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Originally published In Press as doi:10.1074/jbc.M201245200 on March 26, 2002

J. Biol. Chem., Vol. 277, Issue 22, 19339-19345, May 31, 2002
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Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol*

Alexander SchoutenDagger, Jan Roosien, Jaap Bakker, and Arjen Schots

From the Laboratory of Nematology/Laboratory of Molecular Recognition and Antibody Technology, Department of Plant Sciences, Wageningen University, P. O. Box 8123, 6700 ES Wageningen, The Netherlands

Received for publication, February 6, 2002, and in revised form, March 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 non-protein thiol in eukaryotic cells, and the preferential transport of the disulfide form (GSSG) compared with the reduced form (GSH) into the ER1 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). The extracellular protein albumin carries a reactive free cysteine, but when not buried in the tertiary structure, this residue performs a specific physiological function (12, 13).

The general idea is that the typical glutathione redox state of the cytosol in eukaryotic cells does not favor the formation of protein disulfide bonds (2, 11). Also in prokaryotic organisms, intracellular disulfide bond formation seems to be hindered by the redox potential. Many recombinant proteins with disulfide bonds cannot fold properly in Escherichia coli, where the intracellular GSH-GSSG ratio ranges from 50 to 200 (3). On the other hand, it has been suggested that the redox state of the cytosol in eukaryotes is not constant and may change in response to e.g. physiological and metabolic stimuli. Also specialized intracellular compartmentation may provide localized environments within the cell where the thiol-disulfide redox state may be very different from that of the bulk cytoplasm. However, apart from the comparison of the redox state of the ER with the intracellular environment as a whole, techniques to monitor changes or subtle subcellular redox compartmentation are nonexistent. Nevertheless, various lines of evidence indicate that redox modulation for some specific cytosolic proteins is an important regulatory mechanism in eukaryotic organisms (14, 15). In vitro studies demonstrate that various cytosolic enzymes are reversibly activated or inactivated upon incubation with disulfides or thiols (16, 17). Disulfide bridges are often associated with an inactivated protein (1, 18). These observations are consistent with metabolic changes in cell cultures, perfused organs, and whole animals induced by manipulating the cellular thiol to disulfide balance by applying reducing agents (7, 19-21). Although the biological significance of reversible thiol-disulfide exchange reactions for the cytosol remains controversial, in chloroplasts there is little question about the role of redox modulation in regulating the activity of key enzymes (2, 22). Also, for nuclei, evidence has been obtained that redox modulation influences the activity of transcription factors (2).

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. The cytosolic scFv has binding properties that are similar to the scFv located in the ER. Furthermore, the scFvs present in the cytosol are, like the scFvs in the ER, capable of forming intermolecular disulfides through a cysteine present in the linker peptide. Transient expression of the scFv in the ER of tobacco protoplasts also shows intramolecular and intermolecular disulfide bridges. Remarkably, no disulfide bridges are present when the scFv gene is expressed transiently in the cytosol. Although 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines, Strains, and Cloning Vectors-- A scFv directed against a fungal cutinase was derived from mouse immunoglobulin cDNA of the hybridoma cell line 21C5 (23). Cloning procedures were according to Sambrook et al. (24) using the E. coli strains DH5alpha , TG1, and 190 and the scFv cloning vectors pNEM5 and pNEM5K (23). For construction of the various 21C5 anti-cutinase scFv genes, the pNEM-scFv-K (23) was used.

Transgenic tobacco plants expressing scFv-SK and scFv-CK (23, 25) were used for analysis. As a control we used untransformed tobacco (N. tabacum cv. Samsun NN) or a transgenic tobacco plant that was transformed with the empty transformation vector pCPO33T (23). For transient expression assays, tobacco leaf protoplasts were transfected using pRAP-scFv-SK, pRAP-scFv-CK (25), and as a control, the empty transient expression vector pUCAP35S (26) was used.

ScFv Modifications-- To replace the cysteine residue in the linker of the anti-cutinase scFv 21C5 by a serine residue, the XhoI/PvuII fragment in pNEM-scFv was replaced by a suitable adapter (5'-TCGAGTCTGAGGTCCAG-3' and 5'-CTGGACCTCAGAC-3'), resulting in the pNEM-scFvser (Fig. 1). The scFv constructs were cloned as SalI/NotI fragments from pNEM-scFvser into pRAP-scFv-SK, creating and pNEM-scFvser-SK. All modifications were verified by sequencing (27).


