©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
IgA Protease from Neisseria gonorrhoeae Inhibits Exocytosis in Bovine Chromaffin Cells Like Tetanus Toxin (*)

(Received for publication, August 3, 1994)

Torsten Binscheck Frank Bartels Heidrun Bergel (§) Hans Bigalke (¶) Shinji Yamasaki Tetsuya Hayashi Heiner Niemann Johannes Pohlner

From the  (1)From the Institute of Toxicology, Medical School of Hannover, 30625 Hannover, Federal Republic of Germany (2)From the Department of Microbiology, Federal Research Center of Virus Diseases of Animals, 72001 Tübingen, Federal Republic of Germany (3)From the Max Planck Institute for Biology, 72076 Tübingen, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

When tetanus toxin from Clostridium tetani or IgA protease from Neisseria gonorrhoeae is translocated artificially into the cytosol of chromaffin cells, both enzymes inhibit calcium-induced exocytosis, which can be measured by changes in membrane capacitance. The block of exocytosis caused by both proteases cannot be reversed by enforced stimulation with increased calcium concentration. This effect differs from the botulinum A neurotoxin-induced block of exocytosis that can be overcome by elevation of the intracellular calcium concentration. Tetanus toxin is about 50-fold more potent than IgA protease in cells stimulated by carbachol. In this case, the release of [^3H]noradrenaline was determined. Trypsin and endoprotease Glu-C are hardly effective and only at concentrations that disturb the integrity of the cells. Like tetanus toxin, IgA protease also splits synaptobrevin II, though at a different site of the molecule. However, unlike tetanus toxin, it does not cleave cellubrevin. It is concluded that the membranes of chromaffin vesicles contain synaptobrevin II, which, as in neurons, appears to play a crucial part in exocytosis.


INTRODUCTION

Tetanus toxin (TeTx), (^1)IgA protease, and botulinum A neurotoxin (BoNt/A) are bacterial protein toxins with proteolytic activity. TeTx and BoNt/A are zinc-binding metalloproteases (Niemann et al., 1994). Their molecules consist of two chains, which are interconnected by a disulfide bond (DC-TeTx, DC-BoNt/A). The heavy chains mediate the binding to gangliosides and the translocation of the DC-toxins through neuronal membranes (Yavin, 1994). The light chains (LC), which represent the active enzymes, are released from the DC-toxins by reductive cleavage in the cell (Kistner and Habermann, 1992; Bigalke et al., 1993). Purified LC-toxins are untoxic, because they are unable to pass through the neuronal plasma membrane. The site of action of TeTx is inside the neurons where it cleaves synaptobrevin II, which is a fusion complex-forming protein associated with vesicles (Schiavo et al., 1992b). The site of cleavage is located between Gln and Phe (Schiavo et al., 1992a). The substrate for the action of BoNt/A is SNAP 25. This is also a fusion complex-forming protein, but it is located in the plasma membrane (Blasi et al., 1993). Vesicles or plasma membranes damaged by the toxins are unable to fuse with each other. Therefore, the release of various transmitters is inhibited. A second substrate for TeTx is cellubrevin, a ubiquitous protein highly homologous with synaptobrevin II (McMahon et al., 1993). Whereas cellubrevin has no function in the homotypic fusion of early endosomes (Link et al., 1993), it is essential in the recycling of transferrin receptors from endosomes to the plasma membrane, indicating that it might play a similar role to that of synaptobrevin II in constitutive exocytosis (Galli et al., 1994). Unlike the clostridial toxins, IgA protease, an exoenzyme from Neisseria gonorrhoeae, performs its action in the extracellular space where it hydrolyzes IgA molecules in their Fc regions. It recognizes specifically the motif PPXP, where X can represent alanine, threonine, or serine. It cleaves between the second and third proline residue (Simpson et al., 1988).

