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Evaluation of Immunoglobulins from Plant Cells Mich B. Hein,*,+Ying Tang2 Donald A. McLeod,tJ Kim D. Janda,t,s and Andrew Hiattf Departments of Molecular Biology and Chemistry, The Research Institute of Scripps Clinic, La Jolla, California 92037

Expression of cDNA constructs encoding full-length mouse immunoglobulin chains with their native leader sequences or fusion constructs substituting the native leader with a pre-pro sequence derived from Saccharomyces cerevisiae yielded blocked N-termini on the y chain or the correct amino terminal sequence on the mature K chain. Lectin binding assays revealed that assembled immunoglobulin complexes contained a glycosylated heavy chain. The attached glycan was resistant to digestion by endoglycosidase H and its lectin binding pattern was distinguishable from that of the mammalian glycan. The results indicated processing of the immunoglobulin carbohydrate in the tobacco Golgi to yield a complex oligosaccharide. Secretion of antibody by protoplasts isolated from regenerated transgenic plants or from suspension callus cells was demonstrated by pulse-chase labeling experiments. When purified, the tobaccoproduced antibody was found to possess the antigen binding and catalytic properties of the murine monoclonal antibody. Kinetic parameters ( K m ,Ki,Vm,, and kcat)of the tobacco-derived antibody were comparable to those of the mouse-derived antibody. The results in general show that the endomembrane system of tobacco cells possesses cognate mechanisms for the recognition of diverse leader sequences. These signals can be used to initiate the assembly, processing, and secretion by plant cells of complex foreign proteins.

Introduction Plant cells have been evaluated for the production of several mammalian proteins including human serum albumin (Sijmons et al., 19901,enkephalins (Vandekerchove et al., 1989),and antibodies (During et al., 1990; Hiatt et al., 1989). In general these efforts have resulted in the production of low levels of foreign proteins and low levels of transgenic protein secretion. In one instance, high-level expression of a nonmammalian foreign protein (chitinase) has been achieved up to a level of 0.25% of total extractable protein (Jones et al., 1988). Production and secretion of functional antibodies may provide a difficult challenge for plant cells since, unlike single-chain proteins, they require processing and assembly of a heteromultimeric protein complex. As in other eukaryotic cells, secretion of proteins from plant cells is accomplished by an endomembrane system. Recognition and initial processing of proteins destined for secretion occurs in endoplasmic reticulum, and subsequent processing and vesicularization is a function of the Golgi apparatus (Palade, 1975;Farquhar 1985;Walter and Lingappa, 1986). We know little about the fidelity with which secreted proteins from evolutionarily distant organisms can be recognized and processed by a plant host endomembrane system. Although signal sequences for secretion generallypossess common features, the amino acid sequences are not highly conserved (von Heijne, 1985). Data for heterologous in vitro translation of mRNAs has suggested that although there are similarities between animal and plant translocating systems, they are not truly homologous (Meyer, 1985;Duong et al., 1987;Prehn et al., 1987;Campos et al., 1988). In addition, once association of a heterologous protein with endomembrane has occurred, there are a variety of processing alternatives that + Department

of Molecular Biology.

* Department of Chemistry.

8758-7938/9 113007-0455$02.50/0

could yield a protein different from the one synthesized in ita native host (Jacobsen et al., 1988). For example, in the case of immunoglobulins, an unusual glycosylation pattern of an antibody produced in yeast resulted in a structure that did not bind complement (Horwitz et al., 1988). Subsequent to endomembrane association, proteins in plants can be subject to proteolytic processing, assembly with other subunits, and glycosylation (Jones and Robinson, 1989),as well as other possible modifications (Jacobsen et al., 1988). Little is known about the ability of plants cells to perform these functions on gene products derived from other organisms since relatively few transgenic plants have been regenerated that express, in abundance, proteins from animals or microorganisms. For that reason, we have chosen to evaluate some of the characteristics of murine antibodies produced by plant cells that could result from association with the plant endomembrane system. We have previously reported efficient synthesis and assembly of a functional murine antibody by regenerated tobacco plants (Hiatt et al., 1989). This antibody, called 6D4, was raised against a synthetic hapten antigen whose structure mimicked a possible transition state induced by an esterase enzyme (Tramantano et al., 1986 a,b). This antibody was chosen because of its catalytic properties in order to evaluate any modifications of kinetic parameters resulting from expression in plant cells. Production of the functional antibody was found to be completely dependent on the presence of the mouse signal sequence at the amino terminus of the immunoglobulin. In the absence of the signal, immunoglobulinaccumulation was very poor and heavy chain-light chain complexes were not detected. Dramatic increases in immunoglobulin accumulation were observed when signal sequences were included as well as when the individual chains were coexpressed in the same plant. These results suggested that

