Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol

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.Disulfide bond formation and disulfide rearrangements are reversible processes and are kinetically and thermodynamically affected by the redox state of the environment (1-3). In eukaryotic cells, subcellular compartmentation plays an important role in the regulation of these reversible thiol-disulfide exchange reactions. Glutathione is the most abundant nonprotein thiol in eukaryotic cells, and the preferential transport of the disulfide form (GSSG) compared with the reduced form (GSH) into the ER 1 lumen is thought to be responsible in maintaining redox compartmentation between the ER and cytosol. For murine hybridoma cells, it was shown that in the secretory pathway the ratio of reduced glutathione to the disulfide form (GSH/GSSG) ranged from 1 to 3, whereas the overall cellular GSH/GSSG ratio ranged from 30 to 100 (3). In vitro studies show that the redox environment of the ER corresponds with the optimum for refolding of disulfide-bonded proteins (3). In the ER, disulfide formation and rearrangements are further enhanced by protein disulfide isomerase (4).As soon as the N-terminal part of a nascent polypeptide chain enters the oxidizing environment of the ER, folding starts with the aid of a full array of molecular chaperones and folding catalysts. Failure of disulfide formation has an adverse effect on various processes ranging from protein folding, oligomerization, and intracellular transport to secretion (5-10). In extracellular proteins containing cysteine residues, there is rarely more than one free thiol group (11). Free thiols are considered extremely reactive in the oxidizing extracellular environment and may lead to disastrous polymerization or make folding more complex (11). Th...

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