Pentameric C-reactive Protein Research Articles

Pentameric C-reactive protein is a better prognostic biomarker and remains elevated for longer than monomeric CRP in hospitalized patients with COVID-19.

The differing roles of the pentameric (p) and monomeric (m) C-reactive protein (CRP) isoforms in viral diseases are not fully understood, which was apparent during the COVID-19 pandemic regarding the clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Herein, we investigated the predictive value of the pCRP and mCRP isoforms for COVID-19 severity in hospitalized patients and evaluated how the levels of the protein isoforms changed over time during and after acute illness. This study utilized samples from a well-characterized cohort of Swedish patients with SARS-CoV-2 infection, the majority of whom had known risk factors for severe COVID-19 and required hospitalization. The levels of pCRP were significantly raised in patients with severe COVID-19 and in contrast to mCRP the levels were significantly associated with disease severity. Additionally, the pCRP levels remained elevated for at least six weeks post inclusion, which was longer compared to the two weeks for mCRP. Our data indicates a low level of inflammation lasting for at least six weeks following COVID-19, which might indicate that the disease has an adverse effect on the immune system even after the viral infection is resolved. It is also clear that the current standard method of testing pCRP levels upon hospitalization is a useful marker for predicting disease severity and mCRP testing would not add any clinical relevance for patients with COVID-19.

C-reactive protein: a target for therapy to reduce inflammation.

C-reactive protein (CRP) is well-recognized as a sensitive biomarker of inflammation. Association of elevations in plasma/serum CRP level with disease state has received considerable attention, even though CRP is not a specific indicator of a single disease state. Circulating CRP levels have been monitored with a varying degree of success to gauge disease severity or to predict disease progression and outcome. Elevations in CRP level have been implicated as a useful marker to identify patients at risk for cardiovascular disease and certain cancers, and to guide therapy in a context-dependent manner. Since even strong associations do not establish causality, the pathogenic role of CRP has often been over-interpreted. CRP functions as an important modulator of host defense against bacterial infection, tissue injury and autoimmunity. CRP exists in conformationally distinct forms, which exhibit distinct functional properties and help explaining the diverse, often contradictory effects attributed to CRP. In particular, dissociation of native pentameric CRP into its subunits, monomeric CRP, unmasks "hidden" pro-inflammatory activities in pentameric CRP. Here, we review recent advances in CRP targeting strategies, therapeutic lowering of circulating CRP level and development of CRP antagonists, and a conformation change inhibitor in particular. We will also discuss their therapeutic potential in mitigating the deleterious actions attributed to CRP under various pathologies, including cardiovascular, pulmonary and autoimmune diseases and cancer.

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB.

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins.

Fueling the flames of colon cancer - does CRP play a direct pro-inflammatory role?

Systemic inflammation, diagnostically ascribed by measuring serum levels of the acute phase reactant C-reactive protein (CRP), has consistently been correlated with poor outcomes across cancer types. CRP exists in two structurally and functionally distinct isoforms, circulating pentameric CRP (pCRP) and the highly pro-inflammatory monomeric isoform (mCRP). The aim of this pilot study was to map the pattern of mCRP distribution in a previously immunologically well-defined colon cancer (CC) cohort and explore possible functional roles of mCRP within the tumor microenvironment (TME). Formalin-fixed, paraffin-embedded (FFPE) tissue samples from 43 stage II and III CC patients, including 20 patients with serum CRP 0-1 mg/L and 23 patients with serum CRP >30 mg/L were immunohistochemically (IHC) stained with a conformation-specific mCRP antibody and selected immune and stromal markers. A digital analysis algorithm was developed for evaluating mCRP distribution within the primary tumors and adjacent normal colon mucosa. mCRP was abundantly present within tumors from patients with high serum CRP (>30 mg/L) diagnostically interpreted as being systemically inflamed, whereas patients with CRP 0-1 mg/L exhibited only modest mCRP positivity (median mCRP per area 5.07‰ (95%CI:1.32-6.85) vs. 0.02‰ (95%CI:0.01-0.04), p<0.001). Similarly, tissue-expressed mCRP correlated strongly with circulating pCRP (Spearman correlation 0.81, p<0.001). Importantly, mCRP was detected exclusively within tumors, whereas adjacent normal colon mucosa showed no mCRP expression. Double IHC staining revealed colocalization of mCRP with endothelial cells and neutrophils. Intriguingly, some tumor cells also colocalized with mCRP, suggesting a direct interaction or mCRP expression by the tumor itself. Our data show that the pro-inflammatory mCRP isoform is expressed in the TME of CC, primarily in patients with high systemic pCRP values. This strengthens the hypothesis that CRP might not only be an inflammatory marker but also an active mediator within tumors.

