Abstract
Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fragment consists of the variable light and heavy chain domains from a mouse IgG antibody, which are connected by a flexible linker peptide. In each domain one disulfide bridge is present. The functionality of antibodies, which are normally secreted via the oxidizing environment of the endoplasmic reticulum, depends on the formation of intramolecular disulfide bridges. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. In contrast, transient expression in tobacco protoplasts resulted in a cytosolic scFv lacking disulfide bridges, which had a substantially reduced affinity for the antigen. This indicates that functionality rather than stability is determined by the presence of disulfide bridges in the in planta-expressed scFv antibody. The controversial observation of disulfide bond formation in the cytosol is discussed.
Highlights
Disulfide bond formation and disulfide rearrangements are reversible processes and are kinetically and thermodynamically affected by the redox state of the environment [1,2,3]
Presence of Disulfide Bonds in the single-chain Fv (scFv) Located in the ER and Cytosol—We previously generated transgenic tobacco plants expressing functional scFv-CK and scFv-SK antibodies, which were located in the cytosol and ER, respectively [23, 25]
It was calculated that RNase A, which efficiently refolds at a GSH/GSSG ratio of 2 in the presence of protein disulfide isomerase, would not be able to refold at the cytosolic GSH/GSSG ratios both in the presence and absence of protein disulfide isomerase [3]
Summary
In this report the redox states of scFv antibodies located in either the ER or the cytosol of tobacco (Nicotiana tabacum) were compared. We establish that in transgenic plants intramolecular disulfide bridges are present in both the cytosolic scFv and the scFv located in the ER. Transient expression of the scFv in the ER of tobacco protoplasts shows intramolecular and intermolecular disulfide bridges. No disulfide bridges are present when the scFv gene is expressed transiently in the cytosol. The expression levels are comparable with those in transgenic plants, the binding properties of this cytosolic scFv are very poor. The relationships between transformation system, subcellular location, redox potential, and disulfide bridge formation of heterologous proteins in the plant cytosol and the possible consequences are discussed
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