©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of Toxin Sensitivity in Insect Cells by Infection with Baculovirus Encoding Diphtheria Toxin Receptor (*)

Elsa M. Valdizan (§) , Evgenij V. Loukianov (¶) , Sjur Olsnes (**)

From the (1)Institute for Cancer Research, Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The diphtheria toxin receptor (DTR) has been identified as the precursor of heparin-binding epidermal growth factor-like growth factor, which may interact with other membrane proteins to form the functional receptor. To test if mammalian DTR is able to confer toxin sensitivity onto phylogenetically distant cells, we expressed monkey DTR in the baculovirus system and tested infected insect cells for toxin sensitivity. cDNA encoding an epitope-tagged heparin-binding epidermal growth factor-like growth factor precursor (DTR) was inserted into the virus genome by allelic replacement to construct the recombinant virus vAc-DTR. SF9 cells infected with vAc-DTR expressed functional DTR, which could be precipitated from the solubilized membrane fraction of infected cells with Sepharose-immobilized diphtheria toxin. The highest level of expression (about 5 10 receptors/cell) was observed 48 h after infection, at which time the infected cells were highly sensitive to diphtheria toxin. Uninfected SF9 cells and cells infected with the wild type virus were resistant to the toxin. The presence of heparin increased both the binding and the toxin sensitivity of vAc-DTR-infected SF9 cells. Translocation of toxin A fragment was induced when cells with surface-bound toxin were exposed to low pH, and the translocation was optimal at pH 5.5. It was 100 times more efficient at 24 °C than at 4 °C. The data indicate that monkey DTR is fully functional when expressed in insect cells.


INTRODUCTION

Diphtheria toxin (DT)()is a cytotoxic protein (M 58,348) secreted by Corynebacterium diphtheriae(1) as a single polypeptide, which is easily nicked by trypsin-like proteases into the disulfide-linked fragments A (M 21,147) and B (M 37,195)(2) . The toxin binds to cell surface receptors by the B fragment(3, 4) . The DTR has recently been identified as the precursor of HB-EGF-like growth factor(5) . The receptor-bound toxin undergoes endocytosis via clathrin-coated pits (6). The low pH environment in the endosomes induces a conformational change in the B fragment, and the enzymatically active A fragment is translocated to the cytosol(7, 8, 9, 10, 11) . There, the A fragment catalyzes ADP-ribosylation of elongation factor 2, resulting in inhibition of protein synthesis and cell death(2, 12) . Translocation of diphtheria toxin can also be induced directly from the cell surface by exposing cells with bound DT to low pH medium, thereby mimicking the conditions inside the endosome(8, 9, 11, 13) .

While cells of human and monkey origin are in general sensitive to DT to varying extent, cells from rats and mice are toxin resistant(14) . The variation in sensitivity of different cells appears to be largely due to differences in the number of DTRs(15, 16, 17) . Other factors have been implicated, such as different affinity of the receptors for the toxin and the presence of additional proteins associated with the receptor, that could play a role in the translocation process(18, 19) .

Transfection of DTR has been shown to confer toxin sensitivity onto mouse cells that are normally resistant to the toxin. It is not clear if DTR alone is required for toxin translocation or if other proteins, such as CD9, are also required. We therefore decided to test if monkey DTR can confer sensitivity onto a cell of non-vertebrate origin, as the possibility of correct interaction with any required additional membrane proteins was considered to be much lower in these cells than in mammalian cells. We chose to express DTR in the baculovirus system. This system utilizes insect cells that can be infected with the naturally occurring baculovirus AcNPV. During lytic infection, the gene product for the baculovirus polyhedrin is expressed in high amounts and can account for about 50% of the total cellular protein(20) . The polyhedrin gene is not essential for production of infectious virions. Therefore, expression vectors that use the strong AcNPV polyhedrin promoter to drive expression of foreign genes have been developed(21) . By using this approach, large amounts of eukaryotic proteins have been successfully produced, including a variety of receptors. In addition, insect cells can perform many of the post-translational modifications carried out by higher eukaryotes (for review see Ref. 22). These features make the baculovirus system an attractive choice for large scale production of normal and mutant DTRs, as recently reported by Ono et al.(23) . The biological activity as DTR in the infected cells was not tested by these authors.

In the present work, we have studied the expression of DTR by SF9 and HF cells infected with recombinant baculovirus. We have made a construct with an epitope (B3, against which we have an antibody) added to the COOH-terminal (cytoplasmic) end of the receptor to allow immunoprecipitation from lysed cells. Expression of the DTR in COS cells()showed that the presence of this epitope does not impair the ability of DTR to ensure binding and translocation of the toxin.


