Synthesis of Green Fluorescent Protein− Ricin and Monitoring of Its

Mammalian cells incubated with EGFP−ricin showed strong cell surface fluorescence at 4 °C and, on incubation at 37 °C, distributed initially to en...
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Bioconjugate Chem. 1997, 8, 743−750

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Synthesis of Green Fluorescent Protein-Ricin and Monitoring of Its Intracellular Trafficking Edward Tagge,† Billie Harris,† Chris Burbage,‡ Philip Hall,§ Joseph Vesely,| Mark Willingham,| and Arthur Frankel*,‡ Departments of Surgery, Pathology and Laboratory Medicine, Pharmacy, and Medicine, Medical University of South Carolina, Charleston, South Carolina 29425. Received May 8, 1997; Revised Manuscript Received July 23, 1997X

We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC. The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence. Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 °C and, on incubation at 37 °C, distributed initially to endosomes and then to Golgi vesicles. Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing. Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured. Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells. Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells. These data support a model requiring a Golgidependent step in cell intoxication by ricin. The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.

INTRODUCTION

Ricin from the Ricinus communis plant is a frequently used component in antibody-toxin conjugates for therapy of cancer and autoimmune diseases. Thus, knowledge of its mechanism of action is important both for understanding structure-function relationships of toxic lectins and for improved design of human therapeutics. The first necessary step in ricin intoxication is cell surface binding to galactose-terminated oligosaccharides on glycoproteins and glycolipids (1). The second step involves internalization from the cell surface to intracellular compartments. This step is energy-dependent (2) and leads to toxin localization in a compartment which avoids neutralization by anti-toxin antibodies added to the medium (3). Mutant cells resistant to ricin have been isolated with reduced internalization (4). Morphologic studies using ricin labeled with gold, ferritin, and horseradish peroxidase demonstrated endocytosis of ricin from clathrin-coated pits into endocytic vesicles (5-7). The third step in ricin intoxication is transport to the Golgi. This hypothesis is supported by several experiments. Immunoelectron microscopy of cultured cells exposed to ricin showed uptake sequentially in endosomes and then the trans-Golgi network (8). Hybridoma cells producing anti-RTB antibodies were not intoxicated by ricin, suggesting neutralization in the trans-Golgi network, a major membrane compartment shared by the two pathways. Finally, at 19 °C, ricin failed to reach the * Address correspondence to this author at the following address: Hollings Cancer Center, Rm 311, 86 Jonathan Lucas St., Charleston, SC 29425. Telephone: 803-792-1450. Fax: 803792-3200. † Department of Surgery. ‡ Department of Medicine. § Department of Pharmacy. | Department of Pathology and Laboratory Medicine. X Abstract published in Advance ACS Abstracts, September 1, 1997.

S1043-1802(97)00074-8 CCC: $14.00

Golgi and cell death was not observed (9). The fourth intoxication step involves post-Golgi sorting of ricin to a translocation-competent compartment. Brefeldin A blocks ricin toxicity but does not alter trafficking of ricin to the trans-Golgi network (10), suggesting a critical distal compartment. After addition of the ER retention signal KDEL to the C terminus of RTA, both RTA and ricin cytotoxicity were markedly enhanced (11-13), supporting the ER as the distal translocation-competent organelle. Transient expression of transdominant mutants of GTPases involved in vesicular traffic followed by ricin exposure showed reduced intoxication when the GTPases which regulate ER-Golgi traffic (Rab1, ARF1, and Sar1) were modified but no change in ricin cytotoxicity when endosome-related GTPases (dynamin element 1, Rab5, and Rab9) were mutated (14). These results suggest that ricin reaches the Golgi system and yet must proceed further to the ER to produce cell toxicity. The penultimate step in intoxication is membrane translocation. This step requires reduction of the intersubunit disulfide bond (15) and unfolding of RTA (16), but little else is known regarding the molecular mechanism. The final step takes place in the cytosol where RTA catalytically inactivates protein synthesis by hydrolysis of a highly conserved adenosine at the elongation factor binding site on the 60S ribosome (17). Variable sensitivity of tumor cell lines and fresh tumor cells to ricin and ricin-based targeted molecules may in part be due to altered intracellular trafficking. Measurements of premature transport of toxins to lysosomes with subsequent degradation and lack of cytotoxicity have been observed with anti-CD2-RTA, anti-CD3-RTA, and anti-CD5-RTA conjugates (18-20). In each case, the assays were indirect and detected release of free label in the media after incubation of cells with radiolabeled conjugate or increased label in lysosome-enriched sucrose gradient fractions of cell extracts. We sought a labeling method which would permit live cell measurements and not damage the ligand-receptor function of the molecule. © 1997 American Chemical Society

