Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation

Abstract

Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans -cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid ( k ~2000 M −1 s −1 ), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F 1 -F o sub-unit alpha (ATP-α-F 1 ) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F 1 oxidation at evolutionary conserved cysteine residues (i.e., C 244 and C 294 ) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50–60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F 1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F 1 -F o ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.

Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions

“…The necessity for a cytotoxic catalyst is, however, problematic [115]. We extended their work by using Inverse Electron Demand Diels Alder (IEDDA) chemistry to develop catalyst-free Click PEGylation workflows [116]. The following subsections critique the chemistry, promise, and challenges of catalyst-free Click PEGylation.…”

“…Third, newly reduced (i.e., reversibly oxidized) thiols are alkylated with trans-cyclooctene (TCO)-PEG3-maleimide (TPN) via Michael addition. The short PEG3 linker enhances TPN hydrophilicity, flexibility, and accessibility [116]. Excess TPN should be removed via a spin column to prevent unproductive competition with TPN decorated protein thiols.…”

“…Mass shifted band intensity is internally normalized to unshifted band (i.e., reduced) intensity to calculate percent reversible thiol oxidation. No between-lane loading control is required because data are internally normalized [116]. Additionally, the contribution of each mass shifted band (i.e., each individual thiol) to the total reversible oxidized signal can be calculated.…”

“…Additionally, the contribution of each mass shifted band (i.e., each individual thiol) to the total reversible oxidized signal can be calculated. Click PEGylation, therefore, enables reversible thiol oxidation to be quantified by Western blotting [98,116]. Importantly, Click PEGylation is compatible with cell, isolated organelle, and tissue lysate analysis.…”

“…In principle, therefore, IEDDA mediated Click PEGylation enables one to immunologically quantify protein thiol redox state by Western blotting. [116].…”