EXPERIMENTAL PROCEDURES

Materials

[S]Methionine, 1-[3,4,5,-H]leucine, and I were purchased from DuPont NEN. Pronase E (from Streptomyces griseus), trypsin (TPCK-treated), PMSF, N-ethylmaleimide, heparin (sodium salt grade 1A from porcine intestinal mucosa), monensin, Nonidet P-40, sodium deoxycholate, dithiothreitol, Triton X-100, leupeptin, aprotinin, DNase, antipain, and X-gal were from Sigma. Chymostatin and pepstatin A were from Fluka (Buchs, Switzerland). Diphtheria toxin (crude) was purchased from Connaught Laboratories (Willowdale, Canada) and purified as described(9) . Protein-A Sepharose and CNBr-Sepharose were from Pharmacia LKB (Uppsala, Sweden).

Antibodies

Rabbit immune serum against the B3 peptide (GVNEYNEMPMPVN) was prepared by Cambridge Research Biochemicals (Cambridge, United Kingdom).

Buffers

The following buffers were used: PBS (140 mM NaCl, 10 mM NaHPO, pH 7.2); lysis buffer (50 mM Tris-Cl, pH 8, 150 mM NaCl, 1 mM MgCl, 0.02% (w/v) sodium azide, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 1 mM dithiothreitol, 20% (v/v) glycerol, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml chymostatin, 2 µg/ml aprotinin, 1 mg/ml DNase, 1 µg/ml pepstatin A, 2 µg/ml antipain, and 10 IU/ml heparin); Hepes medium (bicarbonate- and serum-free Eagle's minimum essential medium buffered with 20 mM Hepes at pH 7.4); low pH buffer for SF9 cells (TNM-FH medium plus 10 mM sodium gluconate adjusted to the indicated pH); low pH buffer for Vero cells (Hepes medium plus 10 mM sodium gluconate adjusted to the indicated pH).

Cells

The insect cell line SF9 was obtained from Unni Spaeren (Tromsø, Norway), HF insect cells were purchased from InVitrogen Corp. (Oxon, UK). The cells were propagated in suspension at 27 °C in TNM-FH Medium (Sigma) supplemented with 10% fetal calf serum, 10,000 IU/liter penicillin, and 10 mg/liter streptomycin (Life Technologies, Inc., Uxbridge, UK).

Viral and Plasmid DNA

Wild type linearized AcNPV DNA and plasmid pBlueBacIII were purchased from InVitrogen. In the transfer vector, the cloning site downstream of the polyhedrin promoter and the LacZ gene are flanked by two recombinant sequences identical to sequences in the polyhedrin region of the AcNPV DNA.

To construct the recombinant baculovirus expressing DTR (vAc-DTR), the 690-bp segment of the DNA corresponding to the monkey HB-EGF precursor with the COOH-terminal B3 epitope was cloned into the BamHI and PstI sites of the pBlueBacIII. 3 µg of plasmid DNA was cotransfected with 1 µg of linearized wild type AcNPV total viral DNA into 2.5 10 SF9 cells using cationic liposome-mediated transfection as indicated in the instructions from InVitrogen. A double crossover recombinant was insolated by the plaque purification method as described by O'Reilly et al.(22) by screening in the presence of X-gal for blue plaques with the occlusion body negative (polyhedrin negative) phenotype. The chosen plaques were tested by binding of I-labeled DT to infected cells, and those that showed high binding were tested by PCR for the total absence of any wild type AcNPV virus DNA.

In the PCR, a band of about 800 bp is expected for the wild type viral DNA, whereas for the recombinant virus the expected band is about 1,300 bp. If contamination with the wild type viral DNA occurs or if the recombinant virus originated from a single crossover, two bands of about 800 and 1,300 bp appear upon PCR amplification. All the recombinants tested by PCR gave a single band of the expected size (1,300 bp), demonstrating that vAc-DTR but not wild type viral DNA (800-bp band) was obtained. After confirmation, one recombinant was expanded to 3.10 plaque-forming units/ml by infecting SF9 cells at a multiplicity of infection of 0.1 plaque-forming units/cell.

For experiments, SF9 cells were infected with the wild type or the recombinant vAc-DTR at a multiplicity of infection of about 20 plaque-forming units/cell or were mock infected with TNM-FH medium (SF9) for 1 h. Then, complete medium was added, and the infection was followed for the indicated periods of time (hpi).