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  		Fig. 1.   Diagram of the scFv-SK, scFvser-SK and scFv-CK constructs. In the scFvser-SK construct the cysteine residue present in the linker peptide was replaced by serine. The N-terminal signal peptide (SP), the single chain antibody construct (scFv) with the variable light (VL) and heavy (VH) chain domains connected by the linker peptide (L), the location of the two cysteines (boxed C) in each variable domain, which can form intramolecular disulfide bridges (---), and the C-terminal c-Myc tag (t) followed by the KDEL peptide sequence (K) are indicated.

Preparation of Protoplasts and Transient Gene Expression by Transfection-- Protoplasts were obtained from transformed tobacco plants expressing scFv antibodies in the ER and cytosol at a level of 1 and 0.2% of total soluble protein (23) by the method described (28). As a control, protoplasts were isolated from untransformed tobacco leaves (N. tabacum cv. Samsun NN).

For transient gene expression assays the same procedure was used to obtain protoplasts from tobacco leaves (N. tabacum cv. Samsun NN). Then plasmid DNA was transfected to the protoplasts according to the polyethylene glycol procedure as described (28).

Analysis of Intrachain Disulfide Bonds-- The protoplasts from stable transformants or from a transfection assay expressing both scFv-SK and scFv-CK proteins were separated from the incubation medium and lysed by adding an equal volume of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM Pefabloc, 80 mM N-ethylmaleimide (NEM), and subsequent vortexing. The suspension was kept at 0 °C for 5 min and at room temperature for 10 min. The chloroplasts were removed from the lysate by centrifugation, and the supernatant was split into two portions. The first portion received one-third volume of 4× SDS-PAGE sample buffer containing 244 mM Tris-HCl, pH 6.8, 8% (w/v) SDS, 50% (w/v) glycerol, and the second portion received one-third volume of 4× SDS-PAGE sample buffer and dithiothreitol (DTT) to a final concentration of 55 mM. Both samples were boiled for 5 min. To the DTT-containing portion NEM was added to a final concentration of 200 mM and incubated for 15 min at room temperature. The proteins were pelleted by ethanol precipitation and subsequent centrifugation. After drying, the pellet was resuspended in 1× SDS-PAGE sample buffer with 0.008% (w/v) bromphenol blue. Protein samples were loaded on a 13% SDS-polyacrylamide gel (29) (Bio-Rad mini-protean system). After electrophoresis the proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting. For immunodetection, the membranes were incubated with 1:1000-diluted anti-c-Myc monoclonal antibody (9E10) (30) followed by a 1:5000-diluted rat-anti-mouse alkaline phosphatase conjugate (Jackson ImmunoResearch). The blots were stained in 0.1 M ethanolamine-HCl, pH 9.6, supplemented with 4 mM MgCl2, 5-bromo-4-chloro-3-indolyl phosphate (0.06 mg/ml) and nitro blue tetrazolium (0.1 mg/ml). The relative molecular weights of the proteins were estimated with pre-stained low range molecular weight markers (Bio-Rad).

Analysis of Interchain Disulfide Bonds-- Protoplasts transfected with scFv-SK and scFvser-SK were separated from the incubation medium and lysed by adding an equal volume of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM Pefabloc SC (Roche Molecular Biochemicals) and subsequent vortexing. The chloroplasts were removed from the lysate by centrifugation at 13,000 × g for 2 min. For Western analysis one-third volume 4× SDS-PAGE sample buffer was added to an aliquot of the supernatant, equaling 3 × 104 protoplasts. DTT and bromphenol blue were added to final concentrations of 40 mM and 0.008% (w/v), respectively, and the samples were boiled at 100 °C for 5 min. For non-reducing gel electrophoresis, DTT was omitted during sample preparation. The protein samples were analyzed by SDS-polyacrylamide gel followed by electroblotting and immunodetection as described above.

