sites for C1. Subtle structural rearrangements were observed between the conditions tested, including a putative change in histidine protonation that may prime the disulphide bridges for reduction and enhanced ability to activate the immune system. Enzyme-linked immunosorbent assays showed that CRP had markedly increased association to the C1 complex and immunoglobulins under conditions associated with acidosis, whilst a reduction in the Ca2+ concentration lowered this pH-sensitivity for C1q, but not immunoglobulins, suggesting different modes of binding. These data suggest a model whereby a change in the ionic nature of CRP and immunological proteins can make it more adhesive to potential ligands without large structural rearrangements.

Intra-subunit Disulfide Determines the Conversion and Structural Stability of CRP Isoforms.

C-reactive protein (CRP) is a major human acute-phase reactant that is composed of five identical subunits. CRP dissociates into subunits at inflammatory loci forming monomeric CRP (mCRP) with substantially enhanced activities, which can be further activated by reducing the intra-subunit disulfide bond. However, conformational changes underlying the activation process of CRP are less well understood. Conformational changes accompanying the conversion of CRP to mCRP with or without reduction were examined with circular dichroism spectroscopy, fluorescence spectroscopy, electron microscopy, size-exclusion chromatography, and neoepitope expression. The conversion of CRP to mCRP follows a two-stage process. In the first stage, CRP dissociates into molten globular subunits characterized by intact secondary structure elements with greatly impaired tertiary packing. In the second stage, these intermediates completely lose their native subunit conformation and assemble into high-order aggregates. The inclusion of reductant accelerates the formation of molten globular subunits in the first step and promotes the formation of more compact aggregates in the second stage. We further show a significant contribution of electrostatic interactions to the stabilization of native CRP. The conformational features of dissociated subunits and the aggregation of mCRP may have a key impact on their activities.

Caracterización de los pacientes con diagnóstico precoz de aneurisma de la aorta abdominal / Characterization of patients with early diagnosis of abdominal aortic aneurysm

Introducción: la incorporación de la ecografía a los métodos diagnósticos de los aneurismas aórticos abdominales favorece su detección precoz. Objetivo: Describir las características de los pacientes a quienes se les detectó precozmente un aneurisma aórtico abdominal. Métodos: Estudio descriptivo de corte transversal en 243 pacientes pertenecientes al área de salud del Hospital Clinicoquirúrgico "General Freyre de Andrade", clasificados como población de riesgo (hombres > 50 años y mujeres > 60 años). El periodo de estudio: noviembre de 2016 a octubre de 2017. Resultados: Se realizó el diagnóstico de aneurisma aórtico abdominal en 2,1 por ciento (n= 5); en estos pacientes prevaleció el grupo de edad de 70 a79 años (80 por ciento) y el sexo masculino (60 por ciento). Los factores de riesgo predominantes fueron el tabaquismo, la hipertensión arterial y la enfermedad arterial periférica, todos con igual porcentaje (100 por ciento). El segmento aórtico más afectado fue el infrarrenal (100 por ciento), los diámetros aórticos predominantes fueron: transversal (3-3,9 cm), longitudinal (5-10 cm) y antero-posterior (3-3,9 cm). La proteína C reactiva estuvo incrementada (mayor de 5 mg/L) en el 100 por ciento de los casos. La claudicación intermitente fue la más frecuente. Conclusiones: A pesar de la baja prevalencia porcentual encontrada, destaca en todos los pacientes con aneurisma aórtico abdominal la presencia de factores de riesgo relevantes como es el tabaquismo, la hipertensión arterial y la enfermedad arterial periférica; incremento en la concentración de la proteína C reactiva, afectación en el segmento infrarrenal así como alto porcentaje con edades por encima de 70 años(AU)