X-Gal Staining of Infected Cells

At 24, 48, and 72 hpi, the cells were washed with PBS and fixed with 1% (v/v) formaldehyde, 0.2% (v/v) glutaraldehyde in PBS for 5 min. Subsequently, the cells were washed three times with PBS and incubated overnight in 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl, and 0.4 mg/ml X-gal. The reaction was stopped by washing twice with PBS, and the cells were observed under the microscope, screening the number and the intensity of blue cells.

Immunofluorescence Detection of Expressed DTR

At 24, 48, and 72 hpi, HF cells adherent to poly-L-lysine-treated glass coverslip were washed with ice-cold PBS, and then 10 µg/ml DT was added and allowed to bind to the infected cells for 30 min at room temperature as described below. The cells were washed three times with ice-cold PBS and subsequently incubated with anti-DT antibodies and then with FITC-labeled goat anti-rabbit IgG, and the coverslip was mounted on a microscopy slide with Mowiol and examined under UV light.

Radiolabeling of DT

DT or nDT were labeled with I by the IODO-GEN method to a specific activity of about 1 10 cpm/ng as described(24) .

DT Binding Assay

At 48 hpi, SF9 cells were incubated in binding medium (Grace's insect medium plus 10% fetal calf serum and with or without 10 IU/ml heparin as indicated) with increasing amounts of I-labeled DT in the absence or presence of 10 µg/ml unlabeled DT for 30 min at room temperature. The reaction was stopped by filtration through Whatman GF/A filters, which were then washed five times with 10 ml of ice-cold PBS, and the radioactivity associated with the filters was measured in an autogamma spectrometer. The specific binding was calculated as the difference between the radioactivity associated with the filters in the absence and presence of 10 µg/ml unlabeled DT. The unspecific binding to cells infected with the recombinant virus varied from 10 to 40% of the total binding for different experiments, depending of the batch of labeled DT. The specific binding was plotted as described by Scatchard (25) to determine the number and the affinity of DT to the DTR. Binding curves were generated by regression analysis.

Vero cells in 12-well plates were incubated for 30 min at room temperature with I-labeled DT in Hepes medium plus 0.1 mg/ml bovine serum albumin in the absence or presence of 10 µg/ml unlabeled DT. The cells were then washed four times with PBS and lysed in 0.1 M KOH, and the radioactivity associated with the cells was measured.

Nicking of DT

DT at a concentration of 1.1 mg/ml was nicked with 0.5 µg/ml trypsin for 30 min at 37 °C; the reaction was stopped by adding 25 µg/ml soybean trypsin inhibitor.

Cytotoxicity Assay

To assess the rate of protein synthesis, the incorporation of [H]leucine into cellular proteins was measured at 36 or 48 hpi. Although maximum expression of DTR was at 48 hpi, for some of the toxicity experiments binding of the toxin was carried out at 36 hpi to allow the toxin to act overnight to produce maximal effect on protein synthesis without lysis of the cells. At the indicated hpi, the cells were recovered by centrifugation for 5 min at 1000 g at room temperature and then washed with 10 ml of PBS. The cells were resuspended in binding medium and incubated with increasing amounts of nDT for 30 min at the indicated temperatures in the absence or presence of 10 µM of monensin. The medium and unbound toxin was removed, and the cells were recovered by centrifugation and resuspended in buffer at the indicated pH for 5 min at 27 °C to allow translocation of bound toxin to occur; then, 3 ml of complete TNM-FH medium with or without 10 µM monensin and anti-DT serum was added, and the cells were incubated for 2 h at 27 °C to allow the translocated toxin to inhibit protein synthesis. In some experiments, the low pH pulse was omitted. After the incubation, the cells were recovered by centrifugation and washed with 1 ml of leucine-free Grace medium and incubated in 1 ml of leucine-free Grace medium with 1 µCi/ml [H]leucine for 30 min at room temperature. The cells were recovered by centrifugation and treated twice with 1 ml of 5% trichloroacetic acid for 10 min; the pellets were recovered by centrifugation for 10 min at 14,000 rpm and dissolved in 300 µl of 0.1 M KOH, and the radioactivity was measured.

Vero cells were incubated in binding medium with increasing amounts of nDT for 30 min at the indicated temperature in the absence or presence of 10 µM monensin as indicated. Medium and unbound toxin was washed away, and low pH buffer was added for 5 min at 37 °C; then, 3 ml of complete medium (with or without 10 µM monensin and anti-DT serum) was added, and the cells were incubated or for 2 h at 37 °C. The incorporation of [H]leucine was then measured for 30 min at 37 °C in leucine-free Hepes medium supplemented with 1 µCi/ml [H]leucine. The cells were treated twice with 1 ml of 5% trichloroacetic acid for 10 min and dissolved in 300 µl of 0.1 M KOH; the radioactivity was then measured.