Affinity of Oxidized and Reduced Anti-cutinase scFv for Its Antigen-- The affinity of the oxidized and reduced scFv for cutinase was determined by quantitative ELISA. To prevent disulfide bridge formation during sample preparation, alkylated protein samples were prepared as followed. The proteins from transgenic plants were extracted by grinding 0.2-0.4 g of tobacco leaves in liquid nitrogen to a fine powder. The powder was transferred to an Eppendorf tube and mixed 1:2 (w/v) with 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 60 mM NEM, 1 mM Pefabloc SC (Roche Molecular Biochemicals) and subsequent vortexing. As a control, extracts were prepared in the presence of 4% ethanol (the solvent of NEM) instead of NEM. The suspension was kept at 0 °C for 5 min and at room temperature for 10 min. Insoluble material was removed by centrifugation at 13.000 × g. To the supernatant, 25% (v/v) 5× PBS, 0.5% (v/v) Tween, 5% (w/v) skimmed milk powder was added. Serial dilutions were made in 1× PBS, 0.1% (v/v) Tween (PBST), 1% (w/v) skimmed milk powder, and loaded on an ELISA plate.

Proteins from the protoplasts, transfected with pRAP-scFv-CK, pRAP-scFv-SK, and pUCAP35S, were obtained as follows. The cells were separated from the incubation medium and lysed by adding an equal volume of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM Pefabloc SC (Roche Molecular Biochemicals) and subsequent vortexing. The lysate was kept at 0 °C for 5 min and at room temperature for 10 min, and the chloroplasts were removed by centrifugation at 13,000 × g for 2 min. The supernatant was diluted with 5 volumes of PBST-1.25% (w/v) skimmed milk powder and loaded on a 96-well ELISA plate that had been coated overnight with 2.5 µg/ml cutinase in 50 mM sodium carbonate, pH 9.6 (100 µl/well). After blocking for 30 min with 200 µl of PBST, 5% skimmed milk powder per well, the plates were washed, and 100 µl of protein extract per well was added. The plate was incubated for 2 h. After washing with PBST, each well was incubated for another 2 h with 100 µl of anti-c-Myc tag antibody 9E10 (1 ng/µl) in PBST, 1% skimmed milk powder. Then, after washing 3 times with PBST, the wells were incubated for 1 h with alkaline phosphatase-conjugated rat-anti-mouse antibody (Jackson ImmunoResearch) diluted 1:5000 in PBST, 1% skimmed milk powder. Finally the wells were washed five times with PBST. 100 µl of substrate (0.75 mg/ml p-nitrophenylphosphate in 1 M diethanolamine, pH 9.8) was added, and the A405 was monitored. All incubations were carried out at 37 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Presence of Disulfide Bonds in the scFvs 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). The scFv-SK antibody contains a signal sequence for translocation into the ER combined with the ER retention signal KDEL (31). The scFv-CK, located in the cytosol, lacks the ER translocation signal. All scFv antibodies carry the c-Myc tag sequence for detection purposes.

To compare the in vivo redox state of the disulfide bridges in the cytosolic scFv-CK and ER-located scFv-SK in both transgenic plants and transfected protoplasts, we isolated protoplasts from transgenic tobacco leaves according to the procedure used for a transient expression assay. The protoplasts were cooled and immediately lysed in the presence of the sulfhydryl alkylating agent NEM to block free thiols and prevent rearrangements of disulfide bonds. The scFv proteins were analyzed by SDS-PAGE followed by Western blotting and immuno-detection using the anti-c-Myc antibody, 9E10 (Fig. 2). This showed under non-reducing conditions for both scFv-CK and scFv-SK, a protein band at 32 kDa, the estimated molecular mass of the scFv antibodies, and at ~65 kDa (Fig. 2A, lanes 1 and 3). In the presence of the reducing agent DTT only a protein band at 33 kDa was detected (Fig. 2A, lanes 2 and 4). No protein bands were detected in protoplasts obtained from the plants transformed with the empty transformation vector pCPO33T (Fig. 2A, lane 5). Apparently, as reported before (32), the reduction of the two intramolecular disulfide bridges in a scFv antibody results in the shift in migration from 32 to 33 kDa. The 65-kDa band present in both the scFv-CK and scFv-SK is only present under oxidizing conditions (i.e. in the absence of DTT) and, as reported before (23), shows antigen binding capabilities. This suggests it to be a scFv dimer, formed through intermolecular disulfide bridge formation by the cysteine residue present in the linker peptide (Fig. 1). Thus, in transgenic tobacco, both the cytosolic scFv-CK and ER-located scFv-SK are present in an oxidized state.


