Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells

•Disulfide formation of unpaired surface cysteine with free cysteine or glutathione does not utilize oxidoreductase-mediated mechanism.•These so-called Cys-capping modifications do not take place in the lumen of endoplasmic reticulum, but outside mammalian cells.•The discovery has led to a novel process to selectively modify thiol side-chains of surface or secreted proteins in live mammalian cells.•The findings have also provided a novel approach for improving the process and quality of protein therapeutics conjugation.

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.