C-reactive protein isoforms as prognostic markers of COVID-19 severity.

C-reactive protein (CRP), an active regulator of the innate immune system, has been related to COVID-19 severity. CRP is a dynamic protein undergoing conformational changes upon activation in inflammatory microenvironments between pentameric and monomeric isoforms. Although pentameric CRP is the circulating isoform routinely tested for clinical purposes, monomeric CRP shows more proinflammatory properties. Therefore, we aimed to determine the potential of monomeric CRP in serum as a biomarker of disease severity in COVID-19 patients (admission to intensive care unit [ICU] and/or in-hospital mortality). We retrospectively determined clinical and biological features as well as pentameric and monomeric CRP levels in a cohort of 97 COVID-19 patients within 72h of hospital admission. Patients with severe disease had higher levels of both pentameric and monomeric CRP. However, multivariate analysis showed increased mCRP but not pCRP to be independently associated to disease severity. Notably, mCRP levels higher than 4000 ng/mL (OR: 4.551, 95% CI: 1.329-15.58), together with number of co-morbidities, low lymphocyte count, and procalcitonin levels were independent predictors of disease severity in the multivariate model. Our results show the potential of mCRP levels as a marker of clinical severity in COVID-19 disease.

A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein.

C-reactive protein (CRP) is an early-stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro-inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X-ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide invitro and invivo proof-of-concept data demonstrating that the low molecular weight tool compound inhibits CRP-driven exacerbation of local inflammatory responses, while potentially preserving pathogen-defense functions of CRP. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy.

Structurally Altered, Not Wild-Type, Pentameric C-Reactive Protein Inhibits Formation of Amyloid-β Fibrils.

The structure of wild-type pentameric C-reactive protein (CRP) is stabilized by two calcium ions that are required for the binding of CRP to its ligand phosphocholine. CRP in its structurally altered pentameric conformations also binds to proteins that are denatured and aggregated by immobilization on microtiter plates; however, the identity of the ligand on immobilized proteins remains unknown. We tested the hypotheses that immobilization of proteins generated an amyloid-like structure and that amyloid-like structure was the ligand for structurally altered pentameric CRP. We found that the Abs to amyloid-β peptide 1-42 (Aβ) reacted with immobilized proteins, indicating that some immobilized proteins express an Aβ epitope. Accordingly, four different CRP mutants capable of binding to immobilized proteins were constructed, and their binding to fluid-phase Aβ was determined. All CRP mutants bound to fluid-phase Aβ, suggesting that Aβ is a ligand for structurally altered pentameric CRP. In addition, the interaction between CRP mutants and Aβ prevented the formation of Aβ fibrils. The growth of Aβ fibrils was also halted when CRP mutants were added to growing fibrils. Biochemical analyses of CRP mutants revealed altered topology of the Ca2+-binding site, suggesting a role of this region of CRP in binding to Aβ. Combined with previous reports that structurally altered pentameric CRP is generated in vivo, we conclude that CRP is a dual pattern recognition molecule and an antiamyloidogenic protein. These findings have implications for Alzheimer's and other neurodegenerative diseases caused by amyloidosis and for the diseases caused by the deposition of otherwise fluid-phase proteins.

