Loss of Pentameric Symmetry of C-Reactive Protein Is Associated with Promotion of Neutrophil-Endothelial Cell Adhesion

Abstract

The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1–200 μg/ml) down-regulated neutrophil l-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21ras oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.

Loss of Pentameric Symmetry of C-reactive Protein Is Associated with Delayed Apoptosis of Human Neutrophils

“…tyrosine kinases (53). Tyrosine kinase activation can lead to activation of ERK through the Ras/Raf-1/MEK pathway (24) or indirectly through activation of PI 3-kinase and Akt, as was reported for granulocyte macrophage-colony-stimulating factor-activated signaling in neutrophils (49). Phosphorylated Akt has been shown to inactivate procaspase-9, a trigger of the activation of the caspase cascade (54).…”

“…mCRP antigens were detected in inflamed rabbit tissues (22) as well as in the wall of human normal blood vessels (23). Unlike native CRP, mCRP binds to the low affinity IgG immune complex Fc␥RIIIb 2 and enhances neutrophil adhesiveness to endothelial cells via up-regulation of the expression of CD11b/C18 on the neutrophil surface (24). Exposure of CRP to membrane-bound serine proteases leads to cleavage of biologically active peptides, which mimic the actions of native CRP on neutrophil chemotaxis (25) and adhesion to endothelial cells (15).…”

Structural Characterization of PTX3 Disulfide Bond Network and Its Multimeric Status in Cumulus Matrix Organization

“…Emerging evidence suggests that both short and long pentraxins express distinct bioactivities depending on subunit organization. For example, native CRP inhibits platelet activation and prevents platelet capture of neutrophils, whereas monomeric CRP, resulting from loss of the pentameric symmetry of the molecule, displays potent prothrombotic activities (30,31). Moreover disruption of the pentameric structure, as achieved by urea treatment or by site-directed mutagenesis, leads to enhanced CRP binding to C1q and subsequent C1 activation as well as the ability of CRP to interact with complement-regulatory proteins factor H and C4b-binding protein (32).…”

Purification of recombinant C-reactive protein mutants

“…Storage of purified CRP in the absence of Ca 2+ causes spontaneous dissociation of pentameric CRP into monomers Singh et al, 2009;Zouki et al, 2001). We have reported monomerization of pentameric CRP upon prolonged storage even in the presence of Ca 2+ (Singh et al, 2009).…”

“…We have reported monomerization of pentameric CRP upon prolonged storage even in the presence of Ca 2+ (Singh et al, 2009). The functions of monomeric CRP are different than that of pentameric CRP; monomeric CRP is pro-inflammatory while pentameric CRP is not (Agrawal et al, 2014;Eisenhardt et al, 2009;Lane et al, 2014;Mihlan et al, 2011;Potempa et al, 1987;Thiele et al, 2014;Zouki et al, 2001). Therefore, on the day of the experiments, CRP is re-purified by gel filtration to remove any form of modified CRP generated due to storage of CRP.…”