0 199 1 Amerlcan Chemlcal Society and Amerlcan Institute of Chemical Englnwrs

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the mouse signal sequences effected endomembrane localization, assembly, and possibly secretion of the antibody. In order to optimize the secretion of immunoglobulins from plant cells, we designed an expression strategy to exploit secretion pathways known for other eukaryotes. We have replaced the native mouse leader sequence with a yeast signal peptide and pro sequence from the a-mating factor peptide oligomer. To date, there have been few reports of pro sequences as precursors to secreted proteins of plants. Posttranslational processing of large prosequences of cysteine proteinases in barley aleurone layers proceeds in a multistep fashion to produce the mature enzymes (Koehler and Ho, 1990). The pre-pro proteins in barley aleurone may be similar to preprocathepsins of mammals; however, removal of the pro sequence of cathepsins occurs in one step. The pro sequences described in barley are significantly different from pro sequences associated with secretion of mammalian hormones. In mammals and yeast many hormones are derived from precursors containing long pro sequences terminating in Lys-Arg residues, which are removed prior to secretion (Burgess and Kelly, 1987). A specific protease resident in the endoplasmic reticulum is responsible for cleavage (Fuller et al., 1988). In some heterologous systems, such as pituitary cell lines, proinsulin, proenkephalin, and pro parathyroid hormone are processed, suggesting that processing of precursor hormones may not be cell type or hormone specific (Moore et al., 1983; Comb et al., 1985; Hellerman et al., 1984; Warren and Shields, 1984). While plant leaf cells are not known to contain either peptide hormones or specialized cells for protein secretion, our initial observations indicated that these cells were capable of recognizing and assembling with high fidelity specialized mammalian proteins containing leader sequences (Hiatt et al., 1989). In the present report we further explore the assembly of immunoglobulins in tobacco cells in the context of requirements for the correct processing of immunoglobulins chains and the subsequent secretion of functional antibodies.

Experimental Procedures Plant Transformationand Regeneration. Leaf disks were punched from surface-sterilized leaves with a paper punch (6 mm in diameter) and submerged in a culture of recombinant Agrobacterium tumefaciens containing the pMON530 plant expression vector (Rogers et al., 1987) modified by introduction of immunoglobulin cDNAs at the EcoRI site. The Agrobacterium was grown overnight in Luria broth at 28 "C. After gentle shaking to ensure that all edges were infected, the disks were blotted dry and incubated upside-down on culture plates containing medium that induces regeneration of shoots (Horsch et al., 1985). After 1 day, the disks were transferred to the same medium containing carbenicillin (500 pg/mL) and kanamycin (200 pg/mL). After 2-4 weeks, shoots that developed were excised from calli and transplanted to appropriate root-inducing medium containing carbenicillin (500 pg/mL) and kanamycin (100 pg/mL). Rooted plantlets were transplanted to soil as soon as possible after roots appeared. Antibody Purification. Midveins were removed from 10 g of young leaves, which were homogenized by hand in 50 mL of 50 mM Tris-HC1 and 50 mM EDTA, pH 8.0, containing 2 mM PMSF. The homogenate was centrifuged a t lOOOOg, concentrated to 10 mL (Centricon 30),laoded onto a Sephacryl S-300 column, and eluted with 0.1 M sodium acetate buffer, pH 5.0. FPLC fractions were collected and antibody was measured by ELISA (Hiatt et

al., 1989; Engvall and Perlmann, 1972). These fractions were pooled, dialyzed against 1.5 M glycine and 3.0 M NaC1, pH 8.9, and then passed twice over 2 g of protein A-Sepharose (Pharmacia). Antibody was eluted with 10 mL of 0.1 M citrate buffer, pH 6.0, concentrated to 50 gg/mL, and dialyzed against 50 mM phosphate buffer, pH 8.0. Antibody from mouse ascites was purified by the same techniques. Specific antibody concentration was determined by ELISA. N-Terminal Sequence of Purified Immunoglobulin Chains. Sequence analysis was performed on an Applied Biosystems automated sequenator on y and K chains purified from functional antibodies. The immunoglobulin chains were first separated by SDS-PAGE and subsequently blotted onto poly(viny1idene difluoride) membranes as described (Matsudaira, 1987). The membrane sections containing heavy or light immunoglobulin chains were used directly for sequencing. Leader Sequence Replacement. The pre-pro sequence from the Saccharomyces cerevisiae n-mating factor (Kurzan and Herskowitz, 1982) was subcloned into M13mp18 as an EcoRI/HindIII fragment; antibody 6D4 K or y chains from which the endogenous mouse leader had been removed (Hiatt et al., 1989) were then ligated into the same vector as Hind111 fragments. Oligonucleotide-directed mutagenesis was used to remove the sequence between the end of the pre-pro sequence and the glutamine codon (y chain) or aspartate codon ( K chain). The chimeric cDNAs were finally ligated into pMON530 and used to transform leaf disks as described (Horsch et al., 1985). Individual regenerants expressing K chain or y chain with the a-mating factor pre-pro sequence (Kmat, ymat) were crossed to yield y m a t X Kmat progeny. In some cases, plants that have previously been described (i.e., constructs containing no leader sequences or constructs containing the native mouse leader; Hiatt et al. 1989) were used in crosses with plants expressing the mating factor leader construc~ to yield %matx Ynative, Knative x ?"at, K m t x Yno leader, and Kno leader X Ymat. Progeny were screened for antibody production by ELISA. At least 3 plants/genotype were evaluated. The variability in expressionranged from about 0.1 ?6 of extractable protein to about 0.8%. The standard error of the mean was calculated to reflect this variability. Lectin Binding and Endoglycosidase Digestion. One microgram of purified antibody or human transferase and ovalbumin were subjected to SDS-PAGE (Chua, 1980) and blotted onto nitrocellulose (Harlow and Lane, 1988). The blots were blocked in 37% gelatin (Faye and Chrispeels, 1985) and incubated in a buffer containing 0.5 M NaCl, 20 mM Tris-HC1 (pH 7.4), 0.1 mM each CaC12, MgC12, and MnClz (TIBS) and containing 5 pg/mL of a biotinylated lectin. Lectin binding was visualized by incubation with streptavidin-alkaline phosphatase and bromochloroindolyl phosphate (Hiatt et al., 1989). Endoglycosidase H digestion was performed in 50 mL of 200 mM sodium acetate buffer, pH 5.8, with 40 milliunits of enzyme (Sigma) for 2 h at 37 OC. Methyl a-glucopyranoside incubations (15 mM, in 100 mL of TIBS buffer) were performed after Con A binding to filters and were left overnight at room temperature prior to visualization. Protoplast Isolation. Protoplasts were isolated as described (Tricoli et al., 1986). A total of 2 X 106 cells were suspended in 0.5 mL of mannitol medium containing 10 pCi of [3SS]methionine. An aliquot was taken to assess cell viability, with fluorescein diacetate fluorescence as the marker. After 2 h, an aliquot of cells and medium was removed to determine intracellular and extracellular incorporation and cell viability. The medium was then made 100 pM in methionine; after 2 h, cells and medium were harvested to again determine intracellular and ex-