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Green fluorescent protein appeared to offer the ideal properties. Bioluminescent cnidaria, including Aequorea victoria, contain green fluorescent proteins (GFPs) which absorb radiative and nonradiative energy from the photoprotein aequorin and emit green light (21). GFP cDNA has been cloned and expressed in prokaryotic and eukaryotic cells and retains the fluorescent properties of the original compound (22). Recently, the three-dimensional structure of GFP has been solved (23), and chromophore mutants with increased fluorescenceenhanced green fluorescent proteins (EGFPs) have been identified (24). GFP has been fused to several proteins and expressed in mammalian cells (25, 26). Intracellular distributions of the fusion molecules have been measured and depended upon signal sequences in the mammalian peptide. To date, no investigators have reported attachment of GFPs to external proteins which undergo endocytosis and intracellular routing. To obtain more accurate quantitative information on ricin intracellular trafficking, we now report the cloning, expression, and reassociation of an EGFP-ricin molecule and its chemical and biological properties. Our findings support the hypothesis that ricin must be routed to the Golgi network. In addition, the experiments document the ability of EGFP to serve as a tool for measuring the intracellular fate of imported molecules. EXPERIMENTAL PROCEDURES

Construction of the Transfer Vector Encoding EGFP-RTA. The pEGFP-1 plasmid containing DNA encoding EGFP was obtained from Clontech Laboratories (Palo Alto, CA) and propagated in Escherichia coli INVRF′ cells (InVitrogen, San Diego, CA). The plasmid was then prepared using the alkaline lysis method and cesium chloride density gradient centrifugation, and a BamHI DNA cassette encoding EGFP was amplified by polymerase chain reaction (PCR) on a thermal cycler with Taq polymerase following the recommendations of the supplier (Perkin-Elmer, Foster City, CA). Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer and desalted with butan-1-ol. The 5′oligonucleotide contained nine nonsense bases, a BamHI site, and 18 bases from the coding sequence for the N terminus of the EGFP gene. The 3′-oligo contained nine nonsense bases, a BamHI site, and the reverse noncoding strand sequence for two stop codons and the C-terminal amino acid residues of EGFP. The PCR product was subcloned into pCR2.1 (InVitrogen) again as suggested by the manufacturer. PCR2.1-EGFP DNA was restricted with BamHI, and the EGFP fragment was purified by agarose gel electrophoresis and a Prep-AGene matrix (BioRad, Hercules, CA). A cesium chloridepurified preparation of the pGEX2T-RTA plasmid (13) was restricted with BamHI, treated with calf intestinal phosphatase (Boehringer Mannheim, Indianapolis, IN), purified with a Prep-A-Gene matrix, ligated with the EGFP DNA, and used to transform INVRF′ cells. Transformants were analyzed by restriction enzyme digestions with BamHI, EcoRI, and NcoI. The pGEX2T-EGFPRTA plasmid was purified from the appropriate transformant and dideoxy sequenced using the Sequenase kit (U.S. Biochemicals, Cleveland, OH). Expression of GST-EGFP-RTA. The pGEX2TEGFP-RTA construct was transformed into JM105 E. coli cells (Pharmacia Bikotech, Piscataway, NJ), and 1 L cultures were grown at 30 °C with 225 rpm in 2XYT broth containing 100 µg/mL ampicillin induced with 1 mM IPTG (Gibco BRL, Grand Island, NY). After 4 h, the cells were harvested by centrifugation at 5000g for 10 min at 4 °C.

Tagge et al.

