@article{dfcc84aa22874222ab7ef6ebff0c701f,
title = "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 {\textquoteleft}oxidant-mediated thiol-disulfide reactions{\textquoteright} 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.",
keywords = "Aggregation, C-reactive protein, Crosslink, Disulfide, Hypochlorous acid, Peroxynitrite, Protein oxidation",
author = "Shuwen Jiang and Per H{\"a}gglund and Luke Carroll and Rasmussen, {Lars M.} and Davies, {Michael J.}",
note = "Funding Information: Our previous studies, and other research on model systems, have provided evidence for the formation of zwitterion peroxides [RS-S+(-OO-)R] on reaction of both low-molecular-mass and protein disulfides, with 1O2, and thiosulfinates [RS-S(=O)R] with two electron oxidants [ 40?42, 44?48]. These species have modest lifetimes (several hours [42]), but undergo subsequent rapid reaction with other thiols to give thiolated proteins in which the original disulfide bond is cleaved, and a new disulfide generated between the added thiol and one of the Cys residues of the original disulfide. This process has been termed ?oxidant-mediated thiol-disulfide exchange? [42]. Whilst initial studies examined reaction with low-molecular mass thiols (e.g. GSH, N-acetyl cysteine [ 40?42], reaction can also occur with a thiol group present on a second protein (e.g. GAPDH) with this yielding a new protein-protein crosslink [43]. The current study extends this work to show that CRP undergoes this type of reaction with HSA, both in isolated systems and also in fresh human plasma, with reaction occurring between Cys34 on HSA and one of the Cys residues (Cys36) of the former disulfide of CRP. Thus, a new species is observed at an apparent mass of ~75 kDa when the pre-oxidized CRP was subsequently incubated with HSA. The formation of this species was inhibited by pre-treatment of the HSA with NEM to block the free thiol ( Figs. 2, 3). This band was recognized by both anti-CRP and anti-HSA antibodies consistent with the presence of both proteins. The apparent molecular mass of this species (~75 kDa) is lower than that expected from the combined mass of the two parent proteins (23 kDa and 66.5 kDa respectively). The reason for this anomalous apparent mass (commonly called ?gel shifting?) is not completely resolved, but is a common feature of gels run under non-reducing conditions, and is widely observed. It has been proposed to result from altered detergent/surfactant binding, or the pI of the protein [84,85], though it may also be related to altered migration of dimeric species. The LC-MS/MS analyses carried out on the excised gel bands from this species, provide very strong evidence for the presence of both CRP and HSA in this band, and supports its assignment to a novel CRP-HSA dimer.Consistent with our proposed mechanism, pretreatment of the plasma samples with the thiol-blocking reagent NEM significantly inhibited the intensity of the proposed CRP-HSA crosslink species ( Figs. 2, 3), supporting the role of the Cys34 residue on HSA in the generation of these species. Whilst this has not been investigated as yet, it is possible that similar reactions occur with other thiols present on other plasma proteins (although these are present at much lower concentrations), and also with low-molecular-mass thiols present in plasma (e.g. cysteine, cysteinylglycine, glutathione, homocysteine, and ?-glutamylcysteine, which are present at ~12?20 ?M in total [99]). Analogous reactions may also occur with other plasma proteins (e.g. immunoglobulins) that contain disulfide bonds, and this would appear to be worthy of further study in order to determine the generality of the reactions described here. Thus, the chemistry outlined here may be a common mechanism for the formation of reducible protein-protein crosslinks, including with proteins that do not contain initial free Cys residues.The authors are grateful for financial support from the Novo Nordisk Foundation (Laureate grants: NNF13OC0004294 and NNF20SA0064214 to MJD), the China Scholarships Council (PhD scholarship to SJ: 201708340066), a WHRI International Fellowship (to LC) co-funded by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007?2013) under REA grant agreement n? 608765, and an infrastructure grant from the Carlsberg Foundation (CF19-0451 to PH). Publisher Copyright: {\textcopyright} 2021 The Author(s) Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",
year = "2021",
month = may,
doi = "10.1016/j.redox.2021.101925",
language = "English",
volume = "41",
journal = "Redox Biology",
issn = "2213-2317",
publisher = "Elsevier",
}