Bbtechnol. prog., 1991, Vol. 7, No. 5

tracellular incorporation and cell viability. During the labeling period, no reduction in cell viability was observed. On average, between 80 % and 90% of isolated protoplasts were found viable. Radiolabel in the 6D4 antibody was measured as counts adhering to protein A-Sepharose and into 6D4 heavy chain after elution from an SDS-10 96 polyacrylamide gel slice. Heavy chain in the SDS gel was visualized with Coomassie Brilliant Blue R-250 after addition of 1pg of purified 6D4 antibody to the gel sample. Callus cell lines were initiated over a period of 8 weeks by incubating leaf segments on the appropriate growth hormones as described (Hiatt et al., 1986). Liquid suspension cell lines were then initiated from clumps of callus cells. Incorporation of [36S]methionine into callus was performed as described above for protoplasts. In general, we obtained between 50 000 and 100 000 cpm of total incorporation into TCA-precipitable protein of both protoplast and callus at 2 h. Incorporation into the heavychain band ranged from 400 to lo00 cpm above background. Catalytic Assay. Hydrolysis of ester substrate 1 (Table 111)by plant- and ascites-derived antibody was measured on a Hewlett-Packard 8452A diode array spectrophotometer by monitoring the adsorption change at 245 nm. Antibody (approximately 100 nM, average value determined from a bicinchonic acid assay and absorbance at 280 nm and by assuming a molecular weight of 150 000) was preincubated at 25 "C in 50 mM phosphate buffer, pH 8.0. The reactions were initiated by addition of varying amounts of substrate (in dioxane stock solution) to give a substrate concentration of 1-8 mM; the total organic phase was 5% dioxane in each case. The reaction was monitored for 1 h. The absorption change for complete hydrolysis was determined by addition of nonspecific esterase (Sigma). Kinetic parameters were obtained after subtraction of background hydrolysis, by LineweaverBurk treatment of the data. Inhibition constants were determined by plots of slopes obtained with phosphonate 2 (Table 111)present at concentrations of 100 and 300 nM. The data were analyzed by linear regression.

Results and Discussion Dependence of Antibody Expression on Signal or Pro Sequences. In order to investigate the expression of immunoglobulins from cDNAs containing a yeast signal and pro sequence, we removed the native leader sequence from the 6D4 immunoglobulin cDNAs. This consisted of the first 60 nucleotides of the K chain and the first 57 nucleotides of the y chain. To these truncated cDNAs, we ligated the pre-pro sequence from the a-mating factor of S. cereuisiae (Kurzan and Herskowitz, 1982). The resulting y and K chain cDNAs encoded a signal peptide of 20 amino acids and a pro sequence of 66 amino acids (Table IA). The pro segment was designed to terminate with the basic amino acid pair, lysine-arginine,immediately preceding the first amino acid of the mature y or K chain. In the case of y, the N-terminal amino acid sequence of the mature chain is Gln-Val-Gln-Leu,and on K the sequence is Asp-Val-Val-Leu. Our strategy was to transform separate tobacco plants withy- or K-chain cDNA constructs ligated into the pMON530 plant expression vector (Rogers et al., 1987). The regenerated transformants could then be evaluated for expression of y- or K-chain immunoglobulins prior to coexpression of the two chain in the same plant. Coexpression of chains results from sexual recombinations of y and K genes in progeny of a cross of primary transformants (Hiatt et el., 1989). Three pairs of cDNA constructs were used to evaluate the dependence of antibody assembly on the N-terminal

457

Table I. Expression of cDNA Constructs Containing the a-Mating Factor Pre-Pro Sequence (A) Sequence of the a-Mating Factor Leader Ligated to y- or .+Chain cDNAs MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEA

VIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIA AKEEGVSLDLKR

- D V V L ( r chain)

-EV EL

(y chain)

(B)Accumulation of y or K Chains or Antigen Binding Y K Complexesa detection antibody antigen P3 genotype anti-rc anti-y (best plant) 48f8 8 nd Kmat 0 743 f 260 nd Ymat 2410 i 1230 7700 Kmat x Ymat 2280 f 1300 2490 f 1175 nd 8300 K m t X yMtive nd 2615 i 1505 8300 K~ti~XYmat 38 f 8 nd 0 Kmat x Yno leader 705 f 300 0 x.0 leader X Y m t nd a Values are expressed in nanograms of antibody per milligram of total protein (mean f SE) where purified 6D4 antibody from m o w ascites was used as the ELISA standard. Values are derived from at least 2 determinations/plant; a t least 3 planta/genotype were analyzed. Only plants expressing the highest levels of Y Kcomplexes were used in the antigen binding assays (Hiatt et al., 1989). nd, not determined. In some cases, plants that have previouslybeen described (i.e., constructa containing no leader sequences or constructa containing the native mouse leader; Hiatt et al., 1989)were used in crosses with plants expressing the mating factor leader constructs to yield Kmt x Y ~ t i v e ,K M ~ ~ V Ox Ymu, Kmt x Yno laderr, and Kno leader x Yrmt. The variability in expression ranged from about 0.1% of extractable protein to about 0.8%. The standard error of the mean was calculated to reflect this variability.

