©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Furin-induced Cleavage and Activation of Shiga Toxin (*)

Garred (1), Bo van Deurs (2), Kirsten Sandvig (1)(§)

From the (1) Institute for Cancer Research at the Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway and the (2) Structural Cell Biology Unit, Department of Anatomy, Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Shiga toxin has a single A subunit non-covalently associated with a pentamer of B subunits. The toxin has a trypsin-sensitive region near the COOH-terminal end of the A-chain, and upon cleavage, two disulfide bonded fragments, A and A, are generated. These fragments are also formed upon incubation with cells. The disulfide loop contains the sequence (Arg- X-X-Arg), which is a consensus motif for cleavage by the membrane-anchored protease furin. We found that a soluble form of furin cleaves intact A-chain producing A and A fragments, and furin also seems to be responsible for rapid cellular cleavage of Shiga toxin. LoVo cells, which normally do not produce functional furin, cleave intact A-chain very efficiently when transfected with furin (LoVo/fur), whereas a control cell (LoVo/neo) cleaves the toxin very slowly. To investigate the role of this cleavage for intoxication of cells, we studied the ability of unnicked and furin-nicked toxin to inhibit protein synthesis in LoVo/fur and LoVo/neo cells. LoVo/fur cells were intoxicated equally well with unnicked and nicked toxin, whereas in LoVo/neo cells nicked toxin was about 20 times more active than unnicked toxin. The results suggest that cleavage of Shiga toxin is important for intoxication of cells, and they indicate that furin can cleave and thereby activate Shiga toxin in cells.


INTRODUCTION

Shiga toxin produced by Shigelladysenteriae type 1, is composed of an enzymatically active A-subunit in non-covalent association with a pentamer of B-subunits responsible for binding to cell surface receptors (for review, see Ref. 1). The A-subunit is a specific N-glycosidase that cleaves off a single adenine residue from 28 S rRNA of the 60 S ribosomal subunit (2, 3) , resulting in inhibition of the protein synthesis. After binding to cell surface receptors, the toxin is endocytosed from clathrin-coated pits (4, 5) . The functional receptor is a Gal (1, 2, 3, 4) Gal-containing glycolipid, primarily globotriasyl ceramide (4, 6, 7, 8) .

Shiga toxin A-chain (ST-A)() contains 2 cysteines that are linked by a disulfide bond. The loop between the 2 cysteines contains the sequence Arg-Val-Ala-Arg, which is recognized and nicked by trypsin, separating the A-chain into A (27.5 kDa) and A (4.5 kDa) fragments (9) . It has previously been shown that Shiga toxin and Shiga-like toxin type IIv can be nicked by a cellular protease (10) ,() and we have in this study tried to identify this protease.

Both diphtheria toxin and anthrax toxin seem to be activated by furin (11, 12) , which is a Ca-dependent serine protease with a subtilisin-like catalytic domain (13, 14) . Furin is expressed in most cells and tissues (14) , and the enzyme seems to be localized primarily in the trans-Golgi network (TGN) (15, 16) but can also be found in endosomes and at the cell surface in small amounts (11, 12, 16) . Furin has been reported to cleave precursors of various secretory and membrane proteins with the consensus motif Arg- X-Arg/Lys-Arg in the secretory pathway (17, 18) , the cleavage site being at the COOH terminus of this sequence (14) . Substrates with the sequence Arg- X- X-Arg, which is the minimal sequence for recognition by furin, have also been reported to be cleaved by furin, although less efficiently (19) . Shiga toxin contains this motif in the disulfide loop between the A and A fragments. Furthermore, after endocytosis, the toxin can be transported to the TGN in a number of different cell types (4, 20, 21) . Thus, furin or a furin-like enzyme could recognize and cleave Shiga toxin after endocytosis of the toxin.

In the present article, we demonstrate that furin cleaves ST-A into A and A fragments both in vitro and in vivo.We used LoVo cells, a cell line that does not express functional furin (22) , and LoVo cells transfected with furin to provide evidence for the involvement of furin in cellular cleavage and activation of Shiga toxin.