Pronase Protection Experiments

At 48 hpi, 100 ng of I-labeled nDT was bound to vAc-DTR-infected cells for 30 min at 4 °C in presence of 10 µM monensin. Unbound toxin was removed by washing the cells five times with 10 ml of PBS. Then, the cells were exposed for 5 min to the indicated pH values at 27 °C to allow translocation of the bound toxin to occur. The cells were incubated for 10 min at 27 °C with 6 mg/ml Pronase E in the presence of 10 µM monensin and subsequently recovered by centrifugation for 3 min at 14,000 rpm. After washing with PBS containing 1 mMN-ethylmaleimide and 1 mM PMSF, the cells were lysed for 10 min in lysis buffer containing 1 mMN-ethylmaleimide and 1 mM PMSF on ice, and the nuclei were removed by centrifugation. Cellular proteins were precipitated with 5% trichloroacetic acid for 15 min on ice and pelleted by centrifugation. The pellets were washed with ether and subjected to SDS-PAGE under non-reducing conditions.

DTR Precipitation

At 48 hpi, uninfected SF9 cells, SF9 cells, and SF9 cells were labeled with 1 µCi/ml [S]methionine for 4 h and then lysed with 1 ml of lysis buffer for 30 min on ice; the nuclei were removed by centrifugation for 5 min at 14,000 rpm at 4 °C, and the supernatant was used for DTR precipitation. For precipitation of DTR, 500 µl of cell lysate was incubated for 2 h at 4 °C with DT-Sepharose or anti-B bound to protein-A-Sepharose. The pellets were washed three times with lysis buffer, once with PBS, and once with water. They were then treated with sample buffer, and the extracted material was analyzed by SDS-PAGE under reducing conditions.

SDS-PAGE-SDS-PAGE was carried out in 12% gels. The gels were fixed in 4% (v/v) acetic acid, 27% (v/v) methanol for 30 min and then treated with 1 M sodium salicylate, pH 5.8, in 2% (v/v) glycerol for 30 min. To visualize proteins labeled with I or with [S]methionine, dried gels were exposed to Kodak XAR-5 films with intensifying screens at -80 °C for fluorography.


RESULTS

Receptor Expression

Recombinant baculovirus encoding the DTR gene was constructed by standard techniques and checked by PCR as described under ``Experimental Procedures.'' To check the time course of the virus infection as well as the level of viral protein production, we first monitored the expression of the LacZ gene visualized by staining the insect cells with X-gal at different times post-infection. The number of blue cells and the intensity of the blue color reflect the amount of infected cells and the level of viral protein expression. By this method, we found that 24 hpi almost 90% of the vAc-DTR-infected cells were blue, whereas cells infected with wild type virus as well as uninfected SF9 cells remained colorless. At 48 and 72 hpi, 100% of the cells became blue, and also the intensity of the color was increased from 48 to 72 hpi, indicating an increase in the viral protein production (data not shown). About 72 hpi, virus-induced cell lysis starts. As the LacZ gene is produced from an earlier promoter (pEtl promotor) than that of polyhedrin, the X-gal staining of the cells indicates the progress in the virus infection but not the levels of DTR production.

To test directly the expression of DTR, we monitored the ability of the transfected cells to bind I-labeled DT at 24, 48, and 72 hpi. The specific binding increased to a maximum at 48 hpi, and it was again lower at 72 hpi, probably due to beginning cell lysis.

To visualize the expressed DTR by immunofluorescence, we used HF insect cells because these cells were attached more strongly to glass coverslips than SF9 cells. We allowed DT to bind to the cells, which were then incubated with antibodies against DT followed by an FITC-labeled second antibody (Fig. 1). No fluorescence was observed in uninfected cells or in cells infected with wild type virus. In cells infected with recombinant virus, it was possible to detect receptor expression at 24 hpi. The fraction of cells showing fluorescence and the intensity of the fluorescence increased with time from 24 to 48 hpi, whereas at 72 hpi it was not possible to stain the cells because of lysis.


Figure 1: Immunofluorescence staining of vAc-DTR-infected HF cells. 24 h after infection, the cells were incubated for 30 min with 10 µg/ml DT at room temperature, washed to remove unbound toxin, and subsequently incubated with rabbit anti-DT serum for 30 min at room temperature. The bound antibodies were visualized with FITC-labeled anti-rabbit IgG in TNM-FH medium plus 20% aggregated -globulin for 30 min at room temperature. Finally, the cells were washed with PBS, mounted with Mowiol on microscopy slides, and observed in a fluorescence microscope.