Purification of the EGFP-RTA Protein. Purification was accomplished by affinity chromatography utilizing a glutathione-Sepharose matrix and thrombin cleavage. Briefly, cells were resuspended in 50 mL of phosphate-buffered saline (PBS), homogenzied three strokes in a glass homogenizer, sonicated for 5 min total in 30 s bursts, and adjusted to 1% Triton X-100. After gentle mixing on ice for 30 min, and centrifugation at 12000g for 10 min at 4 °C, 1 mL of a 50% slurry of glutathione-Sepharose 4B (Pharmacia) was added and incubation continued for 30 min. This suspension was centrifuged at 500g for 5 min and washed three times with 5 mL of PBS. The matrix was then resuspended in 475 µL of PBS and 25 µL of thrombin (1 IU/mL) added. After overnight cleavage at room temperature, the solution was centrifuged at 500g for 5 min and the supernatant saved. Characterization of EGFP-RTA. The protein concentration was determined by optical density based on absorbance at 280 nm (OD of 1 mg/mL ) 0.77) and a BCA (Pierce, Rockford, IL) protein assay. Tubes containing various stages of purification were subjected to imaging via a 302 nm UV transilluminator for fluorescence. Aliquots of EGFP-RTA at various stages of purification and prestained low-molecular mass standards were subjected to 15% reducing SDS-PAGE, stained with Coomassie Blue R-250 (Sigma, St. Louis, MO), and scanned on an IBAS automatic image analysis system (Kontron, Germany). Additional aliquots of EGFP-RTA were run on 15% reducing SDS-PAGE and proteins transferred to nitrocellulose, blocked with 10% Carnation’s nonfat dry milk/0.1% bovine serum albumin (BSA)/ 0.1% Tween20, washed with PBS plus 0.05% Tween 20, and reacted with either 1:400 rabbit antibody/ricin (Sigma) or 1:1000 rabbit antibody/EGFP (Clontech). The blots were rewashed, incubated with alkaline phosphatase-conjugated goat anti-(rabbit IgG), washed again, and developed with the Vectastain alkaline phosphatase kit (Vector Laboratories, Burlingame, CA) as escribed the manufacturer’s recommendations. The enzymatic activity of EGFP-RTA was quantitated by an in vitro protein synthesis inhibition assay. Dilutions of plant RTA or EGFP-RTA were added to a rabbit reticulocyte lysate protein translation mixture (Promega, Madison, WI) with [3H]leucine (133 Ci/mmol, Du Pont, Wilmington, DE) and the brome mosaic virus mRNA model substrate as specified by the manufacturer. The mixtures were incubated at 4 °C for 30 min and then at 30 °C for 30 min. Protein translation was monitored by following [3H]leucine incorporation into acid-precipitable product according to the manufacturer’s instructions. A 50% inhibition of incorporation (ID50) was determined for each protein. Reassociation of EGFP-RTA with Plant RTB. EGFP-RTA (400 ug) was mixed with a 3-fold molar excess of deglycosylated plant RTB (1.2 mg, Inland Laboratories, Austin, TX) in a total volume of 500 µL of PBS. The excess RTB was added to ensure heterodimer formation of EGFP-RTA. After gentle rocking for 12 h at room temperature under oxidizing conditions that should lead to interchain disulfide bridge formation, aliquots were analyzed to determine heterodimer concentration by a modified ricin ELISA (27). Briefly, wells of an EIA plate (Costar, Cambridge, MA) were coated with 10 µg/mL P2 anti-RTB monoclonal antibody in 100 µL of PBS overnight at 4 °C. The wells were washed with PBS plus 0.1% Tween 20, blocked with 3% BSA in PBS plus 0.02% sodium azide, rewashed, incubated with 12 dilutions of plant ricin (Inland Laboratories) or reassociated heterodimers, washed again, incubated with biotin-