EXPERIMENTAL PROCEDURES

Materials

Brefeldin A (BFA) was purchased from Epicentre Technologies (Madison, WI). Calpain inhibitor I was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Butyric acid, Hepes, diaminobenzidine, and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester were purchased from Sigma. NaI was purchased from Du Pont (Belgium), and [H]leucine was purchased from Amersham International (Amersham, United Kingdom). Shiga toxin was I-labeled according to Fraker and Speck (23) to a specific activity of 30,000-40,000 cpm/ng. A conjugate of Shiga toxin and horseradish peroxidase was prepared by the 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester method as previously described (24) . Shiga toxin was a gift from Dr. J. Kozlov (W. A. Engelhardt Institute of Molecular Biology, Moscow) and Dr. J. E. Brown (U. S. Army Medical Research Institute of Infectious Diseases, Frederick, MD). Furin was a gift from Dr. G. Thomas (Vollum Institute, Oregon Health Sciences University, Portland, OR).

Cell Culture

LoVo cells (obtained from ATCC) were grown in HAM's F-12 media supplemented with 10% fetal calf serum. LoVo cells transfected with mouse furin (LoVo/LF36) and with control vector (LoVo/neo) were a gift from Dr. E Mekada (Kurume University, Kurume, Fukuoka, Japan). LoVo/LF36 cells are called LoVo/fur cells in this paper. Establishment of these cell lines are described elsewhere (25) .

Assay of Shiga Toxin Cytotoxicity

Cells were transferred to 24-well plates at a density of 3 10 cells/well 2 or 3 days prior to the experiments. The cells were incubated with increasing amounts of toxin for the indicated time. Then, the cells were incubated 10 min in Hepes medium with 1 µCi/ml [H]leucine and no unlabeled leucine. The medium was removed, and the cells were washed two times in 5% trichloroacetic acid. Then, the cells were dissolved in 0.1 M KOH, and the radioactivity was measured.

Cleavage of Shiga Toxin by Cultured Cells

Cells were seeded out with a density of 6 10 cells/well in 12-well plates 3 days prior to the experiments. On the second day, 1 mM butyric acid was added to some of the wells, and the cells were incubated further for 48 h before the experiments. The cells were washed in Hepes medium and preincubated with BFA (2 µg/ml) or protease inhibitors for 20 min at 37 °C; then, I-labeled Shiga toxin was added, and the incubation was continued for the indicated period of time. Control cells (no inhibitors) were incubated in parallel. The cells were then washed three times with phosphate-buffered saline and lysed in 1% Triton (1% Triton X-100, 20 mM Hepes, 140 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, pH 7.4) on ice for 20 min. The cell lysates were transferred to Eppendorf tubes, nuclei were removed by centrifugation, and proteins were precipitated for 30 min on ice in the presence of 5% trichloroacetic acid. After centrifugation, the pellet was washed in ether, dissolved in sample buffer containing 2-mercaptoethanol, and then subjected to SDS-PAGE. After autoradiography, the bands corresponding to A and A fragments of Shiga toxin were quantified by densitometry (model 300 A, Molecular Dynamics).

In Vitro Cleavage of Shiga Toxin by Purified Furin

The cleavage was performed in a reaction volume of 25 µl containing 5 mM CaCl, 1 mM 2-mercaptoethanol, 100 mM buffer (sodium acetate, pH 5.0; MES, pH 5.5-7.5; Hepes, pH 8.0), 10 ng of I-Shiga toxin, and 6 ng of purified furin. In a parallel experiment at pH 6.0, 5 mM EDTA was added instead of CaCl. The reaction mixture was incubated for 3 h at 30 °C, and the reaction was stopped by adding SDS sample buffer with 2-mercaptoethanol. The samples were boiled and subjected to SDS-PAGE. For use in toxicity experiments, unlabeled Shiga toxin was cleaved by furin at a toxin concentration of 100 µg/ml at pH 6.0. The reaction mixture was the same as above, but without the addition of 1 mM 2-mercaptoethanol. After the incubation, the sample was frozen at -20 °C before use.

Acrylamide Gel Electrophoresis

Electrophoresis was carried out as described by Laemmli (26) . After electrophoresis, the gels were fixed for 30 min in 4% acetic acid and 27% methanol. For autoradiography, Kodak XAR films were exposed to dried gels at -80 °C.

NH -terminal Sequence Analysis

Shiga toxin cleaved by furin, as described above, was applied to a protein sequenator (Applied Biosystems 477A). As expected, the reaction mixture contained three peptides due to nicking of the A-chain and the presence of the B-chain.

Processing for Electron Microscopy

LoVo/fur cells grown as monolayers in T-24 flasks were incubated 2 h with Shiga toxin horseradish peroxidase conjugate. The cells were fixed and processed for electron microscopy, as earlier described (24) .