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  		Fig. 2.   Determining the redox state of cysteine residues present in scFv-SK and scFv-CK proteins expressed by stably transformed plants and in a transient expression assay. Panel A, Western blot of total protein from stably transformed tobacco plants expressing scFv-CK and scFv-SK in the absence (-) or presence (+) of the reducing agent DTT. The scFv proteins were detected with the anti-c-Myc antibody 9E10. The positions of the reduced (red.) and oxidized (ox.) scFv proteins and the presumed scFv dimer (dimer) are indicated. As a control, a tobacco plant stably transformed with the empty transformation vector pCPO33T was used (Control). Panel B, Western blot of total protein from protoplasts transfected with the vectors containing the scFv-CK and scFv-SK gene cassettes in the absence (-) or presence (+) of the reducing agent DTT. The scFv proteins were detected with the anti-c-Myc antibody 9E10. The positions of the reduced (red.) and oxidized (ox.) scFv proteins and the presumed scFv dimer (dimer) are indicated. As a control, a vector without scFv gene cassette was used (Control).

The transfected protoplasts synthesizing scFv-SK also showed the 32- and 65-kDa protein bands under non-reducing conditions and the 33-kDa band under reducing conditions (Fig 2B, lanes 3 and 4). Both under non-reducing and reducing conditions the protoplasts transfected with scFv-CK showed only the 33-kDa band (Fig 2B, lanes 1 and 2). Only a weak background was detected in the transient expression assay when the empty expression vector pUCAP35S was transfected (Fig. 2B, lane 5). Therefore, in contrast to scFv-SK, the transiently expressed scFv-CK lacks the intramolecular and intermolecular disulfide bridges and, thus, is present in a reduced state. This situation deviates from the oxidized scFv-CK present in stable transformed plants.

Analysis of the scFv Dimer Formation-- To ascertain that the 65-kDa protein observed in the transgenic plants is formed through disulfide bridge formation using the cysteine present in the linker, we modified the linker region in the scFv-SK gene (Fig. 1). The cysteine-coding triplet (TGT) was changed into a serine-coding triplet (TCT), creating the scFvser-SK gene. We anticipated that, if the cysteine residue in the linker peptide was involved in dimer formation, scFvser-SK expression would not result in the formation of the disulfide band under non-reducing conditions. We only modified the scFv-SK gene because we wanted to analyze the 65-kDa band through a transient expression assay. Because as mentioned before the scFv-CK synthesized in a transfection assay does not form disulfide bridges, a transient expression assay with a modified scFv-CK would be useless to obtain the required information.

The scFv-SK and scFvser-SK genes were both transfected to tobacco protoplasts, and the synthesized scFv proteins were analyzed by Western blotting and immuno-detection using the anti-c-Myc antibody, 9E10 (Fig. 3). Under non-reducing conditions scFv-SK expression resulted in a weak 32-kDa and relatively strong 65-kDa protein band, and the scFvser-SK expression resulted only in a 32-kDa protein band (Fig. 3, lanes 1 and 2). Under reducing conditions both scFv-SK and scFvser-SK expression resulted in 33-kDa protein bands (Fig. 3, lanes 4 and 5). Under both conditions, only a weak background was detected in the transient expression assay with the empty expression vector pUCAP35S (Fig. 3, lanes 3 and 6). The 65-kDa band is only formed under non-reducing conditions when the cysteine is present in the linker peptide, indicating that the 65-kDa protein represents a dimerized scFv.


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  		Fig. 3.   Determining the role of the cysteine residue present in the linker peptide of scFv-SK in dimer formation. Western blot of total protein from protoplasts transfected with the vectors containing the scFv-SK and scFvser-SK gene cassettes in the absence (-DTT) or presence (+DTT) of the reducing agent dithiothreitol. The scFv proteins were detected with the anti-c-Myc antibody 9E10. The scFv monomer and dimer proteins are indicated. As a control a vector without scFv gene cassette was used (Control).