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conjugated RBR12 anti-RTA monoclonal antibody at 5 µg/ mL in PBS/0.5% BSA/0.1% sodium azide, washed, incubated with alkaline phosphatase-conjugated strepavidin (Sigma) at 1:1000 in PBS/0.5% BSA/0.1% sodium azide, washed again, and developed with p-nitrophenyl phosphate (Sigma) at 1 mg/mL in 50 mM diethanolamine at pH 9.8. Absorbance at 405 nm was read on a microplate reader. Excess RTB was removed from the EGFP-RTA-RTB by size exclusion chromatography on a Bio-Sil SEC 125 HPLC column with a Bio-Sil SEC Guard column (BioRad) on a Waters 510 pump and 486 absorbance detector using the PBS column eluant. Column fractions were stored at 4 °C. Characterization of EGFP-Ricin. The protein concentration was again determined by absorbance at 280 nm and the BCA protein assay. Aliquots and prestained low-molecular mass protein standards were run on a nonreducing 15% SDS-PAGE and either stained with Coomassie blue R-250 or transferred to immunoblots for reaction with anti-ricin and anti-EGFP antibodies. Immunoblots were performed as described above. The nonreducing SDS-PAGE measured disulfide bond formation between EGFP-RTA and RTB. An asialofetuin ELISA was performed to measure the galactoside avidity of the heterodimer as previously described (27). Cell Cytotoxicity Assays. KB human epidermoid carcinoma cells and Hep3B human hepatocellular carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI1640 with 10% fetal bovine serum (Irvine Scientific, Irvine, CA). Cells were trypsinized and seeded at 1.5 × 104 cells per well in 100 µL of media in 96-well flatbottomed plates. The cells adhered to plates after several hours of incubation. Fifty microliters of 12 different concentrations of castor bean ricin or EGFP-ricin was added in the same medium, and the cells were incubated at 37 °C in 5% CO2 for 48 h. [3H]Leucine, (0.5 µCi per well, 133 Ci/mmol, Du Pont) in 50 µL of the same medium was added and incubation continued for 4 h. Cells were then harvested with a Skatron cell harvester onto glass fiber mats, dried, mixed with 3 mL of liquid scintillation fluid, and counted in an LKB-Wallac liquid scintillation counter gated for 3H. Cells cultured with medium alone served as controls. All assays were performed in triplicate. The IC50 was the concentration of protein which inhibited protein synthesis by 50% compared to control. Cell Fluorescence Assays. Corning 35 mm dishes were seeded with 105 KB or Hep3B cells and the cells grown in RPMI1640 plus 10% fetal bovine serum at 37 °C in 5% CO2 until 70% confluence. The medium was then aspirated, and dishes were blocked for 15 min with 1% BSA in PBS at 4 °C. Dishes were then incubated with 1% BSA in PBS alone or with EGFP-ricin at 10 µg/mL in 1% BSA in PBS at 4 °C for 30 min, washed three times with PBS, fixed with 3.7% formaldehyde in PBS, washed and permeabilized with inclusion of 0.1% saponin in all subsequent incubations, incubated with 1:500 wheat germ agglutinin conjugated to rhodamine (WGA-TRITC, Sigma) in PBS/BSA/saponin, rewashed, mounted under a no. 1 coverslip in glycerol/PBS (90:10), and examined using a Zeiss Axioplan epifluorescence microscope with the fluorescein channel and Texas Red channel. Identical dishes were heated to 37 °C after the 4 °C incubation with EGFP-ricin and, after 30 or 60 min, washed three times with PBS, fixed in 3.7% formaldehyde, rewashed in saponin-containing buffer, incubated with or without WGA-TRITC in saponin/BSA/PBS,

Figure 1. Schematic diagram of the pGEX2T-EGFP-RTA expression plasmid. In pGEX2T-EGFP-RTA, the coding region of GST-EGFP-RTA is under the control of the Ptac promoter (hatched box) and is followed by codons encoding glutathione S-transferase (striped box), a thrombin cleavable linker (LVPRGS) with a BamHI site (white box), EGFP (black box), and RTA (dotted box). Ampr denotes the β-lactamase gene. pBR322 ori is the high-copy number bacterial replicon. lacIq stands for the lac repressor gene. B ) BamHI. E ) EcoRI. arrow ) thrombin cleavage site. The 729 bp EGFP gene and the 810 bp RTA gene add to the 4948 bp pGEX2T vector to produce a total size of 6.487 kb.

Figure 2. Model of EGFP-RTA showing R-carbon backbones of EGFP (bluegreen), junction amino acid residues of GS (purple), and RTA (yellow). All molecules depicted are based on coordinates read from Brookhaven Protein Data Bank files. The PDB abbreviations are 2aa1-ricin and 1-gfp. The MUSC BioMolecular Computing Resource SYBYL molecular modeling software was used to render the protein chains as shaded ribbons derived from cubic spline fits to the R-carbon backbone.

rewashed, mounted under glycerol/PBS, and examined for epifluorescence. RESULTS

Construction of the EGFP-RTA-Encoding Bacterial Expression Plasmid. The EGFP-coding region from pEGFP-1 was modified to create a BamHI cassette with the correct reading frame and subcloned in pGEX2T-

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Figure 3. Nonreducing 15% SDS-PAGE of EGFP-RTA, an EGFP-RTA and RTB mixture, and purified EGFP-ricin. (A) Coomassie-stained gel. (B) Immunoblot using rabbit anti-ricin antibody. (C) Immunoblot using rabbit anti-EGFP antibody: lane 1, low-molecular mass prestained BioRad protein standards; lane 2, EGFP-RTA; lane 3, mixture of EGFP-RTA and plant RTB after 24 h incubation at room temperature; and lane 4, post-HPLC-purified EGFP-ricin. Bands are observed at 55 kDa (EGFP-RTA), 34 kDa (RTB), 65 kDa (RTB homodimers), and 90 kDa (EGFP-ricin). Arrows point to protein standards and show molecular masses in kilodaltons.

