Hypochlorite Action on Plasma Fibronectin Promotes Its Extended Conformation in Complexes with Antibodies

Abstract

We investigated the influence of hypochlorite (HOCl/OCl-) on plasma fibronectin (Fn) aggregation and examined an affinity of Fn aggregates to Fn specific antibodies. Human plasma Fn HOCl/OCl(-)-mediated modification was monitored with differential OD method and with measurements of tryptophan fluorescence followed by acrylamide quenching of tryptophan emission. Antibody fibronectin complex formation was examined in ELISA systems with chemiluminescence (CL) detection. Results were expressed as an average of three experiments performed in triplicate. Fn aggregation/fragmentation was monitored with dynamic light scattering method. It was showed that HOCl/OCl- mediated chlorination promotes Fn aggregation/fragmentation with concomitant oxidation of tryptophan moieties and dichlorotyrosine formation. Quenching experiments revealed that in chlorinated Fn the percentage of intact tryptophan moieties buried in the hydrophobic Fn core increases as compared to unchlorinated Fn. In general, ELISA experiments showed that chlorination of plasma Fn diminished the number of available epitopes but for lower HOCl/OCl- concentrations (1-2 mM) the reverse effect is observed--the number of accessible fibronectin epitopes is increased when Fn adopts extended conformation in complex with antibody. Our results suggest that HOCl/OCl(-)-mediated plasma Fn chlorination leads to the formation of soluble aggregates and is followed by refolding processes. Fn chlorination with low doses of HOCl/OCl- promotes extended Fn conformation which in turn increases affinity toward specific antibodies and may promote Fn-IgG cluster formation. Thus it seems possible that mildly chlorinated plasma Fn promotes formation of IgG clusters which in turn may activate neutrophils.

Detection, identification, and quantification of oxidative protein modifications

“…However, analysis of such data can be complex, as damage may also enhance antibody binding by exposing cryptic epitopes. Thus, low doses of HOCl appear to increase the affinity of anti-fibronectin antibodies to plasma fibronectin, whereas high concentrations have the opposite effect (87). This has been rationalized in terms of the generation of an extended fibronectin conformation at low HOCl doses, and aggregation with high concentrations.…”

Myeloperoxidase-derived damage to human plasma fibronectin: Modulation by protein binding and thiocyanate ions (SCN−)

“…These only occurred in the presence of H 2 O 2 , and hence are not a result of direct MPO binding, and therefore are likely to arise from oxidant-induced reactions. HOCl can modify specific protein side chains, resulting in unfolding or altered protein conformations, that migrate differently on gels, and thereby appear to have an altered molecular mass [ 16 , 77 ]. Oxidation can also increase the molecular mass of proteins as a result of (multiple) oxygen or chlorine atom incorporation [ 78 ], but the apparent mass changes are large, and hence unlikely to arise from this mechanism.…”

Human plasma and cerebrospinal fibronectins differ in the accessibility of the epitopes on the N-terminal domains.

“…Thus the distribution of electrostatic forces stabilizing the protein structures is probably different in the plasma and cerebrospinal fluid. Some in vitro experiments show that native fibronectin can undergo dramatic structural changes from a globular to an extended conformation after treatment with low doses of hypochlorite (Olszowski et al, 2003), or after urea-induced denaturation (Lairez et al, 2003;Patel et al, 2006;Nelea et al, 2008). In such cases the accessibility of fibronectin epitopes for an antibody increases.…”

“…In such cases the accessibility of fibronectin epitopes for an antibody increases. Olszowski et al (2003) have shown that the number of accessible fibronectin epitopes increases when fibronectin adopts an extended conformation in complex with the antibody. In our experiment under the mildly denaturing conditions of 0.5-2 M urea, the hidden epitopes of the N-terminal and some of the CBD domains were probably exposed and made more accessible for reaction with the respective antibodies.…”

“…It has been shown that FN has a globular shape in the blood, but it tends to form a multimeric network of fibers when assembled by cells. Some laboratory experiments have also shown that FN reveals large-scale flexibility and local order and can exist in unfolded and refolded forms (Johnson et al, 1999;Olszowski et al, 2003;Patel et al, 2006). It is claimed that the FN unfolding and extension within the cell matrix is controlled by cytoskeletal tension (Baneyx et al, 2002).…”