sequence: (1)y and K chains containing the native mouse signal sequence (Hiatt et al., 1989), (2) y and K chains with no signal sequence but with an ATG codon in front of the CAG glutamine codon (y chain) or GAT aspartate codon ( K chain), and (3) y and K chains containing the yeast prepro sequence in place of the native mouse signal. Evaluation of regenerated transformants by ELISA assays revealed that the individual y or K chains from constructs containing the yeast signal and pro sequence accumulated to nearly the same levels as constructs expressingthe native mouse leader, previously reported (Hiatt et al., 1989).We have previously observed that constructs containing no leader resulted in very poor y or K accumulation. Clearly, the presence of the yeast signal and pro sequence in the cDNA constructs resulted in enhanced immunoglobulin accumulation relative to the leaderless constructs. This effect is presumably due to targeting the translation of the immunoglobulin transcripts to the endoplasmic reticulum. Further evaluation of the immunoglobulin messenger levels and protein stability will be necessary to obtain a precise understanding of the mechanisms responsible for increased immunoglobulin protein accumulation. We found that assembled immunoglobulin was produced in plants with y and K chains containing the same signal (e.&, Kmat x ymat) Or different Signah (Kmat x ynatiw; Knauw X ymat). In contrast, crosses of plants expressing a leaderless transcript of either K or y did not produce assembled immunoglobulin regardless of the leader present on the complementary chain. Amino-Terminal Sequence Analysis. Antibodies were purified from plants of ynativef Kmat or ymrt/Km~w genotypes. The purification scheme consisted of homogenization of leaves by mortar and pestle, followed by size fractionation on SephacryLS300 FPLC and adsorption to

458

Figure 1. Western blot of antibodies isolated from tobacco leaf. Antibody was purified by adsorption to protein A-Sepharose from leaf of two tobacco genotypes. Approximately 200 ng from each preparation was subjected to SDS-PAGE and Western blotting. The blob were incubated with biotinylated goat antimouse y-chain-specific antibody and goat anti-mouse K-chainspecific antibody, followed by incubation with streptavidin conjugated to alkaline phosphatase. Visualization of binding was as described under Experimental Procedures. Left lane, 6D4 antibody derived from mouse ascites; center lane, antibody ~ ~genotype; ~ ~right/ lane, K antibody ~ ~ from purified from the Y the ymt/x,,.tim genotype; the molecular weight standards shown are phosphorylase b (97 400), catalase (58 loo), carbonic anhydrase (29 OW),and trypsin inhibitor (20 100).

protein A-Sepharose. In some cases, FPLC fractionation was omitted and antibody was purified from the clarified crude homogenate by a single protein A-Sepharose adsorption. y and K chains from the purified antibody as well as from antibody derived from mouse ascites were separated by SDS-PAGE and blotted onto nitrocellulose or PVDF membranes (Matsudaira, 1987). Staining of the PVDF membranes with Coomassie blue (data not shown) or Western blot analysis (Figure 1) showed the ymatand ?native chains comigrated, as did Kmat and Knative chains. The stained bands cut from the PVDF membranes were subjected to automated sequence analysis. In all cases, the K-chain sequence was DVVL, indicating that proteolytic cleavage had occurred in front of the first amino acid of the mature K chain regardless of the leader composition. None of the y-chain bands produced an amino acid sequence. Removal of the yeast sequence by the plant ER might have occurred in the absence of signal peptidase processing of the yeast presequence. The inability to sequence the y chain suggests that the N-terminal glutamine has been chemically modified (Johnston et al., 1975). The nature of the blocking agent on the y chain is currently under investigation. y-Chain Glycosylation. Further evidence for the association of mouse immunoglobulinswith the plant endomembranes was obtained by analyzing y-chain glycosylation. In these assays, purified antibody was blotted onto nitrocellulose and probed with biotinylated lectins (Faye and Crispeels, 1985; Goldstein and Hayes, 1976; Kijimoto-Ochiaiet al., 1989). In some cases, the purified antibody was incubated with endoglycosidase H prior to blotting. The results (Figure 2) showed'that only concanavalin A (specific for mannose and glucose) bound to the plant-derived y chain, whereas the ascites y chain was recognized by concanavalin A as well as the lectins from