RESULTS

Furin Cleaves Intact Shiga Toxin A-chain to A and A Fragments in Vitro

Shiga toxin has a trypsin-sensitive region near the COOH terminus of the A-chain, containing the motif Arg-Val-Ala-Arg, the minimal recognition sequence for furin (19) . Upon cleavage, two disulfide-bonded fragments A (27.5 kDa) and A (4.5 kDa) are generated. The pH optimum for proteolytic activity by furin is variable depending on the substrate (27) , and we therefore measured the ability of furin to cleave Shiga toxin at different pH values. As shown in Fig. 1, a soluble form of furin cleaves intact Shiga toxin with a pH optimum of 5.5-6.0, generating A (27.5 kDa) and A (4.5 kDa) fragments (the A fragment is in the front of the gel). Furin requires calcium for enzymatic activity (13) , and in accordance with this, EDTA completely inhibited the cleavage at pH 6.0 (data not shown). To locate the cleavage site in the toxin, we performed amino-terminal sequencing of furin-nicked A-chain. The resulting sequence was Met-Ala- X-Asp-Glu-Phe-Pro, identical with residues 252-258. This indicates that furin cleaves Shiga toxin at the COOH-terminal side of Arg, as expected.


Figure 1: pH-dependent cleavage of Shiga toxin by furin. I-Labeled Shiga toxin was incubated with a soluble form of furin for 3 h at 30 °C at different pH values (see ``Experimental Procedures''). The reaction products were analyzed by SDS-PAGE (13.5%) and autoradiography. Lane1, pH 7.0 without furin; lane2, pH 5.0; lane3, pH 5.5; lane4, pH 6.0; lane5, pH 6.5; lane6, pH 7.0; lane7, pH 7.5; lane8, pH 8.0.



Proteolytic Cleavage of Shiga Toxin by Cells

To examine whether furin is involved in cleavage of Shiga toxin in vivo, we used a stable, transfected cell line of LoVo cells expressing furin (LoVo/fur) and a control cell transfected with the vector alone (LoVo/neo). Cells were incubated with I-labeled Shiga toxin and then subjected to SDS-PAGE and autoradiography; the A and A fragments were then quantified by densitometry. In LoVo/fur cells, ST-A was cleaved rapidly into A and A fragments. After 1 h of incubation, 40% of the cell-associated toxin was cleaved, while after 5 h, 80% was cleaved (Fig. 2, lanes8 and 9). In LoVo/neo cells, ST-A was cleaved much more slowly and not as extensively. After a 1-h incubation cleavage was not observed, while after 5 h of incubation, 30% of the cell-associated toxin molecules were cleaved (Fig. 2, lanes2 and 3). The data suggest that in vivo, furin is the cellular protease mainly responsible for cleavage of Shiga toxin. However, other cellular proteases can also cleave Shiga toxin in cells without furin, although much less efficiently.


Figure 2: Processing of Shiga toxin in LoVo/neo and LoVo/fur cells. The cells were preincubated with or without calpain inhibitor ( Calp. Inh.) (100 µg/ml) or brefeldin A (2 µg/ml) in Hepes medium for 20 min at 37 °C. Then I-toxin (100 ng/ml) was added, and the incubation was continued for 1 or 5 h more. After the indicated period of time, the cells were washed and lysed in buffer containing 20 mM Hepes, 150 mM NaCl, 1% Triton X-100, and 1 mM phenylmethanesulfonyl fluoride, pH 7.2. Nuclei were removed by centrifugation, and proteins in the supernatant were precipitated with 5% (w/v) trichloroacetic acid, washed in ether, and dissolved in sample buffer containing 6% (v/v) 2-mercaptoethanol and analyzed by SDS-PAGE (13.5%) and autoradiography. Lanes2-7, LoVo/neo cells; lanes8-13, LoVo/fur cells. In lane1, I-labeled Shiga toxin that had not been incubated with cells.