Associations of C-reactive protein isoforms with systemic lupus erythematosus phenotypes and disease activity

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by a large production of autoantibodies and deficient clearance of cellular waste. The disease typically oscillates between episodes of elevated disease activity and quiescent disease. C-reactive protein (CRP) is a pentameric acute-phase protein usually reflecting inflammation and tissue damage. However, despite increased inflammation and elevated interleukin-6, the levels of CRP typically remain low or only slightly raised in SLE. Under certain conditions, pentameric CRP (pCRP) can dissociate into its monomeric isoform (mCRP), which mainly has been ascribed pro-inflammatory properties. The present study aims to investigate the potential relationship between pCRP and mCRP, respectively, with disease activity and clinical features of SLE.MethodsThe levels of pCRP and mCRP were measured, by turbidimetry (high-sensitive) and sandwich enzyme-linked immunosorbent assay (ELISA) respectively, in serum samples from 160 patients with SLE and 30 patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Twenty-two of the SLE cases were selected for analysis at two time-points; quiescent disease and active disease. The two CRP isoforms were evaluated in relation to disease activity and clinical features in the two diseases.ResultsLevels of pCRP and mCRP were significantly lower in SLE than AAV (p < 0.001) and the ratio of mCRP/pCRP was higher in SLE compared to AAV. The mCRP/pCRP ratio was higher for patients in remission and able to significantly separate between active/quiescent disease in paired, but not in non-paired, samples from patients with SLE. Significant correlations were observed with SLICC/ACR damage index for pCRP levels as well as inversely with the mCRP/pCRP ratio. Lower mCRP levels associated with malar rash.ConclusionAs the interrelationship between the two isoforms appear to (a) discriminate between quiescent and active SLE and (b) differ between SLE and AAV, our data indicates that the two CRP isoforms could exert contrasting immunological effects and/or reflect different milieus. Given the biological effects of mCRP, it is possible that altered levels may indicate increased opsonization of immune complexes and apoptotic debris, and thereby prevent their deposition outside the reticuloendothelial system and manifestations such as lupus nephritis and lupus-related skin disease.

Monomeric C-reactive protein induces the cellular pathology of Alzheimer's disease.

IntroductionHuman study shows that elevated C‐reactive protein (CRP) in blood impacts apolipoprotein E (APOE) ε4, but not APOE ε3 or APOE ε2, genotype to increase the risk of Alzheimer's disease (AD). However, whether CRP is directly involved in cellular AD pathogenesis and in which type of neuronal cells of APOE ε4 carriers are unknown.MethodsWe aimed to use different primary neuronal cells and investigate if CRP induces cellular AD pathology depending on APOE genotypes. Here the different primary neuronal cells from the different APOE genotype knock‐in mice cortex were isolated and used.ResultsMonomeric CRP (mCRP) increased amyloid beta production and, in parallel, induced tau phosphorylation in addition to their related proteins in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose‐ and time‐dependent manner. Consistently, mCRP induced the staining of other neurodegenerative biomarkers, including Fluoro‐Jade B stain (FjB), TUNEL and Cleaved Caspase‐3, in primary neurons in a similar pattern of APOE ε4 > APOE ε3 > APOE ε2. In contrast, pentameric CRP (pCRP) had a tendency to induce cellular AD pathology but did not reach statistical significance. On the other hand, it is intriguing that regardless of APOE genotype, mCRP did not influence the expressions of Iba‐1 and CD68 in primary microglia or the expression of glial fibrillary acidic protein in primary astrocytes, and additionally mCRP did not affect the secretions of interleukin (IL)‐1α, IL‐1β, and tumor necrosis factor α from these cells.DiscussionThis is the first report to demonstrate that mCRP directly induces cellular AD pathogenesis in neurons in an APOE genotype‐dependent pattern, suggesting that mCRP plays a role as a mediator involved in the APOE ε4‐related pathway for AD during chronic inflammation.Highlights Pentameric C‐reactive protein (pCRP) can be dissociated irreversibly to form free subunits or monomeric CRP (mCRP) during and after the acute phase.mCRP increased amyloid beta production in the primary neurons in a pattern of apolipoprotein E (APOE) ε4 > APOE ε3 > APOE ε2 in a dose‐dependent manner.mCRP induced the expression of phosphorylated tau in the primary neurons in a pattern of APOE ε4 > APOE ε3 > APOE ε2 in a dose‐ and time‐dependent manner.mCRP plays an important mediator role in the APOE ε4‐related pathway of Alzheimer's disease risk.