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Ricinus communis agglutinin (specific for terminal galactose and N-acetylgalactosamine) and wheat germ agglutinin (N-acetylglucosaminedimers, terminal sialic acid; Kijimoto-Ochiaiet al., 1989). The lectins fromDatura stromonium (N-acetylglucosamineoligomers, N-acetyllactosamine) and Phaseolus vulgaris [galactose(fll,4) N-acetylglucosamine(~l,2)mannose] did not bind to either plant- or ascites-derived y chain. Elution of the lectin from the blots using methyl a-glucoside was used to compare the relative affinity of Con A binding to the plant and ascites heavy chains (Faye and Crispeels, 1985). The results showed that the two antibodies are indistinguishable by Con A affinity as well as by the quantity of Con A bound per microgram of y chain. Blots in which the antibodies were first digested with endoglycosidase H (Kijimoto-Ochiai et al., 1989; Trimble and Maley, 1984) displayed no reduction in Con A binding under conditions where Con A binding to ovalbumin (containing a highmannose-type carbohydrate) was diminished. Since endoglycosidase H resistance is characteristic of complex carbohydrates processed in the Golgi apparatus, these results indicate that the transgenic antibody is processed in a similar fashion to complex mammalian glycoproteins. N-Linked glycosylation of proteins in plants is similar to the glycosylation process in mammals (Jones and Robinson, 1989; Sturm et al., 1987). A core high-mannose oligosaccharide is attached to the asparagines contained within the canonical Asn-X-Ser/Thr sequence. This occurs in the endoplasmic reticulum and can be modified in the Golgi apparatus, where a-mannosidase removes some mannose residues and terminal sugars are attached. In mammals, the predominant terminal residue is N-acetylneuraminic acid (NANA);this carbohydrate has not been identified in plants. Terminal residues in plants have been found to consist of xylose, fucose, N-acetylglucosamine, mannose, or galactose (Sturm et al., 1987). In other respects, such as the size and extent of branching, plant glycans are very similar to mammalian glycans. Although we have not attempted a structural characterization of the oligosaccharides attached to the antibody, the comparative analysis of plant-derived and tobacco-derived glycans by lectin binding has revealed both differences and similarities. Both glycans are resistant to endoglycosidase H under conditions of carbohydrate digestion of the control glycoprotein, ovalbumin, which contains a highmannose carbohydrate structure. This demonstrates processing of the high-mannose carbohydrate to the complex type in the Golgi. In addition, both glycans have approximately the same affinity for Con A since they were not distinguishable by competition with methyl a-mannoside. This type of assay has previously been used to distinguish a variety of plant glycans with respect to their affinity for lectin (Faye and Crispeels, 1985). Two of the lectins used in the binding assays were found to distinguish the plant-derived from the mammalian antibody. R. communis agglutinin, which can bind terminal galactose and N-acetylgalactosamine residues, and wheat germ agglutinin, which can bind to terminal NANA, did not bind to the plant glycan. This suggests a distinct composition of terminal residues on the plant glycan and is consistent with the absence of NANA in plants. Antibody Secretion from Tobacco Cells. y-Chain glycosylation and correct tc-chain N-terminal processing indicated that antibody migrated from endoplasmic reticulum to Golgi and was possibly being secreted through the plant plasma membrane (Palade, 1975;Farquhar, 1985; Walter and Lingappa, 1986). To investigate secretion directly, protoplasts containing no cell walls (Tricoli et al., 1986) were isolated from antibody-producing leaves derived from a Ynative/Knative plant and the antibody was

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Figure2. Glycosylation of 7 chains produced in tobacco. Abbreviations: Con A, concanavalin A; RCA, R.communisagglutinin; WGA, wheat germ agglutinin; DSA, D.stramonium agglutinin; PHA,P. vulgaris erythrolectin; Endo H,endoglycosidase H;a M G , methyl a-glucopyranoside. Lane 1 in each blot except ‘Con A + aMG” contained 1 pg of antibody purified from tobacco; lane 2 contained

1pg of antibody from mouse ascites; lane 3 contained 1 pg each of human transferrin (complex-type carbohydrate) and ovalbumin (high-mannose-type carbohydrate). The ‘Con A + aMG” blot contained 2 pg of plant antibody (lane l),2 pg of ascites antibody (lane 2), and 2 pg each of transferrin and ovalbumin (lane 3).

Table 11. Secretion of Antibodies from Protoplasts and Callus Cells

incorporation ( %) cell type method cells medium [WIMethionine Incorporation into 6D4 at 2 h protoplasts protein A 75 25 SDS-PAGE 76 24 callus suspension protein A 72 28 SDS-PAGE 80 20 [WIMethionine Incorporation into 6D4after 2-h Chase protoplasts protein A 13 87 SDS-PAGE 14 86 callus suspension protein A 27 73 SDS-PAGE 32 68 O Results are expressed as the percentage of total radiolabel in immunoglobulinassociated with cells or medium as determined by bindingto protein A-Sepharose or SDS-PAGE mobilityas described under Experimental Procedures. Equal numbers of cells were used in each labeling experiment. Cell viability of protoplasts was measured by using fluoresceindiacetate as the marker and was found to be 80-W% throughout the labeling period. In general, between 50 OOO and 100 OOO cpm of total incorporation into TCA-precipitable protein of both protoplast and callus was obtained at 2 h. Incorporation into the heavy-chain band ranged from 400 to lo00 cpm above background.