To immunoprecipitate the expressed receptors, we used an anti-B3 serum against the COOH-terminal epitope added to the receptor. We also precipitated the DTR with DT immobilized on Sepharose. As it is shown in Fig. 2(lanes3 and 6), a band of about 25 kDa (arrow) was observed in both cases. When the DTR was precipitated with DT-Sepharose, the amount precipitated was much higher than in the case of immunoprecipitation with anti-B3 serum, indicating that the B3 epitope is either not well exposed or partly removed in the cells. Also, two additional bands of about 20 kDa (asterisks) were precipitated with DT-Sepharose (lane6). These bands were not observed in uninfected SF9 cells (lanes1 and 4) or in cells infected with wild type virus (lanes2 and 5).


Figure 2: Expression of DTR in SF9 insect cells. Uninfected SF-9 cells (lanes1 and 4) and SF9 cells infected with wild type virus (lanes2 and 5) or with vAc-DTR (lanes3 and 6) for 48 h were labeled for 4 h with [S]methionine and lysed in 1 ml of lysis buffer for 30 min. The nuclei were removed by centrifugation, and the supernatant was incubated for 2 h at 4 °C with DT-Sepharose or anti-B3 bound to protein-A-Sepharose as described under ``Experimental Procedures.'' For precipitation, immobilized rabbit anti-B3 serum bound to protein-A-Sepharose (lanes1-3) and DT-Sepharose (lanes4-6) was used. For fluorography, the dried gel from anti-B3 protein-A-Sepharose immunoprecipitation was exposed, as described under ``Experimental Procedures,'' for 8 days. The dried gel from DT-Sepharose precipitation was exposed overnight.



Binding of Radiolabeled DT to Cell Surface Receptors

We have in the following defined specific binding of DT as the difference in binding of I-labeled DT in the presence and absence of excess unlabeled toxin. In uninfected SF9 cells and cells infected with the wild type virus, only marginal binding was detected, indicating that the cells do not express appreciable amounts of receptors for diphtheria toxin.

To measure DT binding to infected cells, we tested SF9 cells 48 h after the infection with vAc-DTR. HB-EGF constitutes part of DTR(26) . We therefore tested if the presence of heparin in the binding medium affected the binding of DT to the receptor. Uninfected SF9 cells and vAc-DTR-infected cells were incubated with 100 ng/ml I-labeled DT and increasing amounts of heparin in the absence and presence of 10 µg/ml unlabeled DT. The results (Fig. 3) showed that heparin induced a considerable increase in the amount of total binding in the vAc-DTR-infected cells but not in uninfected cells, indicating that the binding to the infected cells was of a different kind. The binding increased up to 10-20 IU/ml heparin and then decreased again with higher heparin concentrations. Compared to the increase seen in vAc-DTR-infected cells (Fig. 4A), there was only a small increase in the binding of I-labeled DT in presence of heparin in Vero cells (Fig. 4B).


Figure 3: Effect of increasing amounts of heparin on binding of I-labeled DT to uninfected and vAc-DTR-infected SF9 cells. The cells were incubated with 100 ng/ml I-labeled DT in the absence (top of the bars) or presence (bottom of the bars) of 10 µg/ml unlabeled DT and increasing amounts of heparin for 30 min at room temperature. Then, the cells were washed with ice-cold PBS on Whatman GF/A filters, and the radioactivity associated with the filters was measured.




Figure 4: Effect of heparin on the specific binding of I-labeled DT to vAc-DTR-infected SF9 cells (A) and Vero cells (B). Cells were incubated with increasing amounts of I-labeled DT in the absence and presence of 10 µg/ml unlabeled DT and 10 IU/ml heparin for 30 min at room temperature. Then, the insect cells were washed on Whatman GF/A filters with ice-cold PBS, and the radioactivity associated with the filters was measured. The Vero cells (growing on plastic) were washed with PBS and lysed in 0.1 M KOH, and the radioactivity associated with the cells was measured. The specific binding indicated on the ordinate was calculated as the difference between the radioactivity bound in absence and presence of unlabeled DT. , no heparin; , 10 IU/ml heparin.



The vAc-DTR-infected cells were found to bind I-labeled DT in a highly specific and saturable manner (Fig. 5A). A single binding affinity (K of 3.7 10M) and 5 10 binding sites/cell were calculated. In comparison, Vero cells were found to express 22,000 receptors/cell with an affinity of 2.1 10M (Fig. 5B).