The Redox State Influences Binding Properties of scFv-CK-- We were interested in whether the redox state of the 21C5 anti-cutinase scFv affected binding to the antigen cutinase. To determine the affinity by ELISA, we first prepared samples from transgenic tobacco leaves expressing scFv-CK, scFv-SK, and as a control, untransformed tobacco (N. tabacum cv. Samsun NN) in the presence of NEM. To exclude possible negative effects on the affinity by NEM (33), we also prepared samples in the absence of NEM. The concentration of the scFv in these crude protein homogenates (Fig. 4A, inset) was calculated by comparison on Western blots with different concentrations of purified Ec-scFv, which is the 21C5 anti-cutinase scFv expressed in the periplasm of E. coli (23).


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  		Fig. 4.   Antigen binding activity of scFv antibodies present in crude protein homogenates from leaves of stably transformed tobacco plants, assayed by ELISA. A, Western blot to detect scFv-CK and scFv-SK (Fig. 4A, inset) with the anti-c-Myc antibody 9E10 was used for calculating scFv concentrations by comparison with different concentrations of purified scFv from E. coli. Serial dilutions of the scFv-CK (triangle ) and scFv-SK (diamond ) protein homogenates and purified scFv from E. coli (Ec-scFv) (×, -NEM; , +NEM) were incubated in wells coated with 250 ng of cutinase. Individual points represent mean values of duplicate trials with S.D. (error bars). Protein samples were prepared in the presence (panel A; +NEM) and in the absence (Panel B; -NEM) of the sulfhydryl alkylating agent N-ethylmaleimide.

Serial dilutions in duplicate of the scFv-SK and scFv-CK and untransformed tobacco samples were loaded on an ELISA plate coated with cutinase. As a reference duplicate serial dilutions of NEM-treated purified Ec-scFv were included in the assay. Fig. 4A shows the comparison of the antigen binding activity of scFv-CK and scFv-SK, both in the crude transgenic plant extracts, and purified Ec-scFv. Apparently, the binding properties are very similar. The plant proteins in the crude homogenates did not affect the A405 values, as determined for the control samples, prepared from untransformed plants (not shown). Treatment with NEM does not affect the antigen binding properties of the anti-cutinase scFv since the samples prepared in the absence of NEM also show no significant difference in antigen binding activity in the same experiment (Fig. 4B).

Next NEM-treated samples were prepared from the protoplasts transfected with the scFv-CK and scFv-SK expression vectors and the empty vector pUCAP35S as described before. As shown, the migration rate and the presence of the 65-kDa protein band indicates the redox state of the scFv antibody. Western analysis of the protoplasts showed 32- and 65-kDa bands for scFv-SK, indicating the oxidized nature, and a 33-kDa protein band for scFv-CK, indicating the reduced nature (Fig. 5A). The concentration of the scFv in these crude protein homogenates was calculated at 8 ng/µl for both scFv-SK and scFv-CK by comparison with different concentrations of Ec-scFv on SDS-PAGE followed by Western blotting and immuno-detection using the anti-c-Myc antibody, 9E10. An ELISA binding assay was performed in triplicate using 150 ng of scFv-CK and scFv-SK. To calculate the binding, duplicate serial dilutions of NEM-treated Ec-scFv were included in the assay. Fig. 5 shows the comparison of the antigen binding activity of scFv-CK and scFv-SK, both obtained from transfected protoplasts. Because the antigen binding activity of the Ec-scFv was similar to oxidized scFv-SK and scFv-CK (see Fig. 4), we calculated from the Ec-scFv reference that the A405 value correlated to an amount of 130 ± 5.1 ng of scFv-SK bound/well after correcting for the background caused by aspecific binding of plant proteins from the crude homogenate (Fig. 5B). Of the scFv-CK, only 8.0 ± 0.8 ng/well was bound. It can therefore be concluded that in comparison to the oxidized scFv-SK, less than 10% of the scFv-CK input remained bound to cutinase. The alkylation of the cysteines by NEM did not cause the reduction in binding strength of the reduced scFv-CK as was determined by sample preparation in the absence of NEM (not shown). This indicates that the reduced scFv-CK antibody has a considerable reduced binding strength when compared with the oxidized scFv-SK and scFv-CK. Apparently, the absence of intramolecular disulfide bridges has a considerable negative effect on the binding strength of the anti-cutinase scFv.