labeled by feeding protoplasts with [3!jS]methioninefor 2 h. A t that time, radiolabel in the cell-associated immunoglobulin was compared to radiolabel in extracellular immunoglobulin. A significant fraction of newly synthesized antibody was found in the growth medium (Table 11). After a chase of 2 h with 100 p M methionine, most of the labeled antibody was extracellular, indicating that secretion of the antibody had occurred. Since plant cells are surrounded by a cell wall that restricts the passage of large molecules (Carpita et al., 1979),it is possible that relatively large protein molecules (e.g., immunoglobulins) secreted from plant cells would be restricted from diffusing into the extracellular medium. To evaluate the movement of secreted antibodies from the protoplast through the cell wall barrier, antibody secretion was also measured in established suspension cells that contain a primary cell wall. An identical radiolabeling protocol was employed with the suspension cells as with protoplasts. The comparison of antibody secretion from cells with intact cell walls (callus) and protoplasts indicated that most of the newly synthesized antibody was secreted from the cells and reached the incubation medium (Table 11). A pulse-chase protocol was necessary to characterize active secretion of immunoglobulin in culture, since cell death can also result in accumulation of macromolecules in the growth medium. The results showedthat, after a 2-h chase,

accumulation of the majority of radiolabeled immunoglobulin occurs in the growth medium. In the case of protoplasts, greater than 85% of the labeled antibody was secreted; from callus cells, approximately 70% of the antibody was found in the medium. These results demonstrate antibody secretion through both the plasma membrane and the cell wall of callus cells. This was somewhat surprising since the permeability of plant cells to macromolecules is thought to be very limited. Evaluation of macromoleculemigration through plant cell walls from a variety of sources has demonstrated an exclusion limit equivalent to a 20000-dalton globular protein (Carpita et al., 1979). Secretion of the antibody (Mr = 150 000)through the cell wall of a callus suspension cell may reflect the presence of a small population of large pores in the plant cell wall to allow passage of large proteins or extracellular carbohydrates (Carpita et al., 1979). It is apparent from these observations that the native mouse leader sequences and the yeast leader and pro sequence direct the processing and assembly of functional antibody and its subsequent secretion from plant cells. Other plant signal sequences have been demonstrated to direct the secretion from plant cells of human serum albumin (Sijmons et al., 1990) and a yeast invertase (von Schaewen et al., 1990). This suggests that plant cells possess cognate mechanism for the recognition of diverse leader sequences. However, it has recently been shown that immunoglobulin chains bearing an N-terminal signal peptide from barley a-amylase are assembled into functional antibodies in tobacco that are not secreted (During et al., 1990). Even though the a-amylase leader sequence was derived from a well-characterizedextracellular protein of barley aleurone (Rogers and Milliman, 1983), immunocytolocalizationindicated the presence of the functional antibody associated with chloroplasts and the endoplasmic reticulum but not in the cell wall or intercellular spaces. The isolated light chain of this antibody was processed to the same size as the parental murine light chain. Since the N-terminal sequences were not provided for the individual chains and the efficacy of heavy-chain processing was not demonstrated (During et al., 1990), it is impossible to ascertain whether the failure of tobacco cells to secrete this antibody is related to processing of the barley leader sequence. Binding and Catalytic Activity of the Antibody from Tobacco. Antigen binding of assembled Y K complexes from tobacco leaves was evaluated by ELISA and shows the same antigen specificity as the ascites-produced antibody (Hiatt et al., 1989). In these experiments, antibody isolated from leaves was incubated in microtiter wells coated with antigen linked to BSA (data not shown).

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Table 111. Catalytic Activity of an Antibody Produced in

Tobacco

(A) Structure of Substrate 1 and Inhibitor 2

1

4

(B) Catalvtic Activitv of 6D4

KI

antibody source tobacco ascites 1.41 X 104 M 9.8 X lo4 M 0.31X 10-8M s-l 0.057X 104 M s-l 0.47 X 10-6M 1.06 x 10-6M

kcat

(competitive) 0.0085-l

parameter KM VUl,

(competitive) 0.025 s-l

Binding of the plant-produced antibody was measured by a horseradish peroxidase conjugated secondary antibody recognizing murine immunoglobulin and demonstrated that functional antibody is produced from constructs containing either yeast or mouse leader sequences. Catalytic activity of the antibody purified from leaf was measured by incubation of 10 pg of antibody with the substrate 1 in the presence or absence of the specific inhibitor 2 to derive K M ,K I , Vm,, and k,,,. The time course of production of the reaction product was measured spectrophotometrically. Table I11 demonstrates that, for each catalytic parameter, plant-derived and ascites-derived antibodies differed by less than an order of magnitude. While these differences in activity may represent real differences in functionality of the antibody imparted by processing in the plant or during purification, the measurements are within the observed range for different batches of the ascites-produced antibody (K.D. Janda, unpublished results). Differences may also reflect inaccurate quantitation of the plant-purified antibody due to interference from plant proteins or low molecular weight compounds that were not eliminated from the antibody fraction during purification. The primary reason for choosing the 6D4 catalytic antibody for expression in plants was the availability of kinetic parameters that could be used to determine functionality of the antibody not detected by binding, antigen affinity, or other biochemical assays. Since no other transgenic plants have produced foreign proteins at levels greater than 1% of total protein, there is no information concerning possible modificationsof abundant transgene products. Possible posttranslational changes in addition to glycosylation could consist of fatty acylation, phosphorylation, sulfation, or modifications that change the p l of the protein (Jones and Robinson, 1989). The data presented in Table 111 demonstrate that the catalytic activity of the antibody derived from the plant is comparable to the original catalytic antibody derived from ascites fluid (Tramontano et al., 1986a,b). The Vm,, of the tobacco antibody appears to be lower than that of the mouse antibody; however, the affinity for the substrate 1 and inhibitor 2 is apparently higher. Since this antibody is clearly a poor catalytic reagent, the differences between the two data sets probably do not reflect structural differences derived from synthesis in different cell types

but may be due to the inherent difficulty in measuring catalysis by this antibody even in ideal conditions. Structural Integrity of the Antibody Constant Region. An additional criterion for the conformational integrity of the plant-derived antibody is its affinity for protein A. Whereas catalytic activity indicates an overall structural integrity of the variable region,protein A binding depends on the correct conformation of paired heavy chains in the constant regions. Different isotypes have different affinities for protein A with IgGl having a low affinity (Harlowe and Lane, 1988). High salt conditions are necessary to enhance the binding of protein A to IgG1. During the purification protocol, we found that the plantderived antibody has the same affinity for protein A as the mouse antibody (an IgG1). Protein A can be used effectivelyto isolate antibody from tobacco crude homogenates.