Figure 5: Saturation binding of I-labeled DT to vAc-DTR-infected SF9 cells (A) and to Vero cells (B). Binding was carried out as described in Fig. 4 in the presence of 10 IU/ml heparin. The specific binding () was calculated as the difference between the total binding () and the unspecific binding in presence of 10 µg/ml DT (). The insets represent Scatchard analyses of the data presented in each panel. The data were fitted by regression analysis of the specifically bound I-labeled DT on abscissa and bound/free toxin (B/F) on the ordinate.



Sensitivity of Infected SF9 Cells to Diphtheria Toxin

After binding to DTR on toxin-sensitive cells, DT is normally endocytosed, and, upon acidification, the enzymatically active A fragment is translocated across the endosomal membrane(7, 8, 9, 10, 11) . This normal route can be mimicked at the level of the cell surface membrane by acidification of the culture medium(8, 9, 11, 13) .

Control experiments with uninfected cells showed that SF9 cells are resistant to diphtheria toxin, indicating the absence of functional receptors. Cells infected with the wild type virus were also resistant (data not shown).

We tested the toxin sensitivity of vAc-DTR-infected cells and Vero cells under conditions where the toxin was bound to the cells for 30 min at 4 °C, and then the cells were exposed to either neutral or low pH medium and incubated at neutral pH in the presence of monensin to inhibit toxin entry by the normal route. The rate of protein synthesis was measured 2 h later. When the low pH pulse was omitted, protein synthesis inhibition was not observed in either cell type (Fig. 6). In contrast, when cells with bound toxin were exposed to pH 4.8, protein synthesis in the vAc-DTR-infected SF9 cells was inhibited at very low toxin concentrations. Both Vero cells and vAc-DTR-infected insect cells were more sensitive to DT when incubated with the toxin over night to let the toxin be endocytosed and enter by the normal route than when the toxin entry was induced by acidification of the medium (Fig. 7, A and B).


Figure 6: Effect of pH on the sensitivity to DT of vAc-DTR-infected SF9 cells (A) and Vero cells (B). Increasing amounts of nicked DT were added to cells for 30 min at 4 °C in binding medium with 10 µM monensin. The medium with unbound toxin was removed, and medium adjusted to pH 4.8 () or to pH 6.5 () was added. The incubation lasted for 5 min at 27 °C for SF9 cells and 37 °C for Vero cells. Subsequently, 3 ml of complete medium with 10 µM monensin and 5 µl of anti-DT serum were added, and the cells were incubated for 2 h at 27 or 37 °C for SF9 and Vero cells, respectively. Then, the ability of the cells to incorporate [H]leucine was measured. The results are expressed as percent of the control values (no toxin added).




Figure 7: Comparison of sensitivity of vAc-DTR-infected SF9 cells () and Vero cells () to DT translocated from the plasma membrane at low pH (A) and from endosomes (B). In A, increasing amounts of nicked DT were added to vAc-DTR-infected SF9 and Vero cells for 30 min at 4 °C in the presence of 10 µM monensin. The medium with unbound toxin was removed, and the cells were incubated for 5 min at 27 °C (SF9) or 37 °C (Vero) with medium adjusted to pH 4.8. Then, 3 ml of complete medium with 10 µM monensin were added, and the cells were incubated for 2 h at 27 or 37 °C for SF9 and Vero cells, respectively. In B, the cells were incubated with DT overnight at 27 or 37 °C for SF9 and Vero cells, respectively. Then, the ability to incorporate [H]leucine was measured. The results are expressed as percent of the control values (no toxin added).



The sensitivity to DT of the infected insect cells was 200 times higher than that of Vero cells (Fig. 7), both when the toxin was translocated directly from the plasma membrane and when it was endocytosed by the normal route. This is in accordance with the 250-fold higher number of DTR on the insect cells with somewhat lower affinity as compared to Vero cells.

In the presence of heparin, more I-labeled DT was bound to the vAc-DTR-infected cells than in the absence of heparin, and we tested if this was accompanied by an increase in the sensitivity to DT. This was indeed the case. In contrast, in Vero cells, where toxin binding was not much increased by heparin, the sensitivity was the same in absence and presence of heparin (data not shown).

