A redox switch in C‐reactive protein modulates activation of endothelial cells

Abstract

C-reactive protein (CRP) has been implicated in the regulation of inflammation underlying coronary artery disease; however, little is known about the molecular mechanisms responsible for the expression of its pro- or anti-inflammatory activities. Here, we have identified the intrasubunit disulfide bond as a conserved switch that controls the structure and functions of CRP. Conformational rearrangement in human pentameric CRP to monomeric CRP (mCRP) is the prerequisite for this switch to be activated by reducing agents, including thioredoxin. Immunohistochemical analysis revealed 36-79% colocalization of thioredoxin and mCRP in human advanced coronary atherosclerotic lesions. Nonreduced mCRP was largely inert in activating human coronary artery endothelial cells (HCAECs), whereas reduced or cysteine-mutated mCRP evoked marked release of IL-8 and monocyte chemoattractant protein-1 from HCAECs, with ~50% increase at a concentration of 1 μg/ml. Reduced mCRP was ~4 to 40-fold more potent than mCRP in up-regulating adhesion molecule expression, promoting U937 monocyte adhesion to HCAECs, and inducing cytokine release from rabbit arteries ex vivo and in mice. These actions were primarily due to unlocking the lipid raft interaction motif. Therefore, expression of proinflammatory properties of CRP on endothelial cells requires sequential conformational changes, i.e., loss of pentameric symmetry followed by reduction of the intrasubunit disulfide bond.

Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis

“…This has led us to propose that the dissociation of the CRP molecule is an activating mechanism that is required for the expression of enhanced bioactivities and is also a buffering mechanism that localizes the actions of mCRP into inflammatory loci to prevent the possible global effects directly induced by large-scale alterations in the serum levels of CRP [73]. Consistent with our proposal, immunohistochemical analyses using highly specific antibodies revealed that mCRP, not CRP, was the major isoform present in local lesions, including atherosclerotic plaques [55,76], diabetic kidneys [77] and stroke neovessels [78].…”

“…Comparable results were obtained with Cys-mutated mCRP, in which the two cysteines were mutated to alanines. These findings thus indicate the intra-subunit disulfide bond as an important switch modulating the activities of mCRP [55].…”

“…These lines of evidence thus identify lipid rafts as the major cellular sensors of reduced mCRP [92] and reveal that the unlocking of CBM in mCRP after reduction underlies the enhanced capacity of reduced mCRP to interact with lipid rafts and to stimulate cellular responses [55]. Interestingly, rabbit mCRP also showed enhanced capacity to activate EC after reduction [55]. This observation provides evidence that cholesterol binding and redox regulation of mCRP are functionally conserved.…”

“…In addition, the specific antibody 8C10 against the epitope containing the CBM markedly repressed lipid raft insertion of Cys-mutated mCRP. These lines of evidence thus identify lipid rafts as the major cellular sensors of reduced mCRP [92] and reveal that the unlocking of CBM in mCRP after reduction underlies the enhanced capacity of reduced mCRP to interact with lipid rafts and to stimulate cellular responses [55]. Interestingly, rabbit mCRP also showed enhanced capacity to activate EC after reduction [55].…”

“…"mCRP" is also used as the abbreviation for "modified CRP" because the conversion to mCRP is usually accompanied by significant alterations in the native subunit structure and epitope expression. Accordingly, a significant loss of secondary structural elements and a marked relaxation of the tertiary packing have been demonstrated upon dissociation of CRP to mCRP [54][55][56]. Presumably due to the prominent conformation rearrangement, mCRP is unable to spontaneously re-associate back to the cyclic pentameric CRP [57]; rather, this molecule is prone to aggregating into high-order oligomers composed of tens of monomers [58].…”