Abstract
Intrabodies, antibodies expressed within cells, offer an interesting way to target intracellular molecules, making them potentially useful for biotechnology and medicine. However, it remains controversial whether full-size IgG intrabodies expressed in the reducing environment of the cytosol of mammalian cells are workable and structurally sound. Herein, we settle this issue with a systematic investigation of the structure and functionality of four chimeric IgG1s with distinct variable (V) domains but identical constant (C) domains. Full-size IgGs expressed in the cytosol of HEK293 cells were either assembly-competent or -incompetent, depending on the intrinsic properties of the V regions. Structural integrity of the C region is required for H:L association and the formation of a functional antigen-binding site. Partial intrachain disulfide bond formation occurs in both H and L chains of cytosolic IgG intrabodies, whereas interchain disulfide bond formation was absent and dispensable for functional assembly. IgG1s expressed in the cytosol and via the ER were shown to assemble differently. Our findings provide insight into the features and possible utilization of full-size IgGs as cytosolic antibodies in biotechnological and medical applications.
Highlights
The cytoplasm and nucleus disfavor the oxidation of cysteine thiols in proteins because the thiol-disulfide redox potential is too low to drive formation of stable disulfide bonds, and there is a deficiency in enzymes that catalyze protein thiol oxidation in these cellular compartments[1,2,3,4]
We aimed to investigate the functional and structural features of IgGs expressed in the cytosol of HEK293 cells using four chimeric IgG1 antibodies with distinct variable heavy chain (VH) and variable light chain (VL) mouse antibody domains and identical C regions (Cγ1 and Cκ) from human IgG1
These results demonstrate that the H:L association of IgG1s expressed in the reducing cytosol of mammalian cells is determined by the intrinsic sequence of the V regions
Summary
The cytoplasm and nucleus disfavor the oxidation of cysteine thiols in proteins because the thiol-disulfide redox potential is too low to drive formation of stable disulfide bonds, and there is a deficiency in enzymes that catalyze protein thiol oxidation in these cellular compartments[1,2,3,4]. Transient formation of disulfide bonds is found in a few redox-sensitive cytoplasmic proteins such as oxidoreductases and transcription factors, these transient disulfide bonds are related to the control of various metabolic and signaling pathways upon sensing oxidants under oxidative stress conditions[6,7]. Most intrabodies employed to date are monovalent recombinant single chain variable fragment (scFv) antibodies composed of a variable heavy chain (VH) domain and a variable light chain (VL) domain connected by a peptide linker. This simple structure has only two intrachain disulfide bonds. The findings expand our understanding of functional and structural aspects of full-size IgG intrabodies, and their potential uses
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