Figure 4. Fluorescence of EGFP-RTA. Eppendorf tubes (1.5 mL) containg 0.5 mL of various fractions with EGFP-RTA illiminated with a 302 nm UV light: tube number 1, induced E. coli pellet postsonication; tube number 2, glutathioneSepharose matrix after binding-induced bacterial extract; tube number 3, eluant from matrix prior to thrombin cleavage; and tube number 4, thrombin-cleaved eluant containing EGFPRTA.

RTA (Figure 1). The final construction was verified by sequencing. Production of EGFP-RTA. Yields of 0.375 + 0.025 mg/(L of culture medium) were obtained on the basis of absorbance measurements and Bradford assays. The predicted folding of the molecule based on published coordinates in the Brookhaven Protein Data Bank is shown in Figure 2. Characterization of EGFP-RTA. SDS-PAGE followed by Coomassie staining showed a single band of 55 kDa (Figure 3A). On the basis densitometry of the Coomassie-stained gel, the purity was in excess of 90%. Immunoblots with rabbit anti-ricin and rabbit anti-EGFP showed similar reactivity (Figure 3B,C). Green light emission after UV illumination at 302 nm was observed in both the GST fusion and the final EGFP-RTA preparation (Figure 4). The enzymatic activity of the EGFP-RTA was indistinguishable from that of previous preparations of bacterial RTA and plant RTA. All three proteins inhibited in

Figure 5. In vitro rabbit reticulocyte lysate protein synthesis inhibition assay. The assay was as described in text. Each experiment was performed in triplicate: (b) plant RTA, (2) bacterial recombinant RTA, and (9) EGFP-RTA. ID50 ) 2 × 10-11 M for each.

vitro protein synthesis of rabbit reticulocyte lysates with ID50s of 2 × 10-11 M (Figure 5). Reassociation of the Recombinant Protein with Plant RTB. After reassociation with a 3-fold molar excess of plant RTB, 31 + 6% of the EGFP-RTA formed heterodimers, based on ricin ELISA and yields from HPLC. As shown in lane 3 of Figure 3C, no EGFP-RTA remained unreassociated after oxidation with excess RTB. Properties of EGFP-Ricin. Size exclusion chromatography of the EGFP-RTA-RTB plus RTB mixture revealed a single fraction eluting at 90 kDa with green fluorescence (Figure 6). SDS-PAGE followed by Coomassie staining and immunoblots revealed a single band at 90 kDa which reacted with both anti-EGFP antibodies and anti-ricin antibodies (Figure 3). The heterodimer bound asialofetuin with that same avidity as plant ricin (Kds ) 10-9 M). The EGFP-ricin was cytotoxic to mammalian cells with IC50s of 6 × 10-11 M on KB cells and 7 × 10-13 M on Hep3B cells (Figure 7). Plant ricin had IC50s of 5 × 10-13 and 5 × 10-14 M on KB and Hep3B cell lines, respectively. Thus, the fusion protein is approximately 10-fold less cytotoxic for Hep3B and 100-fold less cytotoxic for KB cells than ricin.

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Figure 8. Fluorescence of the EGFP-ricin pathway in mammalian cells. KB cells were attached to tissue culture dishes, incubated with EGFP-ricin for varying times, fixed with 3.7% formaldehyde in PBS, and examined for fluorescence on the fluorescein channel. (A and A′) Incubation at 4 °C for 30 min. (B and B′) Incubation at 37 °C for 60 min. (A and B) Phase contrast. (A′ and B′) Fluorescein channel. The arrowhead in panel A′ shows surface localization of fluorescence; the arrowhead in panel B′ shows endosomal distribution, and the long arrow in panel B′ shows Golgi localization. Magnification ) 280 times; bar ) 20 µm.