The observed processing could occur at the cell surface, in a membrane-bounded organelle, or in the cytosol. To study whether cleavage could occur at the cell surface, the cells were depleted for ATP by incubation with NaN and 2-deoxyglucose. Endocytosis is then inhibited, but the toxin molecules can still bind to cell-surface receptors (data not shown). In the presence of ATP-depleting reagents, there was essentially no cleavage of cell-surface bound Shiga toxin in LoVo/neo or LoVo/fur cells (data not shown), indicating that Shiga toxin is cleaved after endocytosis. To further localize the site of cleavage, we used the drug BFA, which disrupts the Golgi stacks in a number of different cell lines (28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and which also protects these cells against Shiga toxin (34) . In BFA-treated cells, toxin molecules can still be endocytosed and exposed to proteases in endosomes and TGN, whereas retrograde transport through the Golgi cisterns and translocation to the cytosol cannot occur. BFA protected also LoVo/neo and LoVo/fur cells against Shiga toxin (data not shown), and importantly BFA treatment strongly inhibited the processing of Shiga toxin in LoVo/neo cells (Fig. 2, lanes4 and 5), whereas there was no effect on the processing in LoVo/fur cells (Fig. 2, lanes10 and 11). Thus, processing by furin seems to occur in endosomes and/or TGN, while furin-independent cleavage seems to occur in another location in the cells.

We have shown earlier that a cell-permeable inhibitor of the cytosolic enzyme calpain protected Vero cells against a trypsin-resistant mutant (R248H/R251H) of Shiga toxin and inhibited the cell-mediated cleavage of this mutant toxin, while the inhibitor had no effect on the intoxication and processing of wild type toxin. We therefore decided to investigate whether calpain inhibitor would interfere with cleavage of wild type Shiga toxin in the LoVo cells. The inhibitor in fact strongly reduced the processing of Shiga toxin in LoVo/neo cells (Fig. 2, lanes6 and 7), whereas in LoVo/fur cells the processing was not inhibited (Fig. 2, lanes12 and 13). The calpain inhibitor has also been reported to inhibit cathepsin B and L (38) , which are also inhibited by leupeptin (39) . Indeed, leupeptin partly inhibited the processing of Shiga toxin in LoVo/neo cells (data not shown), suggesting that cathepsins are responsible for some of the nicking activity seen in the absence of furin.

Furin-induced Nicking of Shiga Toxin Is Important for Intoxication of Cells

To test whether cleavage of ST-A with furin is important for intoxication of cells, we measured the ability of intact and furin-nicked Shiga toxin to intoxicate Vero cells and LoVo cells. Vero cells are highly sensitive to Shiga toxin and cleave intact toxin very efficiently (10) , whereas LoVo cells lack functional furin (22) . In Vero cells (Fig. 3 A), there was no detectable difference between unnicked and nicked toxin, while in LoVo cells (Fig. 3 B), nicked toxin was 30-50 times more active than unnicked toxin, suggesting that cleavage is important for intoxication of cells. We also studied the ability of unnicked and furin-nicked Shiga toxin to inhibit protein synthesis in LoVo/neo and LoVo/fur cells. In LoVo/neo cells, nicked toxin was 20-30 times more active than unnicked toxin (Fig. 4 A), in accordance with the results in Fig. 3 B. In LoVo/fur cells, where the toxin is efficiently cleaved by furin, there was no difference between unnicked and furin-nicked toxin (Fig. 4 B). Unfortunately, LoVo/fur cells were less sensitive to Shiga toxin than LoVo/neo cells. Such clonal difference in sensitivity to Shiga toxin has been observed previously in other cell populations.() Since butyric acid is known to sensitize A431 cells to Shiga toxin and induce increased transport of Shiga toxin to the TGN and the endoplasmic reticulum (20, 40) , from where translocation to the cytosol may take place (20, 40, 41) , we tested if butyric acid could sensitize LoVo/neo and LoVo/fur cells. As shown in Fig. 5, that was indeed the case, and both cell types became equally sensitive to nicked Shiga toxin. In butyric acid-treated LoVo/neo cells, furin-nicked toxin was still about 20 times more active than unnicked toxin (Fig. 5 A), whereas in the sensitized LoVo/fur cells, there was (as seen in the untreated cells) no difference between unnicked and nicked toxin (Fig. 5 B). Electron microscopical investigations confirmed that butyric acid induced Shiga toxin transport to the Golgi apparatus in LoVo/fur cells (data not shown). It should be mentioned that during culture, the LoVo/fur cells occassionally became quite sensitive to Shiga toxin even without treatment, and also then nicked and unnicked toxin were equally toxic to the cells (data not shown). In parallel experiments, we also showed that Shiga toxin was cleaved to about the same extent in butyric acid-treated cells as in untreated cells and that protease inhibitors inhibited cleavage in a similar way as in cells without butyric acid treatment.