Introduction: The incidence of abdominal aortic aneurysms in the population benefits from the incorporation of ultrasound to diagnostic methods due to the importance of their early detection. Objective: To describe the characteristics of patients who are early detected with an abdominal aortic aneurysm. Method: A descriptive, cross-sectional study was conducted in 243 patients belonging to the health area of "General Freyre de Andrade" Clinical- Surgical Hospital that were classified as a risk population (men > 50 years and women > 60 years). The study was conducted from November 2016 to October 2017. Results: The diagnosis of abdominal aortic aneurysm was performed in 2.1 percent (n= 5); in these patients predominated the age group of 70 to 79 years (80 percent), male sex (60 percent) and white skin color (80 percent). The predominant risk factors were smoking habit, arterial hypertension and peripheral arterial disease; all with equal percentage (100 percent). The most affected aortic segment was the infrarenal (100 percent), the predominant aortic diameters were: transversal (3-3.9 cm), longitudinal (5-10 cm) and anterior-posterior (3-3.9 cm). The C-reactive protein increased (greater than 5 mg/L) in 100 percent of the cases. Intermittent claudication was the most frequent. Conclusions: Although the percentage prevalence of the disease was low, it was present in all the patients with abdominal aortic aneurysm, risk factors that are favorable to aneurysms, high concentrations of C-reactive protein, affectation in the infrarenal aortic segment, and high percentage of ages of more than 70 years(AU)

Infection surveillance in transsphenoidal pituitary surgery - comparison of lipopolysaccharide-binding-protein, interleukin 6, C-reactive protein, white blood cell count, erythrocyte sedimentation rate and body temperature.

BACKGROUND: One of the major concerns in transsphenoidal surgery are infections because the approach to the pituitary includes a route of microbial colonization. To minimize the associated morbidity and mortality, a surveillance program is crucial to monitor for perioperative infections. METHODS: For 1 year, we analysed body temperature (BT), erythrocyte sedimentation rate (ESR), white blood cell count (WBC), C-reactive protein (CRP), interleukin 6 (IL-6) and lipopolysaccharide-binding-protein (LBP) following elective transsphenoidal pituitary surgery. Samples were collected on admission, day 1, 3 and 7 as well as 3 months postoperatively. RESULTS: In 116 patients, all data were available. No postoperative infections occurred within the first postoperative week. BT (37.6 ± 0.6, baseline 37.0 ± 0.5 °C), WBC (11,366 ± 2,541, baseline 6,861 ± 2,123/µl), CRP (25.3 ± 22.6, baseline 3.1 ± 6 mg/l), IL-6 (12 ± 13, baseline 2.7 ± 2.6 pg/ml), and LBP (11.3 ± 4.9, baseline 5.7 ± 2.7 µg/ml) peaked on day 1 postoperatively (each p = 0.001), while ESR peaked on day 3 (25 ± 16, baseline 13 ± 11 mm/h, p = 0.001). BT and IL-6 normalized by day 3 and CRP by day 7, while ESR (23 ± 16 mm/h, p = 0.001), WBC (7,807 ± 2,750/µl, p = 0.001) and LBP (7.3 ± 2.6 µg/ml, p = 0.028) were still increased by day 7. CONCLUSION: The present study establishes normative values for an infection surveillance following transsphenoidal pituitary surgery. CRP, a convenient and reasonable priced parameter, is affected by the procedure for the first postoperative week. IL-6 is more robust and allows a close monitoring on the expense of additional pricing. ESR, WBC and LBP are sustained affected by surgery, and do not offer any advantage. Since no infections were observed, we were unable to calculate the respective sensitivity and specificity.

Significance of the pH-induced conformational changes in the structure of C-reactive protein measured by dual polarization interferometry.

Emerging evidence indicates that the conformation of C-reactive protein (CRP) plays important roles in human inflammation and cardiovascular disease (CVD). The different conformations in the structure of CRP under different pH conditions remain an important issue to be investigated for explaining various functions of CRP under certain physiologic and pathologic conditions. We directly measured the pH-induced conformational changes in the structure of CRP by dual polarization interferometry (DPI). The CRP was attached to an aldehyde-functionalized DPI sensor chip at a concentration of 50 µg/ml, and attained 2.019 ng/mm2 to form a surface coverage with a 1.71×10(-14) mol/mm2 CRP monolayer. A pentagonal structure with an average monolayer thickness value of 5.70±0.12nm and a layer density of 0.374±0.058 g/cm2 was obtained at pH 7.0. Moreover, the DPI biosensor signals directly reflected the considerable structural parameters and phenomena of conformational changes of CRP in a pH range of 2.0-10.0. The results obtained showed that the pentameric structure of CRP might dissociated into monomers or monomer aggregates as the pH shifts toward both acidic and alkaline conditions, but only partial rearrangements of CRP subunits might occur at extremely acidic physiological conditions. Considering the proinflammatory effect and subclinical chronic inflammation, pH-induced conformational changes in the structure of CRP between monomeric and pentameric formations may strongly relate to vascular atherosclerosis and subsequent CVD.