Figure 6. Elution profie from a HPLC Bio-sil SEC125 column with PBS elution. OD280 is on the y-axis, and the time for elution is in minutes on the x-axis. Elution times for BioRad gel filtration protein standards are shown as arrows, including thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), and equine myoglobin (17 kDa). The pooled sample with fluorescence is shown in brackets. Delayed elution of EGFP-ricin and RTB fractions was observed.

Figure 9. Fluorescence distribution of EGFP-ricin after 60 min of incubation at 37 °C in ricin-sensitive (Hep3B) and -resistant (KB) cell lines. (A and A′) Hep3B. (B and B′) KB. (A and B) Phase contrast. (A′ and B′) Fluorescence. Arrows ) Golgi region fluorescence. Magnification ) 202.5 times; bar ) 25 µm.

Figure 7. Cell cytotoxicity of ricin and EGFP-ricin. Cells were exposed at the dilutions indicated for 48 h at 37 °C in 5% CO2. Incorporation of [3H]leucine was assayed after a 4 h incubation and compared against untreated cell incorporation: (b) ricin on Hep3B cells, (9) EGFP-ricin on Hep3B cells, and (O) ricin on KB cells, (0) EGFP-ricin on KB cells.

Immunofluorescent Detection of EGFP-Ricin. Cells incubated with EGFP-ricin at 4 °C showed diffuse plasma membrane fluorescence indicating surface localization (Figure 8). After the cells were warmed to 37 °C for 30 min, fluorescence on surface membranes decreased and an intracellular pattern of fluorescence was seen which was consistent with endosomes. After 1 h at 37 °C, EGFP-ricin was detected in both endosomes and in the Golgi network (Figure 8). Comparative studies were done with KB cells and Hep3B cells (Figure 9). After 1

h incubation of cells with EGFP-ricin at 37 °C, much more Golgi fluorescence was detected in Hep3B cells than in KB cells. Control experiments with wheat germ agglutinin conjugated to tetramethylrhodamine isothiocyanate (WGA-TRITC) confirmed colocalization with EGFP-ricin in Golgi organelles (Figure 10). DISCUSSION

EGFP-RTA was produced in bacteria as a glutathione S-transferase (GST) fusion protein. The GST-EGFPRTA lost fluorescence properties when bacteria were grown at 37 °C instead of at 30 °C. A similar loss in fluoroscence was seen on lyophilization of EGFP-RTARTB. The sensitivity of the EGFP chromophore to temperature and high-ionic strength autoxidation has been previously reported (28). The GST-EGFP-RTA produced in E. coli cells at 30 °C was soluble in the bacterial cell and readily released by sonication. After binding to glutathione-Sepharose

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Figure 10. KB cells imaged in a Zeiss Axioplan epifluorescence microscope with a DAGE/MTI 100 integrating CCD with separate Texas Red and FITC filters (Chroma). Cells were magnified 100 times with a Plan-Neofluar NA1.3 objective lens. (A-C) FITC channel. (A′-C′) Texas Red channel. Specimens were prepared as described in the text. Colocalization of EGFP-ricin and WGA-TRITC is shown with arrows. (A and A′) At 4 °C, 30 min of incubation with EGFP-ricin at 10 µg/mL. (B and B′) At 4 °C, 30 min of incubation with WGA-TRITC (1:500, Sigma). (C and C′) At 37 °C, 20 min of incubation with EGFP-ricin and WGA-TRITC.

and cleavage with thrombin, reasonable yields of 0.35 mg/ (L culture medium) were obtained. These were 10-fold lower than that with RTA or RTA-KDEL reported previously with this expression vector and cell line (13), but well within the range of workable concentrations for purification and biological assays. The observed extinction coefficient of 0.77 is similar to that of RTA alone (29) and suggests similar absorbance characteristics for the amino acid residues of EGFP and RTA at 280 nm. The molecular mass of EGFP-RTA deduced from reducing SDS-PAGE was 55 kDa which is identical to that expected from the fusion of the 27 kDa Mr EGFP (22) and the 28 kDa Mr RTA peptides (13). The reactivity of the hybrid molecule with antibodies to EGFP and ricin suggests, epitopes for each domain are accessible on the molecular surface. This would be expected from the three-dimensional structure of each polypeptide. The C terminus of EGFP is freely mobile and remote from the β-barrel (23), and the N terminus of RTA also has significant mobility (30, see Figure 2). Thus, the individual domains should retain their native folding without steric hindrance from the other component. Further,