Figure 3: Effect of unnicked and furin-nicked Shiga toxin on Vero cells and LoVo cells. The cells were incubated in the presence of increasing concentrations of Shiga toxin. The protein synthesis was measured after 2 h in Vero cells and after 3 h in LoVo cells, as described under ``Experimental Procedures.'' A, Vero cells; , unnicked toxin; , nicked toxin. B, LoVo cells; , unnicked toxin; , nicked toxin. In A, bars indicate deviations between two parallels.




Figure 4: Effect of unnicked and furin-nicked Shiga toxin on LoVo/neo and LoVo/fur cells. The cells were incubated in the presence of increasing concentrations of Shiga toxin. After 3 h incubation at 37 °C, the protein synthesis was measured as described under ``Experimental Procedures.'' A, LoVo/neo cells; , unnicked toxin; , nicked toxin; B, LoVo/fur cells; , unnicked toxin; , nicked toxin.




Figure 5: Effect of unnicked and furin-nicked Shiga toxin on butyric acid-sensitized LoVo/neo and LoVo/fur cells. Cells were incubated in the presence of 1 mM butyric acid for 48 h. Then, the cells were incubated with or without calpain inhibitor (50 µg/ml) in Hepes medium for 20 min at 37 °C. Increasing concentrations of Shiga toxin was added at 37 °C, and the protein synthesis was measured after 3 h as described under ``Experimental Procedures.'' A, LoVo/neo cells; , unnicked toxin; , unnicked toxin and calpain inhibitor; , nicked toxin; , nicked toxin and calpain inhibitor. B, LoVo/fur cells; , unnicked toxin; , unnicked toxin and calpain inhibitor; , nicked toxin; , nicked toxin and calpain inhibitor.



As shown in Fig. 5 A, the calpain inhibitor described above protected the butyric acid-treated LoVo/neo cells only against unnicked toxin, and it had no effect on the intoxication of LoVo/fur cells. On the other hand, the protease inhibitor leupeptin, which similarly to the calpain inhibitor reduced nicking somewhat in LoVo/neo cells, had no effect on the toxicity (data not shown).


DISCUSSION

The main finding in this paper is that the enzyme furin is responsible for cellular cleavage of Shiga toxin and that cleavage is essential for efficient intoxication of cells. Furin has been reported to nick proteins with the sequence Arg- X- X-Arg (19) . Indeed, Shiga toxin A-chain contains this motif (Arg-Val-Ala-Arg) in the COOH-terminal region in the loop bridged by a disulfide bond. Here, we provide evidence that purified furin cleaves Shiga toxin A-chain between Arg and Metin vitro, generating A (27.5 kDa) and A (4.5 kDa) fragments, and we show that the pH optimum for this reaction is slightly acidic, pH 5.5-6.0. In LoVo cells that do not express functional furin (22) , Shiga toxin was cleaved very slowly and to a small extent into A and A fragments, and furin-nicked toxin was 20-30 times more toxic than unnicked toxin, whereas in furin-transfected LoVo cells (LoVo/fur), Shiga toxin was efficiently cleaved, and there was no difference in toxicity between unnicked and nicked toxin. These results suggest that furin is responsible for cleavage and activation of Shiga toxin in vivo but also implicate that other cellular proteases can cleave Shiga toxin in cells without furin activity, although less efficiently.

LoVo/fur cells were less sensitive to nicked Shiga toxin than LoVo/neo cells. In the case of A431 cells, we have previously shown that these cells can be sensitized to Shiga toxin by butyric acid treatment, and following this treatment more Shiga toxin is transported to the Golgi apparatus, and the toxin can be visualized not only in the TGN but also in the endoplasmic reticulum (20, 40) , where translocation to the cytosol may take place (20, 40, 41) . Similarly, butyric acid treatment sensitized both LoVo/neo cells and LoVo/fur cells to Shiga toxin, and the toxicity of nicked toxin was then similar in LoVo/neo and LoVo/fur cells.

Furin is expressed in a wide variety of cells (14) , whereas other members of the family of subtilisin-like proteases, including PC 1, PC 2, and PC 4, are expressed in specialized cells, like neuroendocrine cells, and recognize dibasic sequences (14) . Shiga toxin does not have any such sequence in the region where cleavage occurs. Therefore, furin or a furin-like enzyme may cleave and activate Shiga toxin in many different cell types.