Isolation of two C-reactive protein homologues from cod (Gadus morhua L.) serum.

Pentraxins are important molecules in innate defence and play a role in the acute phase response of both mammals and fish. Isolation of cod pentraxins by affinity chromatography using phosphorylcholine agarose revealed two pentraxin-like proteins, referred to as PI and PII proteins. These varied in their overall charge, pentameric and subunit molecular size, glycosylation and N-terminal amino acid sequences. The PI protein was homologous with the CRP-like pentraxin previously described in cod whereas the PII protein was a new CRP homologue, which was characterized by substantial individual heterogeneity with regard to subunit size and relative density. The results indicate considerable genetic variations in the cod pentraxins.

Measurement of dimensions of pentagonal doughnut-shaped C-reactive protein using an atomic force microscope and a dual polarisation interferometric biosensor.

In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. A single pentagonal structure was directly visualized by AFM, and quantitative measurements of the dimensions of the protein were provided. The average height calculated for each pentagonal CRP particle was approximately 3.03+/-0.37 nm, which basically corresponds to that (36 A in protomer diameter) previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, a experiment using dual polarization interferometric (DPI) as a biosensor was then performed, and the average monolayer thickness value (3.18+/-0.43 nm) that was calculated basically corresponds to that obtained from the experimental value (3.03+/-0.37 nm) of the height measured by an AFM method for CRP. Further investigations will be performed to study the surface ultrastructure of a single pentagonal CRP molecule, and for this purpose a CRP sample (at low concentration) was scanned in vacuum by AFM. The higher-resolution images clearly revealed the presence of doughnut-shaped CRP molecules. In addition, phase images of CRP molecules were captured simultaneously with their height images, and the lateral dimensions of the doughnut-shaped CRP molecules were then measured. It was found that the average values calculated for the outer diameter (11.13+/-1.47 nm) and pore diameter (3.52+/-0.42 nm) are respectively close to those (102 A in outer diameter and 30 A in pore diameter) previously obtained from the structure of CRP determined by X-ray crystallography. This study represents the first direct characterization of the surface ultrastructure and dimensional measurement of the CRP molecule on the sensor chip.

Detection of C-reactive protein utilizing magnetic permeability detection based immunoassays.

A new sensing technology platform integrating magnetic permeability detection and a two-site heterogeneous immunoassay in a one-step analysis is described. As a platform model, measurements of C-reactive protein (CRP), a cardiac and inflammation marker, were performed in a rapid (11.5 min) high-sensitivity (hs) procedure with a low detection limit (0.2 mg/L) and accuracy (CV = 11%). The two-site heterogeneous immunoassay was performed in 1.2-mL disposable reagents vials containing solid phase (polyclonal anti-CRP conjugated silica microparticles), labeling agent (monoclonal anti-CRP conjugated superparamagnetic nanoparticles), and reaction buffer. Whole blood (20 muL) was assayed by introducing the sample into a reagent vial using a glass capillary and mixing its contents by hand for 30 s. After a 11-min sedimentation step, the vial was placed into the coil of the magnetic permeability detector, which measured the enrichment of superparamagnetic nanoparticles in the solid-phase sediment. Magnetic permeability detection and quantification is based on the principle that when paramagnetic materials are placed inside a coil, the inductance of the coil is influenced. Screening of CRP on whole blood patient samples showed good correlation with central hospital measurements for hsCRP (y = 1.018x - 0.021, R(2) = 0.980, n = 103) and normal range CRP (y = 1.02x + 2.53, R(2)= 0.991, n = 33) analyses. The mean differences of the two methods according to the Bland and Altman plots were -0.03 +/- 1.12 mg/L for hsCRP analysis and -3.4 +/- 8.64 mg/L for normal range CRP analysis.