Conclusions Little is known about the requirements for the accumulation of foreign proteins in transgenic plants. With the exception of antibodies, no transgenic foreign proteins have been reported to accumulate to levels greater than 0.25% of total soluble protein (Jones et al., 1988; Hiatt et al., 1989; Sijmons et al., 1990; During et al., 1990; Vandekerckhove et al., 1989). Results that could aid in optimization of transgenic protein accumulation in plants were obtained by using murine immunoglobulin cDNAs containing native leader sequences or yeast pro sequences. The results demonstrate that either signal sequence was effective in enhancing the accumulation of the individual chains and were essential for assembly and secretion of functional antibodies. The resulting antibody possesses functional equivalence with the mammalian antibody as well as the integrity of the structural requirements for purification by protein A. Although our results have not eliminated the possibility of undetected modifications of the antibody due to production in tobacco cells, the antibodies produced in tobacco are processed and secreted in a fashion that is mechanistically the same as in mammalian cells. Future experiments should address the potential for large-scale production of antibodies in plants or cultured plant cells as well as the immunogenicity of plant-derived antibodies in mammals in order to evaluate their potential for therapeutic applications. BSA Con A EDTA PMSF SDSPAGE

Notation bovine serum albumin concanavalin A ethylenediaminetetraacetic acid phenylmethanesulfonyl fluoride sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Acknowledgment We acknowledge the expert technical assistance of Ruth Pinney and Regina Durango. Literature Cited Burgess, T. L.; Kelly, R. B. Constitutiveand Regulated Secretion of Proteins. Annu. Bev. Cell Biol. 1987, 3, 243-293. Campos, N.; Palau, J.; Torrent, M.; Ludevid, D. Signal Recognition-like particles are Present in Maize. J . Biol. Chem. 1988, 263,9646-9650.

Carpita, N.;Sabularse, D.; Montezinos, D.; Helmer, D. P. Determination of the Pore Size of Cell Walls of Living Plant Cells. Science 1979, 205,1144-1147.

Biotechnol. prog., 1991, Vol. 7, No. 5

Chua, N. H. Electrophoretic Analysis of Chloroplast Proteins. Methods Enzymol. 1980,69,434-446. Comb, M.; Liston, D.; Martin, M.; Rosen, ti.; Herbert, E. Expression of the Human Pro-enkephalin Gene in Mouse Pituitary Cells: Accurate and Efficient mRNA Production and Proteolytic Processing. EMBO J . 1985,4,3115-3122. Duong, L. T.; Caufield, M. P.; Rosenblatt, M. Synthetic Signal Peptide and Analogs Display Different Activities in Mammalian and Plant in Vitro Secretion Systems. J . Biol. Chem. 1987,262,6328-6333. During, K.; Hippe, S.; Kreuzaler, F.; Schell, J. Synthesis and Self-Assemblyof a Functional MonoclonalAntibody in Transgenic Nicotiana tabacum. Plant Mol. B i d . 1990,15,281-293. Engvall, E.; Perlmann, P. Enzyme-linked Immunosorbent Assay, ELISA 111. Quantitation of Specific Antibodies by Enzymelabeled Anti-Immunoglobulin in Antigen-Coated Tubes. J . Immunol. 1972,109,129-135. Farquhar, A.G. Progress in UnravellingPathways of GolgiTraffic. Annu. Rev. Cell Biol. 1985,1 , 447-488. Faye, L.; Crispeels, M. J. Characterization of N-Linked OligoSaccharides by Affinoblotting with Concanavalin A-Peroxidase and Treatment of the Blots with Glycosidases. Anal. Biochem. 1985,149,218-224. Fuller, R. S.; Stene, R. E.; Thorner, J. Enzymes Required for Yeast Prohormone Processing. Annu. Rev. Physiol. 1988,50, 345-362. Goldstein, I. J.; Hayes, C. E. Lectins-Carbohydrate-Binding Proteins of Plants and Animals. Adv. Carbohydr. Chem. Biochem. 1978,35,120-340. Harlow, E.; Lane, D. Antibodies, A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1988. Hellerman, J. G.; Cone, R. C.; Potts, J. T., Jr.; Rich, A.; Mulligan, R. C.; Kronenberg, H. M. Secretion of Human Parathyroid Hormone from Rat Pituitary Cells Infected with a Recombinant Retrovirus Encoding Preproparathyroid Hormone. Proc. Natl. Acad. Sei. U.S.A. 1984,81, 5340-5344. Hiatt, A. C.; McIndoo, J.; Malmberg, R. L. Regulation of Polyamine Biosynthesisin Tobacco. J . Biol. Chem. 1986,261,12931298. Hiatt, A. C.; Cafferkey, R.; Bowdish,K. Production of Antibodies in Transgenic Plants. Nature 1989,342,76-78. Horsch, R. B.; et al. A Simple and General Method for Transferring Genes into Plants. Science 1985,227,1229-1231. Horwitz, A. H.; Chang, C. P.; Better, M.; Hellstrom, K. E.; Robinson, R. R. Secretion of Functional Antibody and Fab Fragment from Yeast Cells. Proc. Natl. Acad. Sci. U.S.A. i988,85,8678-8682. Jacobsen, J. V.; Bush, D. S.; Sticher, L.; Jones, R. L. Evidence for Precursor Forms of the Low Isoelectric Point Alpha-Amylase IsozymesSecreted by Barley Aleurone Cells. Plant Physiol. 1988,88,1168-1174. Johnston, S.L.; Abraham, G. N.; Welch, E. H. Structural Studies of Monoclonal Human Cryoprecipitable Immunoglobulins. Biochem. Biophys. Res. Commun. 1975,66,843-847. Jones, D. G.; Dean, C.; Gidoni, D.; Gilbert, D.; Bond-Nutter, Do; Lee, R.; Bedbrook, J.; Dunsmuir, P. Expression of Bacterial Chitinase Protein in Tobacco Leaves Using Two Photosynthetic Gene Promoters. Mol. Gen. Genet. 1988,212,536-542. Jones, R. L.; Robinson, D. G. Protein Secretion in Plants. Tansley Rev. 1989,17,567-588. Keohler, S.M.; Ho, T.-H. D. Hormonal Regulation, Processing, and Secretion of Cysteine Proteinases in Barley Aleurone Layers. Plant Cell 1990,2,769-783. Kijimoto-Ochiai, S.; Katagiri, Y. U.; Hatae, T.; Okuyama, H. Type Analysis of the Oligosaccharide Chains on Microheter-