they should have multiple epitopes on each domain accessible to immunoglobulin in the solvent. The ability of EGFP-RTA to reassociate with RTB was expected because of the extensive ionic and hydrophobic bonds in the RTA-RTB interface which promote reassociation and the disulfide bond between RTA Cys-259 and RTB Cys-4 (31). Our observation of 30% reassociation compares reasonably well with the 50% reassociation for plant RTA-RTB under identical conditions (32). The RTA amino acid residues which interact with RTB include H40, E41, Q182, Y183, L207, R234, R235, F240, I247, I249, P250, I251, I252, R258, C259, and A260, which are remote from the N terminus (31). The purification of the EGFP-ricin by HPLC Bio-Sil SEC125 size exclusion chromatography yielded one-step removal of contaminating subunits with excellent recovery and maintenance of activities. We have observed a lack of detectable cell surface binding of EFGP-ricin in the presence of excess RTB ligand (unpublished observations); hence, the purification step was necessary for subsequent studies. Cell fluorescence experiments required high EGFPricin concentrations (20 µg/mL or 500 nM). This concen-

Synthesis of Green Fluorescent Protein−Ricin

tration is 10-fold higher than the available receptor sites at 107/cell and 40-fold above the Kd of 1 nM. Thus, a relatively weak signal was observed relative to immunofluorescence assays with ricin (27) where multiple antibody molecules each with several fluoresceins amplify the signal. Further, the fluorescein channel on the epifluorescence microscope (309 nm) is designed for fluorescein absorption and emission. We did not optimize the detection system for EGFP (489 nm). The lower sensitivity of KB cells relative to Hep3B cells to ricin-based toxicity may be due to altered receptor density, altered intracellular compartmentalization, altered rates of degradation, different efficiencies of translocation, differing rates of cytosolic RTA metabolism, or varying ribosome inactivation. KB and Hep3B cells have different genetic backgrounds, and any of these events may, in principle, affect the sensitivity of these cells with respect to ricin. Nevertheless, our results of different accumulated concentrations of ricin in the Golgi network are consistent with observations from other laboratories. Antibody-RTA conjugates with similar cell surface binding to different epitopes on T cell antigens (CD2, CD3, and CD5) showed variable cytotoxicity with respect to T cells which was related to excessive lysosomal transport and degradation (18-20). Toxin routing in such cells should lead to reduced concentrations in the Golgi cisternae. Further, the reduced toxicity of ricin at 19 °C where Golgi uptake is blocked (9) and the reduced sensitivity of myeloid leukemia cell lines to antibodyRTA conjugates with increased lysosomal trafficking of ligands (33) are additional examples of the role of Golgi transport in ricin intoxication. This report is the first demonstration of the use of EGFP in following the endocytic pathway of surfacebound polypeptide ligands. ACKNOWLEDGMENT

We thank Starr Hazard for the molecular graphics analysis and James Nicholson for imaging analysis. This study was supported in part by NIH Grant R29CA74677 (E.T.). LITERATURE CITED (1) Baenziger, J., and Fiete, D. (1979) Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. J. Biol. Chem. 254, 9795-9799. (2) Sandvig, K., and Olsnes, S. (1982) Entry of the toxic proteins abrin, modeccin, ricin and diphtheria toxin into cells. J. Biol. Chem. 257, 7504-7513. (3) Olsnes, S., Refsnes, K., and Pihl, A. (1974) Mechanism of action of the toxic lectins abrin and ricin. Nature 249, 627631. (4) Ray, B., and Wu, H. (1982) Chinese hamster ovary cell mutants defective in the internalization of ricin. Mol. Cell. Biol. 2, 535-544. (5) van Deurs, B., Pedersen, L., Sundan, A., Olsnes, S., and Sandvig, K. (1985) Receptor-mediated endocytosis of ricincolloidal gold conjugate in Vero Cells. Exp. Cell Res. 159, 287304. (6) Nicholson, G. (1974) Ultrastructural analysis of toxin binding and entry into mammalian cells. Nature 251, 628-629. (7) Gonatas, J., Stieber, A., Olsnes, S., and Gonatas, N. (1980) Pathways involved in fluid phase and adsorbtive endocytosis in neuroblastoma. J. Cell Biol. 87, 579-588. (8) van Deurs, B., Tonnessen, T., Petersen, O., Sandvig, K., and Olsnes, S. (1986) Routing of internalized ricin and ricin conjugates to the Golgi complex. J. Cell Biol. 102, 37-47.

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