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.

Extracellular vesicles are associated with C-reactive protein in sepsis

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP+ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP+ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP+ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP+ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

Association between microvesicles bearing monomeric C-reactive protein and platelet reactivity. Relationship with low response to antiplatelet drugs?

In cardiovascular disease pathogenesis, key roles are played by hypercoagulation disorders mainly associated with platelet activation, aggregation processes, and endothelial dysfunction. Current research is studying new molecular species that might interfere with both previously known and new biochemical pathways, leading to the activation and aggregation of platelets, and growing evidence suggests close links between inflammation and thrombosis. It has been suggested that, in cardiovascular disease, circulating pentameric C-reactive protein (CRP) can dissociate into the monomeric form on activated platelets and the surface of circulating microvesicles, and this could potentially accelerate thrombogenesis and link platelet hemostatic processes with the immune response. Increased microvesicles level has confirmed the link between microvesicles and the inflammatory process in diseases with a strong inflammatory reaction. The treatment of coronary artery disease is partly based on properly selected pharmacotherapy. Despite the significant progress that has recently been made in recognizing and combatting abnormal response to oral antiplatelet agents, it is still unknown why some patients who receive dual antiplatelet therapy do not benefit from expected therapeutic effects. Part of the antithrombotic action that antiplatelet agents exert in different clinical settings may be explained by a modulation in the generation of platelet-derived microvesicles bound monomeric CRP. It is suggested that markers of inflammation, such as monomeric CRP or platelet-derived microvesicles, may provide markers of disease activity or response to treatment and may be used in the prognosis of the course of various diseases. This paper aims to provide an overview of the relationship between the monomeric form of C-reactive protein, associated with membrane microvesicles, higher platelet activation, and the possible association with lower antiplatelet response.

In vitro effects of azide-containing human CRP isoforms and oxLDL on U937-derived macrophage production of atherosclerosis-related cytokines.

Atherosclerosis is an inflammatory chronic disease of the arterial wall. Monomeric (m) and pentameric (p) C-reactive protein (CRP) and oxidized low density lipoproteins (oxLDL) seem to affect the pattern of cytokine production by macrophages, thus playing an important role in atherogenesis. Azide, the commercial preservative of CRP, may influence its action in vitro. The present study aimed to determine the effects of both isoforms of azide-containing CRP (mCRP and pCRP) with and without oxLDL on cytokine production by U937-derived macrophages. U937 monocytes were cultured and differentiated into macrophages and treated with mCRP, pCRP, oxLDL and azide individually and in combination. ELISA were performed to measure the levels of interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, IL-10 and tumor necrosis factor (TNF)-α in culture supernatants collected from U937-derived macrophages following their respective treatments. Most single and combined treatments, especially in triple combination, were able to downregulate the levels of IFN-γ and IL-6 compared with control untreated cells, whilst the combination of mCRP and pCRP increased IL-4 levels. Regarding IL-10, except for an increase induced by mCRP, no significant effect was caused by any treatment compared with the control. On the other hand, the levels of TNF-α were not significantly affected by any treatment except for a decreasing trend that was observed with mCRP/oxLDL treatment compared with control. By contrast, double azide caused a significant decrease in the levels of IFN-γ and IL-6. The results of the present study indicated that mCRP, pCRP, oxLD and possibly azide, individually or in different combinations, had the tendency to upregulate the expression of IL-4 and to downregulate that of the pro-atherogenic cytokines, IFN-γ and IL-6, suggesting that the intima microenvironment serves a crucial role in atherogenesis.

Activation of C-reactive protein proinflammatory phenotype in the blood retinal barrier in vitro: implications for age-related macular degeneration.