Although chromaffin cells possess a neuron-like exocytotic machinery, they are basically insensitive to clostridial DC-toxins. However, the toxins will inhibit carbachol- and calcium-induced release of noradrenaline if they gain access to the cytosol by binding to gangliosides previously incorporated into the plasma membrane (Marxen et al., 1989; Marxen and Bigalke, 1989). In addition to DC-toxins, the purified light chains also block exocytosis when they diffuse into the cytosol through artificial pores generated in the plasma membrane by electroporation (Bartels and Bigalke, 1992; Bartels et al., 1994). The substrates of the enzymes in chromaffin cells are unknown, but their functions have been located beyond the rise in cytosolic calcium concentration during stimulus-secretion coupling (Penner et al., 1986). Synaptobrevin II, but neither cellubrevin nor SNAP 25, carries a putative cleavage site for IgA protease (Pro-Pro-Ala-Pro). When we introduce this enzyme by electroporation into chromaffin cells, we observe an inhibition of exocytosis similar to that shown for TeTx.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium and fetal calf serum were obtained from Life Technologies, Inc., Eggenstein, FRG; cell culture plastic materials were from FALCON Division, Becton Dickinson (Heidelberg, FRG) and NUNC GmbH (Wiesbaden, FRG). Collagenase (0.71 units/ml), bovine serum albumin, HEPES, EGTA, collagen, antibiotics, and cytostatics were purchased from SERVA (Heidelberg, FRG). All buffers and solutions were prepared with analytical grade chemicals from MERCK (Darmstadt, FRG). Tetanus toxin was a gift from U. Weller (Mainz, FRG), and BoNt/A was donated by J. Frevert (Flörsheim, FRG). N. gonorrhoeae IgA protease from Escherichia coli was obtained from Boehringer (Ingelheim, FRG). Levo-[7-^3H])noradrenaline (14.2 Ci/mmol) was from DuPont NEN (Dreieich, FRG). Trypsin (2.5%) was obtained from Boehringer (Mannheim, FRG).

Cell Preparation, Purification, and Cultivation

Bovine adrenal glands were obtained from the local abattoir, and chromaffin cells were prepared as described previously (Marxen et al., 1989; Livett, 1984). For release experiments, cells were seeded onto collagen-coated Multiwellplates (3 times 10^5 cells/cavity) in 100 µl of Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 6 mg of glucose/ml, 100 IU of penicillin/ml, 100 µg of streptomycin/ml, ciprofloxacin, cytosine arabinoside, fluorodeoxyuridine, and uridine (10M each). The cultures were maintained at 37 °C in a humidified atmosphere of 90% air and 10% CO(2).

For electrophysiological experiments the cell suspension was purified from contaminating cell debris, fibroblasts, and smooth muscle cells by isopycnic density gradient centrifugation using a mixture of 10 parts Percoll and 12 parts cell suspension (v/v). It was centrifuged for 25 min at approximately 20,000 times g at 4 °C in a Beckman 70 TI fixed angle rotor. Bands containing chromaffin cells were collected from the gradient, and after washing they were ready for culturing or electroporation. They were seeded onto 35-mm Primaria dishes at a density of 2 times 10^6 cells/dish.

Internalization of Proteins into Chromaffin Cells by Electroporation

Cells suspended in a sterile electroporation cuvette were exposed to an electric field (625 V/cm) by a Bio-Rad Gene Pulser. The built-in capacitor (960 µF) was discharged, and the electric field decayed with a first order kinetic (time constant 12.5 ms). The percentage of viable cells as estimated by neutral red staining was 50%. They were diluted with an appropriate amount of growth medium and seeded as described above. For details see elsewhere (Bartels and Bigalke, 1992). For internalization of DC-TeTx into intact chromaffin cells via gangliosides see Marxen and Bigalke(1989).

[^3H]Noradrenaline Release Experiments

Chromaffin cells were preloaded with 0.125 µCi/ml of L-[^3H]noradrenaline in growth medium (250 µl/well) for 3 h. Then the cells were washed three times at 10-min intervals in a buffer containing (in mM): 125.6 NaCl, 4.8 KCl, 2.2 CaCl(2), 1.2 MgSO(4), 1.2 KH(2)PO(4), 5.6 glucose, 25 HEPES, 1 sodium ascorbate, 0.2% bovine serum albumin, pH 7.3. Basal release was determined within the next 8-min period during incubation in the same buffer. Exocytosis was stimulated by carbachol (5 times 10M) during the following 8-min period, and the amount of supernatant radioactivity was determined. The basal release was subtracted from the stimulated release, and exocytosis was expressed as a percentage of total radioactivity, i.e. the sum of basal release, stimulated release, and radioactivity remaining in the cells (approximately 5000 dpm, determined after extraction from the cells with 0.2% (w/v) sodium dodecyl sulfate).