Narp and NP1 form heterocomplexes that function in developmental and activity-dependent synaptic plasticity.

Narp is a neuronal immediate early gene that plays a role in excitatory synaptogenesis. Here, we report that native Narp in brain is part of a pentraxin complex that includes NP1. These proteins are covalently linked by disulfide bonds into highly organized complexes, and their relative ratio in the complex is dynamically dependent upon the neuron's activity history and developmental stage. Complex formation is dependent on their distinct N-terminal coiled-coil domains, while their closely homologous C-terminal pentraxin domains mediate association with AMPA-type glutamate receptors. Narp is substantially more effective in assays of cell surface cluster formation, coclustering of AMPA receptors, and excitatory synaptogenesis, yet their combined expression results in supraadditive effects. These studies support a model in which Narp can regulate the latent synaptogenic activity of NP1 by forming mixed pentraxin assemblies. This mechanism appears to contribute to both activity-independent and activity-dependent excitatory synaptogenesis.

Dissociation and subunit rearrangement of membrane-bound human C-reactive proteins.

As one of the most important acute-phase reactants in human serum, C-reactive protein plays its physiological roles mainly on membranes. Here we show that the human C-reactive protein is two-dimensionally crystallized upon specific adsorption on the phosphorylcholine ligand containing membranes by monolayer approach. The 2.0-nm resolution projection structure of the two-dimensional crystals analyzed by electron microscopy and image reconstruction reveals open-ring-like pentamers in the crystals. The electron microscope graphs also show that the dissociated pentamers with open-ring-like structure occur in a closed packing region (not two-dimensionally crystallized). These results indicate a membrane-induced dissociation and rearrangement of hCRP, which may relate to the variety of hCRP's physiological functions.

Pentameric two-dimensional crystallization of rabbit C-reactive protein on lipid monolayers.

As a member of the pentraxin family, C-reactive protein plays various roles in the nonspecific immunity of animals. Though soluble, C-reactive protein always functions on membranes. In order to study the structure of the membrane-bound protein and the reaction between protein and membranes, two-dimensional (2D) crystallization of rabbit C-reactive protein on lipid monolayers was performed. The 2D crystals composed of pentameric proteins were obtained on lipid monolayers by specific adsorption for the first time. The projection map at 26-A resolution is presented, which exhibits P2 symmetry with lattice parameters a = 158(+/-3) A, b = 92(+/-1) A, and gamma = 107(+/-1) degrees. The current work may give a basis for the further study on the structure of complexes made up of C-reactive protein with its functional binding molecules on membranes.

Female protein, amyloidosis, and hormonal carcinogenesis in Turkish hamster: differences from Syrian hamster.

The Syrian hamster (Mesocricetus auratus) has been widely used as an experimental animal and is a unique model for three sex hormone-regulated events: 1) estrogen-initiated renal carcinogenesis, 2) sex-limited expression of amyloidosis, a ubiquitous disease, and 3) sex hormone control of a serum amyloid P component (SAP) called female protein (FP). In this study, we evaluated the closely related Turkish hamster (Mesocricetus brandti) for these three events and found some very different responses: 1) estrogen-initiated renal carcinogenesis was not found in Turkish hamster, 2) amyloidosis was not sex limited and actually was a rare disease in the Turkish hamster, and 3) Turkish hamsters did express a sex-limited SAP-FP in serum that was antigenically identical and structurally very similar (97.5%) to Syrian hamster SAP-FP. However, acute phase regulation of SAP-FP synthesis was different, and serum levels of this pentraxin were much lower than those found in the Syrian hamster. On the other hand, in contrast to findings in the Syrian hamster, hepatic tumors were relatively common in normal and especially in estrogen-treated Turkish hamsters. Therefore, although they are closely related, these two Mesocricetus hamster species have markedly dissimilar responses to sex hormones.

