title: Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells.
abstract: 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.
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States. Electronic address: Xiaotian.Zhong@pfizer.com.
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhong
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Tao
authors.initials: T
authors.lastname: He
authors.affiliation: Pharmaceutical Sciences, Medicinal Sciences, Pfizer Worldwide R&D, Pearl River, NY 10965,United States.
authors.forename: Amar S
authors.initials: AS
authors.lastname: Prashad
authors.affiliation: Andover, MA 01810,United States.
authors.forename: Wenge
authors.initials: W
authors.lastname: Wang
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Justin
authors.initials: J
authors.lastname: Cohen
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Darren
authors.initials: D
authors.lastname: Ferguson
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Tam
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Sousa
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Laura
authors.initials: L
authors.lastname: Lin
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Tchistiakova
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Scott
authors.initials: S
authors.lastname: Gatto
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Aaron
authors.initials: A
authors.lastname: D'Antona
authors.affiliation: Andover, MA 01810,United States.
authors.forename: Yen-Tung
authors.initials: YT
authors.lastname: Luan
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Weijun
authors.initials: W
authors.lastname: Ma
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Richard
authors.initials: R
authors.lastname: Zollner
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Pharmaceutical Sciences, Medicinal Sciences, Pfizer Worldwide R&D, Pearl River, NY 10965,United States.
authors.forename: Bo
authors.initials: B
authors.lastname: Arve
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.affiliation: BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, Cambridge, MA 02139, United States.
authors.forename: Ronald
authors.initials: R
authors.lastname: Kriz
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004136:Dinitrobenzenes; D057809:HEK293 Cells; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000906:Antibodies; D004136:Dinitrobenzenes; D011994:Recombinant Proteins; C010936:2,4-dinitrothiocyanatobenzene; D003545:Cysteine
keywords: Antibody; Cys-capping modification; Free thiol; Live mammalian cells; Selective chemical engineering; Unpaired surface cysteine