Pseudomonas toxin, diphtheria toxin, and anthrax toxin clearly require cleavage for successful translocation to the cytosol and for intoxication of cells, and furin seems to be the enzyme responsible for cleavage (11, 12, 27, 42) . In contrast, it has been questioned whether cleavage of Shiga toxin is important for intoxication of cells. Olsnes et al.(9) showed that Shiga toxin nicked between A and A with trypsin had increased enzymatic activity when inhibition of protein synthesis was measured in a cell-free system, whereas there was no difference in cytotoxic effect on HeLa S cells. Kongmuang et al.(43) reported similar findings with Shiga-like toxin on Vero cells and suggested that cleavage did not affect the biological properties of the toxin. Similarly, we found no difference between furin-nicked and unnicked Shiga toxin on Vero cells. This lack of difference can be explained by the ability of these cells to cleave Shiga toxin. It was recently shown that Shiga toxin and Shiga-like toxin type IIv are easily nicked in Vero cells (10) . Furthermore, we have produced mutants of Shiga toxin where the furin-sensitive site has been eliminated. These mutants were less efficient than wild type Shiga toxin in provoking rapid intoxication in Vero cells, and the mutants where cleaved inefficiently compared to wild type Shiga toxin. Similar results were obtained by Burgess and Roberts (44) . All of these results indicate that cleavage of Shiga toxin is important for intoxication.

Furin is reported to be localized primarily in the TGN (15, 16) but may also be present in small amounts in endosomes and at the cell surface (11, 12, 16) . In the presence of ATP-depleting agents, which inhibited the internalization of Shiga toxin, there was essentially no cleavage at the cell surface of LoVo/fur cells, whereas in the presence of BFA, which disrupts the Golgi cisterns, processing occurred at a normal rate, indicating that nicking of Shiga toxin may take place in TGN and/or in endosomes. Diphtheria toxin has recently been reported to be nicked at the cell surface by furin in transfected LoVo cells. This difference from our results can be explained by the different pH optima for furin cleavage of diphtheria toxin and Shiga toxin in vitro. Diphtheria toxin is in contrast to Shiga toxin cleaved efficiently at neutral pH (12, 27) . In LoVo/neo cells, cleavage of Shiga toxin was much less extensive than in LoVo/fur cells. Also in these cells, we could not detect any cleavage at the cell surface, and BFA in this case partly inhibited the cleavage. To further characterize the inefficient cleavage taking place in LoVo/neo cells, we tested the effect of a cell-permeable inhibitor of the cytosolic enzyme calpain and the effect of leupeptin, a protease inhibitor taken up by endocytosis and reported to inhibit the lysosomal proteases cathepsin B and L (39) . Leupeptin inhibited the cleavage extensively but did not protect against Shiga toxin, suggesting that some cleavage occurs in a compartment from where the toxin cannot translocate to the cytosol, most likely in the lysosomes. In contrast, the calpain inhibitor inhibited both cleavage and toxicity, indicating that a fraction of the toxin molecules can be cleaved and activated by another cellular protease than furin, possibly by the cytosolic enzyme calpain. However, from the experiments shown, it seems clear that furin, in contrast to other enzymes in LoVo cells, efficiently cleaves and activates Shiga toxin.


FOOTNOTES

*
This work was supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, Torsteds legacy, Rachel and Otto Kr. Bruuns legacy, Blix legacy, the Jahre Foundation, the Danish Cancer Society, the Danish Medical Research Council, the Novo Nordisk Foundation, and NATO Collaborative Research Grant CRG 90057. 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.

§
To whom correspondence should be addressed. Tel.: 47-22934294; Fax: 47-22508692.

The abbreviations used are: ST-A, Shiga toxin A-chain; BFA, brefeldin A; TGN, trans-Golgi network; MES, 2-( N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

Garred, Ø., Dubinina, E., Holm, P. K., Olsnes, S., van Deurs, B., Kozlov, J. V., and Sandvig, K. (1995) Exp. Cell Res., in press.

Ø. Garred and K. Sandvig, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Anne-Grethe Myrann, Tove Lie Berle, Marianne Lund, Anne Mette Ohlsen, Keld Ottesen, and Kirsten Pedersen for excellent technical assistance. We thank Dr. Eisuke Mekada for providing LoVo/neo and LoVo/fur cells, Dr. Gary Thomas for providing purified furin, and Dr. Knut Sletten (Oslo University) for performing protein sequencing. We are also grateful to Dr. Markus Lanzrein and Dr. Sjur Olsnes for critical reading of the manuscript.


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