Pronase Protection of the Translocated Toxin

Translocation of DT from the plasma membrane can be assayed directly by treating the cells with Pronase E after the low pH pulse to remove surface-bound toxin. Successful translocation yields two protected polypeptides of 21 and 25 kDa. The 21-kDa band represents the toxin A fragment translocated to the cytosol, whereas 25-kDa polypeptide is part of the B fragment inserted into the membrane(11) . In Vero cells, translocation of nicked DT from the plasma membrane occurs only at pH <5.3(9) .

To test to what extent the translocation process in the vAc-DTR-infected insect cells resembles that in Vero cells, we analyzed the amount of protected 21- and 25-kDa polypeptides obtained at different pH values. As shown in Fig. 8, also in insect cells the translocation was a function of the pH during the exposure to low pH medium. The toxin entry was maximal at pH 4-4.8, as in Vero cells, but whereas pH <5.3 is required for translocation in Vero cells(27) , some protected material was observed even at pH 6.5 in the insect cells (Fig. 8, lanes3 and 4). It should be noted that some whole toxin bound to the cells was protected against Pronase, the amount of which increased with pH. This effect, which is not seen in Vero cells, could be due to reduced accessibility of the toxin on the insect cells, possibly by efficient pinching off of DTR-containing coated pits, a process that could be inhibited by low pH(28) .


Figure 8: Effect of pH on Pronase protection of cell-bound DT in vAc-DTR-infected SF9 cells. vAc-DTR-infected cells at 48 hpi were incubated with 100 ng I-labeled nicked DT for 30 min at 4 °C in presence of 10 µM monensin. Unbound toxin was removed by washing with PBS, and the cells were exposed for 5 min to pH 4 (lane1), pH 4.8 (lane2), pH 5.5 (lane3), and pH 6.5 (lane4) at 27 °C to allow translocation of the bound toxin to take place. The cells were then incubated for 10 min with 6 mg/ml Pronase E in the presence of 10 µM monensin. The medium with enzyme was removed, and the cells were washed with medium and lysed in lysis buffer. Proteins in the lysate were precipitated with 5% trichloroacetic acid and analyzed by SDS-PAGE under non-reducing conditions.




DISCUSSION

To test if DTR is able to promote toxin translocation in non-vertebrate cells, the cDNA encoding the monkey HB-EGF precursor was expressed in insect cells using the baculovirus system. Binding and immunofluorescence experiments indicate that the insect cells were able to synthesize and process the DTR so that it was inserted in the correct transmembrane orientation and recognized by diphtheria toxin. Also, the expression of the receptor conferred very high sensitivity to diphtheria toxin of otherwise resistant SF9 cells.

The time course experiments showed that 48 hpi is the best time to carry out functional studies of the expressed DTR, as at this time a high number of receptors is obtained and virus-induced cell lysis has not yet started. This time course is typical for proteins expressed under the control of the polyhedrin promoter(29) , and the time period between 48-72 hpi has been used for the study of other receptors expressed in the baculovirus system, such as the platelet-derived growth factor receptor(30) , the -adrenergic receptor(31, 32, 33) , and the human follicle-stimulating hormone receptor(34) .

Specific binding of I-labeled DT can be demonstrated readily in highly sensitive cells such as Vero, but not in cells with intermediate DT sensitivity such as HeLa cells, or in resistant mouse cells(15) . Naglich et al. (18) and Brown et al. (19) showed specific and saturable binding in mouse LM cells transfected with DTR from Vero cells. In these cells, the number of DTR was about 50,000 receptors/cell, which is twice that in Vero cells, but the affinity was lower in the transfected cells (Kof 5.8-15 10M) than in Vero cells (5.9-12 10M). Iwamoto et al. (35) found that the K of DT to recombinant HB-EGF immobilized on heparin-Sepharose was 7.7 10M. The same authors found 8.1 10 receptors/cell with a K of 10M in mouse L cells transfected with a DTR cDNA. In our expression system, we have obtained the highest density of DTR per cell so far reported, viz. about 5 10 receptors per cell with an affinity (K 3.7 10M) similar to that reported by Brown et al. (19) in transfected mouse cells. The lower affinity of the expressed receptor as compared to Vero cells has been attributed to the absence of proteins associated with the receptor in the transfected cells(18) . In the insect cells, it could also be due to different post-translational processing of the receptor as compared to Vero cells.