The retinal pigment epithelium (RPE) is considered one of the main targets of age-related macular degeneration (AMD), the leading cause of irreversible vision loss among the ageing population worldwide. Persistent low grade inflammation and oxidative stress eventually lead to RPE dysfunction and disruption of the outer blood-retinal barrier (oBRB). Increased levels of circulating pentameric C-reactive protein (pCRP) are associated with higher risk of AMD. The monomeric form (mCRP) has been detected in drusen, the hallmark deposits associated with AMD, and we have found that mCRP induces oBRB disruption. However, it is unknown how mCRP is generated in the subretinal space. Using a Transwell model we found that both pCRP and mCRP can cross choroidal endothelial cells and reach the RPE in vitro and that mCRP, but not pCRP, is able to cross the RPE monolayer in ARPE-19 cells. Alternatively, mCRP can originate from the dissociation of pCRP in the surface of lipopolysaccharide-damaged RPE in both ARPE-19 and primary porcine RPE lines. In addition, we found that the proinflammatory phenotype of mCRP in the RPE depends on its topological localization. Together, our findings further support mCRP contribution to AMD progression enhancing oBRB disruption.

Monomeric C-reactive protein in coronary artery disease

Aim. To develop a method for measuring circulating monomeric C-reactive protein and to determine the levels of C-reactive protein isoforms (native pentameric C-reactive protein and monomeric C-reactive protein) in patients with stable coronary artery diseases and acute myocardial infarction. Methods . Plasma and blood serum samples were collected from 22 patients with stable coronary artery disease (CAD), 14 patients with acute myocardial infarction at days 2-3, and 11 healthy volunteers. The analysis was performed using flow cytofluorometry FACS Cantoll and a set of functional particles Cytometric Bead Array (BD Biosciences, USA). Antibodies for CRP (clones 328 and 372 from ImTek, Russia, and clone 8C8 from Sigma-Aldrich, USA), гСКР-GAH (with antibodies for CRP), native CRP (ImTek, Russia) and recombinant monomeric CRP (received as a gift from Dr. L. Potempa) were used in the analysis. Results. A novel laboratory kit for measuring blood levels of monomeric CRP has been developed. Patients with CAD demonstrated elevated plasma levels of CRP, whereas healthy subjects reported the levels below the lower cut-off. Patients with stable CAD had mCRP concentration of 2.34 (1.42; 3.27) pg/L, whereas patients with acute MI had the highest level of 16.76 (3.65;54.83) pg/L (p = 0.0002, Kruskal-Wallis One Way Annova). There were no any correlations between mCRP levels and nCRP, hs-CRP and IL-6. Conclusion . The novel laboratory kit allows measuring monomeric C-reactive protein and provides novel data on the role of mCRP in the development and progress of CAD. Obtained data suggest that monomeric C-reactive protein can play no less important role than native C-reactive protein in the diagnosis and progress of cardiovascular diseases as well as other diseases associated with the inflammatory process.

Monomeric C-reactive protein affects cell injury and apoptosis through activation of p38 mitogen-activated protein kinase in human coronary artery endothelial cells.

C-reactive protein (CRP) is an important predictor of cardiovascular events and plays a role in vascular inflammation and vessel damage. The aim of this study was to investigate the effect of pentameric CRP (pCRP) and monomeric CRP (mCRP) on the production of atherosclerosis-related factors in cultured human coronary artery endothelial cells (HCAECs). HCAECs were treated with pCRP, mCRP, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, or transfected with p38 MAPK siRNA. Western blotting was performed to detect the expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-2 (ICAM-2) and vascular cell adhesion molecule-1 (VCAM-1). Proliferation, damage, and apoptosis of HCAECs were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, lactate dehydrogenase (LDH), and flow cytometry, respectively. mCRP suppressed VEGF and COX-2 expression and enhanced ICAM-2 and VCAM-1 expression in HCAECs, in both dose-dependent and time-dependent manner. Except at 100 µg/ml concentration and 20-hour or 24-hour incubation, pCRP had no apparent effects. mCRP but not pCRP induced HCAEC injury and phosphorylation of p38 MAPK, and the inhibitor SB203580 reversed the effects of mCRP. mCRP promotes injury and apoptosis of HCAECs through a p38 MAPK-dependent mechanism, which provides a new therapy for the injury of HCAECs in atherosclerosis.

Celluar Folding Determinants and Conformational Plasticity of Native C-Reactive Protein.