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  		Fig. 5.   Binding of transiently expressed scFv-SK and scFV-CK antibodies to cutinase. Panel A, Western blot of total protein homogenates prepared in the presence of NEM from protoplasts, transfected with the vectors containing the scFv-CK and scFv-SK gene cassettes in the absence of the reducing agent DTT. The blot was used for calculating scFv concentrations by comparison with different concentrations of purified scFv from E. coli. The scFv proteins were detected with the anti-c-Myc antibody (9E10). Panel B, protein homogenates containing 150 ng of the scFv-CK and scFv-SK (black bars), and serial dilutions of scFv purified from E. coli were incubated in wells coated with 250 ng of cutinase in triplicate. The serial dilutions were used to calculate the amount of bound scFv-SK and scFV-CK to cutinase (spotted bars). The dotted bars represent the mean values of bound scFv-SK and scFV-CK with S.D (error bars), corrected for the protoplast homogenate transfected with the empty vector pUCAP35S. The antibodies were detected with the anti-c-Myc antibody (9E10).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The capability of a scFv antibody to form disulfide bridges in the cytosol is remarkable. Protein disulfide isomerase, a catalyst in the oxidative folding, is not present in the cytosol, and the estimated GSH/GSSG ratios of 30-100 do not favor disulfide bridge formation. 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). This demonstrates that disulfide bridge formation in proteins is very much dependent on the GSH/GSSG ratio of the subcellular compartment. Cell-free translation assays in the presence of protein disulfide isomerase and added bacterial chaperones showed that a scFv antibody fragment folds better at mildly reducing conditions, i.e. a GSH/GSSG ratio of 10 at 1.1 mM total glutathione, than at oxidizing conditions (34). Bacterial chaperones did not promote the proper disulfide bridge formation, and it was suggested that protein disulfide isomerase merely served in disulfide rearrangements rather than in disulfide formation. Our results indicate that in vivo disulfide bridge formation in scFvs is possible at GSH/GSSG ratios found in the plant cytosol. Consequently, if these ratios correspond to the ratios found in the cytosol of cultured murine hybridoma CRL-1606 cells (3), the scFv antibody is capable of forming disulfide bridges at GSH/GSSG ratios of 30 or higher. The GSH/GSSG ratio and the GSH concentration in the cytosol of tobacco leaf cells are unknown and will be difficult to determine due to the presence of the large central vacuole. However, it is possible that this ratio deviates or that the GSH concentration is relatively low. The total GSH/GSSG ratio of the phloem exudates of Cucurbita (35) and Ricinus (36) was calculated at 13 and 3, respectively. Because phloem exudates represent the cytosol of a plant cell, this total GSH/GSSG ratio represents the cytosolic ratio. In addition, in pumpkin leaves, low concentrations of total GSH were measured. The concentration of GSH also has great influence on disulfide bridge formation in proteins (2).

The presence of certain eukaryotic cytosolic chaperones (37) may facilitate the efficient folding of the VL and VH domains and prevent aggregation through erroneous disulfide bridge formation. Once folded into the native structure, the disulfide bridges are inaccessible for reduced glutathione due to a tight surrounding structure (33, 38). However, proper folding does not always guarantee disulfide bridge formation, as illustrated by the absence of disulfide bridges in two other functional cytosolic scFv antibody fragments in stably transformed tobacco and petunia plants (39, 40). That folding in our scFv antibody is accurate is emphasized by the 202' linker derivative with the cysteine residue. Even this extra cysteine does not result in an aberrant disulfide bridge formation and results in a partial but rather organized dimerization.

As demonstrated by the transfection experiments, the plant cytosol can under circumstances also be reducing enough to prevent disulfide bridge formation. This confirms that the redox state of the plant cytosol is not fixed and that redox modulation may play an important role in the physiology of the plant cell as suggested before. Why the cytosol of protoplasts is more reducing remains a question. Incubation of the protoplasts after transfection occurs in the dark, and therefore, the cytosol is presumed to be more oxidized when compared with illuminated protoplasts (22). This should favor disulfide bridge formation, which would contradict our observations. It may well be that the protoplasts, which are kept under non-natural conditions, protect themselves against an oxidative stress situation. The levels of GSH in foliar tissues have been shown to increase under various oxidative stress conditions (41). Furthermore, extracellular added GSH was also shown to act as an activator of the transcription of genes encoding the cell wall hydroxyproline-rich glycoproteins and phenylpropanoid biosynthetic enzymes in cultured suspension cells or protoplasts of bean, soybean, and alfalfa (42, 43).

