Crosslinking of human plasma C-reactive protein to human serum albumin via disulfide bond oxidation

Abstract

Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by ‘oxidant-mediated thiol-disulfide reactions’ via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.

Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification

“…This complexity can be overcome by using two-dimensional gel electrophoresis, with proteins separated firstly on the basis of their isoelectric point, and subsequently by molecular mass. Both methodologies have been successfully employed in combination with MS-based strategies [ 54 , 56 , 80 , 84 , 93 , 94 , 141 , 185 ], with isolation and enrichment of fractions containing dimers (and species with higher degrees of oligomerization) from non-crosslinked monomers before MS analysis. This approach has been used successfully to characterize crosslinked forms of IgG, α-synuclein and lysozyme [ 56 , 84 , 141 ].…”

“…Rapid formation of new disulfide bonds has also been reported in so-called 'oxidantmediated thiol-disulfide exchange reactions', where the original disulfide is initially oxidized, by a range of different species, to a more reactive intermediate (e.g., a thiosulfinate or peroxidic intermediate) that subsequently undergoes rapid reaction with another RS − [92-94] (Figure 2, lower section). This type of process gives rise to glutathione (and other thiol) adducts to disulfide-containing peptides and proteins (i.e., glutathionylated species [92]), and also new (intermolecular) protein-protein crosslinks [93,94]. In some cases, the residues involved and the exact sites of the new disulfides have been determined by LC-MS peptide mass mapping [93,94] (see also below).…”

“…This complexity can be overcome by using two-dimensional gel electrophoresis, with proteins separated firstly on the basis of their isoelectric point, and subsequently by molecular mass. Both methodologies have been successfully employed in combination with MS-based strategies [54,56,80,84,93,94,141,185],…”

“…This type of process gives rise to glutathione (and other thiol) adducts to disulfide-containing peptides and proteins (i.e., glutathionylated species [ 92 ]), and also new (intermolecular) protein–protein crosslinks [ 93 , 94 ]. In some cases, the residues involved and the exact sites of the new disulfides have been determined by LC-MS peptide mass mapping [ 93 , 94 ] (see also below). The more rapid rate of these reactions relative to ‘native’ thiol–disulfide exchange arises from the activation of the disulfide via the initial oxidation, which typically involves the formation of an electron-deficient center.…”

Thiol-sensitive probe enables dynamic electrochemical assembly of serum protein for detecting SARS-Cov-2 marker protease in clinical samples

“…The probe molecules are immobilized on a conductive glass slide. Previous reports have successfully used a serum disease marker protein to crosslink the most abundant serum species, the albumin, together( Jiang et al 2021 ). Therefore, using similar methods, the virus protease covalently captured on the sensing surface can serve as the covalent anchoring point of the crosslinked serum proteins (mainly albumin).…”