C-reactive protein (CRP) is an acute phase reactant secreted by hepatocytes as a pentamer. The structure formation of pentameric CRP has been demonstrated to proceed in a stepwise manner in live cells. Here, we further dissect the sequence determinants that underlie the key steps in cellular folding and assembly of CRP. The initial folding of CRP subunits depends on a leading sequence with a conserved dipeptide that licenses the formation of the hydrophobic core. This drives the bonding of the intra-subunit disulfide requiring a favorable niche largely conferred by a single residue within the C-terminal helix. A conserved salt bridge then mediates the assembly of folded subunits into pentamer. The pentameric assembly harbors a pronounced plasticity in inter-subunit interactions, which may form the basis for a reversible activation of CRP in inflammation. These results provide insights into how sequence constraints are evolved to dictate structure and function of CRP.

Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein.

A monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks period. Dissociated samples were purified via size exclusion chromatography and characterized by western blot, phosphocholine affinity chromatography and CWPS precipitation. Human serum samples from patients with raised CRP levels (>100 mg/L as determined by the clinical laboratory assay) were purified by affinity and size exclusion chromatography and analyzed (n = 40) to determine whether circulating monomeric CRP could be detected ex vivo. All 40 samples tested positive for pentameric CRP via western blot and enzyme linked immunosorbent assay (ELISA) analysis. Monomeric C-reactive protein was also identified in all 40 patient samples tested, with an average level recorded of 1.03 mg/L (SE = ±0.11). Both the in vitro monomeric C-reactive protein and the human serum monomeric protein displayed a molecular weight of approximately 23 kDa, both were recognized by the same anti-CRP monoclonal antibody and both reversibly bound to phosphocholine in a calcium-dependent manner. In common with native pentameric CRP, the in vitro mCRP precipitated with CWPS. These overlapping characteristics suggest that a physiologically relevant, near-native monomeric CRP, which retains the structure and binding properties of native CRP subunits, has been produced through in vitro dissociation of pentameric CRP and also isolated from serum with markedly elevated CRP levels. This provides a clear route toward the in-depth study of the structure and function of physiological monomeric CRP.

C - REACTIVE PROTEIN: AN INFLAMMATORY BIOMARKER PRESENT IN MULTIPLE DISEASE PATHOGENESIS

C-reactive protein (CRP) is an acute-phase protein belongs to pentraxins family with a feature of ve identical globular subunits. Two different isoforms i.e. pentameric CRP (pCRP) and monomeric CRP (mCRP) have different key role in regulation. The key biological function of CRP is host defense using classical complement pathway against bacterial infection and involved in removal of apoptotic and necrotic cells. Although CRP is mainly associated with inammation in bacterial infection such as tuberculosis or Pneumococcus infection, however it is also presence in atherosclerosis, cardiovascular disease (CVD) and in various cancers which make it good candidate for prognostic study in different clinical conditions.

Functionality of C-Reactive Protein for Atheroprotection.

C-reactive protein (CRP) is a pentameric molecule made up of identical monomers. CRP can be seen in three different forms: native pentameric CRP (native CRP), non-native pentameric CRP (non-native CRP), and monomeric CRP (mCRP). Both native and non-native CRP execute ligand-recognition functions for host defense. The fate of any pentameric CRP after binding to a ligand is dissociation into ligand-bound mCRP. If ligand-bound mCRP is proinflammatory, like free mCRP has been shown to be in vitro, then mCRP along with the bound ligand must be cleared from the site of inflammation. Once pentameric CRP is bound to atherogenic low-density lipoprotein (LDL), it reduces both formation of foam cells and proinflammatory effects of atherogenic LDL. A CRP mutant, that is non-native CRP, which readily binds to atherogenic LDL, has been found to be atheroprotective in a murine model of atherosclerosis. Thus, unlike statins, a drug that can lower only cholesterol levels but not CRP levels should be developed. Since non-native CRP has been shown to bind to all kinds of malformed proteins in general, it is possible that non-native CRP would be protective against all inflammatory states in which host proteins become pathogenic. If it is proven through experimentation employing transgenic mice that non-native CRP is beneficial for the host, then using a small-molecule compound to target CRP with the goal of changing the conformation of endogenous native CRP would be preferred over using recombinant non-native CRP as a biologic to treat diseases caused by pathogenic proteins such as oxidized LDL.