Our results also raise the question of whether such oxidative stress reactions also apply for other types of eukaryotic cell cultures. In other words, is the GSH/GSSG ratio in the cytosol of cultured animal cells higher than the ratio in the cytosol of the tissue from which the cells are derived? If in all cases the scFv functionality is indeed a marker for the presence of disulfide bridges, the reported successes with respect to cytosolic expression of scFvs for introducing resistance against pathogens, like viruses (44-48), or modifying phenotypic traits (49-53) would imply the presence of disulfide bridges. As in stably transformed plant cells, this may not always be the case, since Biocca et al. (54) report that a functional cytosolic anti-p21ras scFv, expressed in Xenopus oocytes, lacked disulfide bridges. To date, nothing is known about disulfide bridge formation in scFv antibodies in the cytosol of other eukaryotic cell culture types.

As our results show, scFv antibodies can also form intermolecular disulfide bridges in the plant cytosol. Maciejewski et al. (55) report the functional expression of an anti-human immunodeficiency virus1 reverse transcriptase Fab fragment in the cytosol of lymphoid cell lines, leading to a blockade of virus replication. Although it was demonstrated that uninfected cells expressed unassembled Fab fragments, i.e. without intermolecular disulfide bridges, it may have been that assembly may have occurred during infection since HIV infection strongly decreases the intracellular reduced glutathione concentration (21).

Our results also demonstrate that scFv antibodies without disulfide bridges can be found as a soluble and poorly functional protein in the plant cytosol. However, its stability does not seem to be affected. This is in contrast to heterologous scFv expression in E. coli (38), where the formation of the intramolecular disulfide bonds in both the variable heavy and light domains are considered crucial for stability. Apparently, next to the redox potential, the environment of the plant cytosol differs in other aspects from the E. coli cytosol. It was reported by Freund et al. (56) that in vitro a scFv antibody folds through an early and fast folding intermediate followed by a slow folding process into the final structure. As mentioned, certain chaperones not present in the cytosol of E. coli may play an important role in stabilizing and maintaining a folding intermediate of the scFv antibody. It may therefore be that the disulfide bridge formation required for the final folding steps to acquire the fully folded structure is prevented due to the reducing environment.

This report illustrates that it is impossible to predict a priori to what extent a given protein sulfhydryl will remain reduced in the cytosol. This was also suggested by Piñeiro et al. (57). Bacterial genes coding for the cytosolic enzymes neomycin phosphotransferase II and beta -glucuronidase were transiently expressed in the cytosol of tobacco protoplasts. Because the typical GSH/GSSG ratio in E. coli ranges from 50 to 300 they reasoned that the GSH/GSSG ratio of 30-100 in the plant cytosol was not sufficiently reducing to prevent disulfide bridge formation in these enzymes, resulting in a reduced stability. Thus, in cases in which disulfide bridges determine functionality and/or stability of a protein, the result of heterologous expression of the coding gene in the cytosol may be difficult to foresee.

Expression in plant cytosol of functional scFvs has proven to be feasible and, therefore, offers good perspective with respect to their use in intracellular immunization and immuno modulation approaches. However, the choice of the expression system should be made carefully and guarantee a redox state of the heterologously expressed protein, which represents the situation in the natural tissue. Transient expression assays by transfecting cell suspensions may, therefore, be only representative for protein expression levels rather than functionality in the cytosol.

    FOOTNOTES

* This research was supported by grants from the Netherlands Technology Foundation (STW) coordinated through the Foundation for Life Sciences (SLW). Additional support was obtained from European Community grants BIO2-CT92-0239 and FAIR1-CT95-0905.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratory of Phytopathology, Dept. of Plant Sciences, Wageningen University, P. O. Box 8025, 6700 EE Wageningen, The Netherlands. Tel.: 31-317-485813; Fax: 31-317-483412; E-mail: sander.schouten@fyto.dpw.wau.nl.

Published, JBC Papers in Press, March 26, 2002, DOI 10.1074/jbc.M201245200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; scFv, single-chain Fv; NEM, N-ethylmaleimide; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PBST, PBS with 0.1% (v/v) Tween.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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