Naglich et al. (18) and Brown et al. (19) have shown that LM mouse cells transfected with Vero DTR expressed a higher number of DTR than Vero cells, but the transfected cells were not more sensitive to DT than Vero cells. The lack of increased sensitivity was attributed to the lower affinity of the DTR in the transfected LM cells. Also, it could be due to other rate-limiting steps in the intoxication process such as the presence of DRAP27(36) , a 27-kDa membrane protein corresponding to CD9(37) . In fact, Vero cells express 7.5 10 copies/cell of DRAP27, while LM cells display only 1,900 molecules/cell(36) . Transfection of DRAP27/CD9 was found to increase the DT binding and sensitivity in a human-mouse hybrid cell line that expresses DTR(37) . Also, Brown et al. (19) and Iwamoto et al. (35) have demonstrated an increase in the number of DT binding sites and DT sensitivity in mouse cells expressing DTR and CD9. In both cases an increase in the receptor number, but not in the receptor affinity, was observed. In our system, the vAc-DTR-infected cells express about 250 times more receptors than Vero cells, and the cells were about 200 times more sensitive to DT than Vero cells despite the observation that the affinity of the expressed receptor was lower than in Vero cells. The very high concentration of receptors under our test conditions (2 10 cells with 5 10 receptors each) reduces the importance of the high affinity of the receptors. It can not be excluded that proteins present in insect cells can increase the sensitivity of the infected cells to the toxin. Conversely, inhibitory factors of the translocation mechanism could exist in Vero cells and be absent in insect cells.

The effect of heparin on binding and toxicity of DT to vAc-DTR-infected cells was unexpected, since heparin did not increase much the binding and toxin sensitivity in Vero cells. It is not clear if this is due to DTR-associated proteins that may differ between Vero cells and SF9 cells. Also, it could be due to differences in the amount and types of proteoglycans expressed at the surface. On Vero cells, most of the DTR are associated with surface heparans,()and this may not be the case in the insect cells.

Upon immunoprecipitation with antibodies against the tag at the carboxyl-terminal end of DTR and by precipitation with DT immobilized on Sepharose, a protein of about 25 kDa was obtained. The apparent molecular weight, which exceeds the molecular weight calculated from the polypeptide size, indicates that the expressed receptor is glycosylated by the insect cells. The receptor was recognized by the anti-B3 serum, but DT-Sepharose was much more efficient in precipitating the receptor. Recently, Iwamoto et al.(35) found a higher amount of DTR precipitated with antibodies against DT bound to the receptor than with antibodies against the receptor as such.

Two additional bands were also detected in lysate precipitated with DT-Sepharose. Additional bands have been observed both after precipitation with anti-HB-HGF antibodies and with anti-DT(35) . These different bands could represent different processing in the amino terminus of the receptor, as described by Higashiyama et al. (26) in mature HB-EGF, and in insect cells infected with a recombinant virus encoding human recombinant transmembrane HB-EGF, as described by Ono et al.(23) .

The high efficiency expression of functional DTR with the baculovirus expression system reported here represents a convenient model to study the role of the DTR in toxin translocation to the cytosol. The possibility of simultaneously infecting with two recombinant viruses allows concomitant expression of other proteins that could be involved in the translocation process.


FOOTNOTES

*
This work was supported by The Norwegian Cancer Society, The Norwegian Research Council for Science and Humanities, The Novo Nordic Fund, The Family Blix' Legat, Otto and Rachel Bruun's Legat, and The Jahre Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral fellow of the European Community Program ``Human Capital and Mobility.''

Present address: Dept. of Internal Medicine, Division of Cardiology, University of Cincinnati, 231 Bethesda Ave., P. O. Box 670542, Cincinnati, OH 45267-0542.

**
To whom correspondence should be addressed: Biochemistry Dept., Institute for Cancer Research, Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. Tel.: 47-22-93-5640; Fax: 47-22-50-8692.

The abbreviations used are: DT, diphtheria toxin; nDT, nicked diphtheria toxin; DTR, diphtheria toxin receptor; HB-EGF, heparin-binding epidermal growth factor; AcNPV, Autographa californica nuclear polyhedrosis virus; vAc-DTR, recombinant baculovirus expressing DTR; SF9, uninfected Spodoptera frugiperda cells; HF, high five uninfected cells; PMSF, phenylmethylsulfonyl fluoride; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactoside; hpi, hours post-infection; PBS, phosphate-buffered saline; bp, base pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.

E. M. Valdizan, E. V. Loukianov, and S. Olsnes, unpublished data.

Lanzrein, M., Garred, Ø., Olsnes, S., and Sandvigk, K. (1995) Biochem. J., in press.


ACKNOWLEDGEMENTS

We are grateful to Unni Spaeren for help with the baculovirus expression system, to Ramon Ruiz for excellent technical assistance, and to Drs. Markus Lanzrein and Harald Stenmark for critical reading of the manuscript.


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