Electrophysiological Measurement of Exocytosis

High resolution measurement of the cell's membrane capacitance was performed in the whole cell configuration under voltage clamp conditions on the stage of an inverted phase contrast microscope (Zeiss, magnification times 400) at room temperature (Neher, 1988; Lindau and Neher, 1989). Cells were incubated in a bath solution consisting of (in mM) 140 NaCl, 2.8 KCl, 2.5 CaCl(2), 0.8 MgCl(2), 10 HEPES, and 5 glucose at pH 7.35 and 315 mosm. Exocytosis was stimulated by using patch pipettes filled with calcium-buffered solutions. One solution contained 1 µM ionic Ca, and (in mM) 140 KCl, 12 NaCl, 10 HEPES, 10 EGTA, 0.86 CaCl(2), 3.04 MgCl(2), 0.5 ATP at pH 7.2. The composition of the other solution was 100 µM Ca plus (in mM) 140 KCl, 12 NaCl, 10 HEPES, 10 EGTA, 1.14 CaCl(2), 2.6 MgCl(2), 0.5 ATP at pH 7.2. Patch pipettes were produced from filamented borosilicate capillaries (Hilgenberg, Malsfeld, FRG) in a three stage pulling process on a computer-controlled BB-CH PC pulling device (Mecanex, Geneva, Switzerland) and backfilled with pipette solutions. An EPC 7 patch clamp amplifier (List Electronics, Darmstadt, FRG) was used to clamp the membrane potential at -70 mV. A sinusoidal voltage of 800 Hz frequency and 10 mV amplitude was superimposed on this holding potential. The resulting sinusoidal pipette current was low-pass filtered at 3 kHz and fed to a phase-sensitive detector (ELGE, Electronic, Hardegsen, FRG). This device allowed the phase-dependent separation of the current components caused by the conductance (G(m)) and the capacitance (C(m)) of the cell membrane. It provided two output voltages, which were proportional to changes in G(m) and C(m), respectively. Immediately after the membrane patch rupture under the pipette tip, the initial membrane capacitance was compensated by the patch clamp amplifier. After adjusting the offset conductance at the phase-sensitive detector, the capacitance compensation was transiently misadjusted by -1 pF to determine the proper phase angle between conductive and capacitive current components at the phase-sensitive detector. Signals for G(m) and C(m) were low-pass filtered at 10 Hz and digitized using a digital oscilloscope equipped with a diskette-storing facility (Nicolet 310; Nicolet, Madison, WI). Only cells exhibiting access resistance to the cytoplasm below 5.0 M were used for the membrane capacitance measurement.

In Vitro Cleavage of Synaptobrevin II and Cellubrevin

Rat brain vesicles were prepared according to Hell et al. (1988). Vesicles were incubated for 60 min with LC-TeTx (100 nM) and IgA protease (1 µM), respectively. Samples were processed by SDS-polyacrylamide gel electrophoresis and immunoblotting using the ECL Western blotting detection system (Amersham Buchler, Braunschweig, Germany) (for details see Yamasaki et al., 1994). In vitro translation of synaptobrevins and cellubrevin was performed in reticulocyte lysate in the presence of [S]methionine according to Mayer et al.(1988). The recombinant proteins were incubated in the absence or presence of 1 µM IgA protease in 20 mM HEPES/NaOH, pH 7.0, containing 100 mM NaCl for 60 min at 37 °C. The material was separated by 15% SDS-polyacrylamide gel electrophoresis according to Laemmli(1970), and bands were visualized by autoradiography.


RESULTS

Intracellular TeTx and IgA Protease Block Membrane Capacitance Increase

Chromaffin cells were electroporated in the presence or absence of 6.6 nM LC-TeTx or 166 nM IgA protease. After an incubation period of 2 h, they were challenged with 1 µM Ca applied through a patch pipette in the whole cell configuration. The increase in membrane capacitance was measured simultaneously during the next 200 s. Exocytosis caused by the artificial rise of cytosolic calcium was significantly reduced by LC-TeTx and IgA protease ( Fig. 1and Fig. 2). An increase in the free calcium concentration of the pipette solution up to 100 µM did not reverse the block of exocytosis in cells treated either with LC-TeTx or IgA protease, even though the exocytosis block was merely partial. In contrast to this finding, the almost complete block caused by dithiothreitol-reduced BoNt/A (6.6 nM) could be lifted in its early stage if the calcium concentration was elevated. But the BoNt/A-induced block also developed into an irreversible block 24 h later (Fig. 3). The lower capacitance increase in control cells after 2 h as compared with 24 h is caused by the electroporation procedure that damaged cells reversibly (see also Bartels and Bigalke(1992)).


Figure 1: LC-TeTx-induced block of exocytosis resists enforced stimulation. a, chromaffin cells were electroporated in the absence (control) or presence of 33 nM LC-TeTx. After 2 h cells were perfused with pipette solution containing 1 or 100 µM Ca for 6 min, and the relative increase in membrane capacitance was determined (ordinate). Values are the means of five recordings ± S.D. b, of the four recordings from cells stimulated with 100 µM Ca, one representative trace is shown on the right. The capacitance of the unstimulated cell and the cell perfused for 6 min with 100 µM Ca is given at the beginning and the end of each trace.




