Monomeric C-reactive protein regulates fibronectin mediated monocyte adhesion
Graphical abstract
Circulating CRP dissociates into monomeric CRP (mCRP) and deposits in inflamed loci. mCRP interacts with FN and enhances monocytes adhesion, upregulates adhesion molecules, and mediates the inflammatory process.
Introduction
C-reactive protein (CRP) is a major human acute phase reactant that has been highly conserved in sequence and structure during evolution (Casas et al., 2008; Pepys and Hirschfield, 2003). Similar to SAP and PTX3, CRP belongs to the pentraxin family, which consists of five identical subunits that associate via non-covalent interactions (Casas et al., 2008; Du Clos, 2013; Pepys and Hirschfield, 2003; Schwedler et al., 2006). CRP can recognize multiple endogenous and exogenous molecules, such as the plasma membrane of apoptotic or necrotic cells, the cell walls of bacteria and polysaccharides through its ligand-binding surface. CRP activity quite resembles that of pattern recognition receptors, which is essential for innate defense (Agrawal et al., 2014; Bottazzi et al., 2010; Du Clos, 2013).Conformational rearrangement of pentameric CRP (pCRP) leads to the formation of monomeric CRP (mCRP) (Eisenhardt et al., 2009; Ji et al., 2007; Thiele et al., 2014). In addition to exhibiting unique antigenicity, mCRP has biological activities distinct from those of CRP (Potempa et al., 1983). mCRP can recognize ligands more efficiently and flexibly than CRP (Ji et al., 2006b) and, regulate the activation of the complement system (Ji et al., 2006a) as well as LDL metabolism (Ji et al., 2006b). In addition, mCRP serves as a potent activator of endothelial cells, neutrophils, and platelets (Eisenhardt et al., 2009; Ji et al., 2009, 2007; Khreiss et al., 2004, 2005; Slevin et al., 2010), and it can significantly upregulate the expression of inflammatory cytokines at low concentrations in a short time period (Ji et al., 2007; Khreiss et al., 2004). Therefore, mCRP may be a more functionally active CRP isoform.
Fibronectin (Fn) is a multifunctional glycoprotein and a main component of the extracellular matrix (ECM), which is also found at high concentrations in plasma (Graille et al., 2010). Fn plays key roles in cell adhesion and migration processes, thrombosis and cancer (Magnusson and Mosher, 1998; Zhou et al., 2015). In inflamed tissue loci and the tumor microenvironment, whether CRP dissociates into mCRP and whether mCRP directly interacts with FN, and affects related physiological and pathological functions remain unclear. Thus, understanding the mechanism underlying the interaction of monomeric CRP with FN and its impact on each other’s functions is of importance.
In previous studies, some features of the CRP-Fn interaction have been reported (Agrawal et al., 1992; Suresh et al., 2004). The maximal interaction of CRP with FN occurs at pH = 5 (Salonen et al., 1984). At physiological pH, the binding of CRP to FN was inhibited by Ca2+ (Salonen et al., 1984; Suresh et al., 2004). In vivo, CRP in the Ca2+- bound form could interact with FN only at inflammatory loci or in the tumor microenvironment, which has low pH levels (Suresh et al., 2004). Interestingly, all these conditions could trigger the dissociation of pentameric CRP (pCRP) to mCRP (Eisenhardt et al., 2009; Ji et al., 2007; Li et al., 2018). These evidences hint that mCRP may be the primary isoform that interacts with FN in vitro and in vivo. In this study, we investigated the interaction of mCRP with FN and its effect on FN-mediated monocyte adhesion as well as the underlying mechanisms. Furthermore, we aimed to identify the CRP dissociation process as a therapeutic target.
Section snippets
Reagents
Human native pentameric CRP (purity>99 %; purified from ascites) was purchased from Binding Site (Birmingham, UK; catalog number: BP300.X).Recombinant mCRP was prepared as described (Khreiss et al., 2004; Wang et al., 2011). Urea denatured mCRP was produced by dissociation of pentameric CRP in 8 M urea/10 mM EDTA for 2 h at 37 °C as described previously (Wang et al., 2011). Proteins were dialyzed to remove NaN3, and passed through Detoxi-Gel Columns (Thermo Fisher Scientific, Rockford, IL;
FN selectively binds to mCRP
Circulating CRP is composed of five identical subunits that associate via non-covalent interactions, but dissociates into the monomeric isoforms (mCRP) in inflamed tissue or local tumor lesions(Thiele et al., 2014, 2015; Wu et al., 2015). We first confirmed using ELISA with mCRP/CRP specific mAb or SRP binding assaysthat FN selectively binds to mCRP instead of pentameric CRP (Fig. 1). The binding of mCRP to FN was concentration dependent and saturable, fitting well to a 1:1 Langmuir binding
Discussion
The importance of the conformational transition of CRP in its interaction with ligands and involvement in disease processes, such as inflammation and cancer, is often overlooked, and it is generally assumed that CRP can bind to FN or some other ligands only under acidic pH and without Ca2+. Coincidentally, these are also the in vitro conditions that can induce the conformational change of CRP. In this study, we tested when CRP dissociated into mCRP, the binding characteristics and molecular
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgements
We thank Dr. Lawrence A. Potempa for the gift of mCRP antibodies. This work was supported by grants from NSFC [31800654], the Fundamental Research Funds for the Central Universities [sxzy012019076] and the China Postdoctoral Science Foundation [2018T111039, 2016M602799].
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These authors contributed equally to this work.