Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation

Abstract

Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen ( 1 O 2 )-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1 O 2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18 O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ‘proof-of-concept’ for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.

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).…”

“…Crosslinking can occur via the thioether group of methionine in the enzymatic (peroxidasin)-induced crosslinking of the NC1 domains of collagens. Reaction of a specific Met residue (Met 93 ) with HOBr formed by peroxidasin generates a transient bromosulfonium ion [-S(Br) + -] that reacts with the amine group of a suitably positioned hydroxy-Lys residue (Hyl 211 ) to give an interchain sulfilimine (-S=N-) crosslink. These crosslinks are critical for the generation of functional extracellular matrices in many organisms, with an absence of this enzymatic activity causing tissue dysfunction that is embryonically lethal in some species [16,17].…”

“…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.…”