C-Reactive protein: re-evaluation of a diagnostic laboratory test

C-reactive protein has been a measure of acute phase reactions to inflammatory for 40 years. Recently improved quantitative assays in serum and cerebrospinal fluid (CSF) have allowed a re-evaluation of its potential as a diagnostic laboratory test. The main advances in the newer methods have been that they can provide rapid (hours) information on the hepatocyte synthesis of this molecule during immune response. We have tested its value in patients with presumed bacterial meningitis. Based on our experience, newer standardized, quantitative assessments of C-reactive protein can be very useful in distinguishing between bacterial and other forms of meningeal irritation during during the first few days of hospitalization. Other investigators have indicated that by serial measurement important information on the resolution or continuation of inflammatory processes can be obtained. We recommend improved standardization of this test, and recalculation of its usefulness as a diagnostic laboratory test.

Two-dimensional crystallization of rabbit C-reactive protein on lipid monolayers.

Two-dimensional (2D) crystals of rabbit C-reactive protein (CRP) have been obtained by protein binding on lipid monolayers at the air/water interface. Two different types of crystalline arrays of CRP were obtained, by specific binding and non-specific adsorption to the lipids. Electron crystallographic analysis of the negatively stained specimens showed that the unit cell parameters of the CRP 2D crystals formed by specific binding were a=81 angstroms, b=78 angstroms, gamma=118.35 degrees, and those formed by nonspecific adsorption were a=74 angstroms, b=67 angstroms, gamma=95.5 degrees, both with the layer group p1. Projection maps were obtained at a resolution of 26 angstroms and 22 angstroms respectively. They showed that only the monomers of the CRP were packed in the 2D arrays and the orientations of the monomers on the lipid monolayers were different in the two types of crystals. By comparing the two projection maps, a preliminary shape of the CRP monomer has been derived. A model of the pentameric structure of the oligomeric CRP has been proposed.

Binding of model soluble immune complexes to modified C-reactive protein.

We have identified a binding interaction between a modified form of C-reactive protein (mCRP) and model immune complexes. We have characterized these interactions in terms of their relative affinity, specificity, pH dependence, and recognition site on mCRP. First, binding isotherms were generated for the reaction of immobilized mCRP with heat-aggregated IgG (HAG) which gave an estimate for the value of the dissociation constant, Kd, of 1.6 nM. Second, competitive binding assays were performed using immobilized HAG to determine the relative affinity (IC50) for the interaction between HAG or monomeric IgG and mCRP in the fluid phase. While the binding of HAG to mCRP occurred with high affinity (IC50=0.72 nM), the binding of monomeric IgG to mCRP occurred with >2000-fold lower affinity (IC50>1.66 muM). Third, immune complex binding to immobilized mCRP was studied using defined ratios of horseradish peroxidase and rabbit anti-peroxidase Ab. The binding of these complexes to mCRP was strongly pH dependent, with a maximum at pH 5.5. At this optimal pH, preformed peroxidase-antiperoxidase immune complexes (approximately 2:3 Ab:Ag molar ratio) were shown to bind immobilized mCRP with a Kd of 111 nM. Fourth, using mCRP-specific mAbs, HAG and peroxidase- antiperoxidase binding sites were localized to CRP sequences to 130 to 138 and 200 to 206. Since inflammatory processes often cause microenvironments to become acidic, and since mCRP selectively binds immune complexes at acidic pH, we propose that mCRP mediates the specific binding of immune complexes in vivo and potentiates effector reactions for immune complex removal.

Complement activation induced by human C-reactive protein in mildly acidic conditions.

Human C-reactive protein (CRP) is known to activate the C system upon reaction with phosphocholine-containing or polycation-containing ligands. We found that, even in the absence of these ligands, CRP caused C activation in mildly acidic conditions. The optimum pH for the activation was 6.3, which is within a physiologic range normally found at an inflammatory locus. In this activation, C components C1 and C4 were extensively consumed, C2 and C3 were moderately consumed, and C5 was only slightly consumed. These results indicate that the activation is mediated via the classical pathway, but is restricted to the early stage of the C cascade. As with the plasma contact system, the reaction proceeded in glass tubes but not in polypropylene tubes. However, even in polypropylene tubes, the reaction proceeded after the supplement of kaolin particles to the system. Probably the C activation induced by CRP at a mildly acidic pH required negatively charged surfaces. Analyses of circular dichroism and fluorescence spectra indicate that CRP undergoes a pH-dependent conformational change, thus affecting the reactivity of CRP with the C system.