461

ogeneous Components of Bovine Pancreatic DNAase by the Lectin-Nitrocellulose Sheet Method. Biochem. J. 1989,257, 43-49. Kurzan, J.;Herskowitz, I. Structure of a Yeast Pheromone Gene (MFa): A Putative a-Factor Precursor Contains Four Tandem Copies of Mature a-Factor. Cell 1982,30, 933-943. Matsudaira, P. Sequence from Picomole Quantities of Proteins Electroblotted onto Polyvinylidene Difluoride Membranes. J . Biol. Chem. 1987,262,10035-10038. Meyer, D. I. Signal RecognitionParticle (SRP)Does Not Mediate a Translational Arrest of Vascent Secretory Proteins in Mammalian Cell-free Systems. EMBO J . 1985,4,2031-2033. Moore, H. P. H.; Walker, M. D.; Lee, F.; Kelly, R. B. Expressing a Human Pro-insulin cDNA in a Mouse ACTH-Secreting Cell. Intracellular Storage, Proteolytic Processing, and Secretion on Stimulation. Cell 1983,35,531-538. Palade, G. Intracellular Aspects of the Process of Protein Synthesis. Science 1975,189, 347-358. Prehn, S.;Weidmann, M.; Rapoport, T. A,; Zwieb, C. Protein Translocation Across Wheat Germ Microsomal Membranes Requires an SRP-like Component. EMBO J. 1987,6,20932097. Rogers, S.G.; Klee, H. J.; Horsch, R. B.; Fraley, R. T. Improved Vectors for Plant Transformation: Expression Cassette Vectors and New Selectable Markers. Methods Enzymol. 1987, 153,253-276. Sijmons, P. C.; Dekker, B. M. M.; Schrammeijer, B.; Verwoerd, T. C.; van den Elzen, P. J. M.; Hoekema, A. Production of Correctly Processed Human Serum Albumin in Transgenic Plants. BiolTechnology 1990,8, 217-221. Sturm, A.; Kuik, A. V.; Vliegenthart, J. F. G.; Crispeels, M. J. Structure, Position, and Biosynthesis of the High Mannose and the Complex Oligosaccharide Side Chains of the Bean Storage Protein Phaseolin. J . Biol. Chem. 1987,262,1339213403. Tramontano, A.;Janda, K. D.; Lerner, R. A. Catalytic Antibodies. Science 1986a,234, 1566-1570. Tramontano, A.; Janda, K. D.; Lerner, R. A. Chemical Reactivity at an Antibody Binding Site Elicited by Mechanistic Design of a Synthetic Antigen. Proc. Natl. Acad. Sei. U.S.A. 1986b, 83,6736-6740. Tricoli, D. M.; Hein, M. B.; Carnes, M. G. Culture of Soybean Mesophyll Protoplasts in Alginate Beads. Plant Cell Rep. 1986,5,334-337. Trimble, R. B.; Maley, F. Optimizing Hydrolysis of N-Linked High-Mannose Oligosaccharides by Endo-b-N-acetylglucosaminidase H. Anal. Biochem. 1984, 141, 515-522. Vandekerckhove, J.; et al. Enkephalins Produced in Transgenic Plants using Modified 2s seed Storage Proteins. Biol Technology 1989, 7,929-932. von Heijne, G. Signal Sequences-The Limits of Variation. J. Mol. Biol. 1985,184,99-105. von Schaewen, A.; Stitt, M.; Schmidt, R.; Sonnewald, U.; Willmitzer, L. Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and stronglyinfluencesgrowth and phenotype of transgenic tobacco plants. EMBO J. 1990,9,3033-3044. Walter, P.; Lingappa, V. R. Mechanism of Protein Translocation Across the Endoplasmic Reticulum Membrane. Annu. Rev. Cell Biol. 1986,2,499-516. Warren, T. G.; Shields, D. Expression of Preprosomatostatin in Heterologous Cells: Biosynthesis, Post-translational Processing, and Secretion of Mature Somatostatin. Cell 1984,39, 547-555. Accepted July 12,1991.