Figure 2: IgA protease-induced block of exocytosis resists enforced stimulation. a, chromaffin cells were electroporated in the absence (control) or presence of 16.6 nM IgA protease. After 2 h they were perfused with pipette solution containing 1 or 100 µM Ca for 3 min, and the relative increase in membrane capacitance was measured. Values are the means of six recordings ± S.D. b, of the six recordings in each group one representative trace is shown on the right. The capacitance of the unstimulated cell and the cell perfused for 3 min with 1 µM or 100 µM Ca is given at the beginning and the end of each trace.




Figure 3: BoNt/A-induced block of exocytosis can transiently be reversed by enforced stimulation. a, chromaffin cells were electroporated in the absence (control) or presence of 6.6 nM BoNt/A (reduced with dithiothreitol prior to application). After 2 and 24 h, respectively, cells were perfused with pipette solution containing 1 or 100 µM Ca for 12 min, and the relative increase in membrane capacitance was determined. Values are the means of seven recordings ± S.D. b, of the seven recordings in each group one representative trace is shown on the right. The capacitance of the unstimulated cell and the cell perfused for 12 min with 1 µM or 100 µM Ca is given at the beginning and the end of each trace.



Concentration-dependent Effect and Time-dependent Decrease of Inhibition

Chromaffin cells were electroporated in the presence and absence of DC-TeTx, IgA protease, endoprotease Glu-C, and trypsin, respectively. 48 h later the cells were preloaded with [^3H]noradrenaline, and the release experiment was performed (Fig. 4). Both DC-TeTx and IgA protease suppressed the release of [^3H]noradrenaline in a dose-dependent manner. DC-TeTx was 50-fold more potent than IgA protease. Both proteases, however, had the same efficacy. In contrast, endoprotease Glu-C and trypsin were unable to mimic the effect of DC-TeTx, indicating that proteolytic activity is not sufficient to suppress exocytosis.


Figure 4: Concentration-dependent inhibition of exocytosis by various proteases. Chromaffin cells were electroporated in the presence of the indicated concentrations of DC-TeTx, IgA protease, trypsin, and endoprotease Glu-C. 2 days later cells were preloaded with [^3H] noradrenaline. Exocytosis was induced by stimulation with carbachol. The inhibition of exocytosis was calculated from [^3H]noradrenaline release by control cells and cells treated with the respective toxin. Each toxin concentration was tested in triplicate.



IgA protease caused a block of exocytosis that decreased spontaneously within 3 days, whereas TeTx maintained the block over several days (Fig. 5). However, when chromaffin cells were electroporated in the presence of specific anti-TeTx antibodies 2 days after TeTx incorporation, the restoration of exocytosis followed the same time course as observed with IgA protease-treated cells (Fig. 5).


Figure 5: Time-dependent restoration of exocytosis. Chromaffin cells were preloaded with gangliosides and then incubated with 66 nM TeTx (circle, bullet). 24 h later the cells were electroporated in the presence of 166 nM IgA protease (box), 50 units/ml anti-TeTx antibodies (bullet), or plain poration medium (circle) and further maintained in culture. The growth medium was changed twice a week. Exocytosis was determined after different periods of time (abscissa). Inhibition of exocytosis by DC-TeTx or IgA protease, expressed as a percentage of [^3H]noradrenaline release from toxin-untreated control cells, was approximately 70 and 60%, respectively. The inhibition, as measured 48 h after permeabilization, was normalized to 1.0, and the other values were expressed as a fraction of it.



Cleavage of Vesicular Proteins by LC-TeTx and IgA Protease

Synaptic vesicles from rat brain were incubated for 60 min in the presence and absence of LC-TeTx and IgA protease, respectively. Alternatively, cellubrevin, synaptobrevin I, and synaptobrevin II were translated from their respective mRNA in vitro in the presence of [S]methionine. Aliquots of recombinant proteins were incubated with or without IgA protease. Subsequently, vesicle-associated and recombinant proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Radioactive cellubrevin, synaptobrevin I, and synaptobrevin II were determined by film exposure (Fig. 6b). Vesicular proteins were detected by an immunoblot using mouse monoclonal antibodies directed against Rab3A, synaptotagmin, synaptophysin, and synaptobrevin II. Detection of antibody was performed by chemoluminiscence (Fig. 6a). Both LC-TeTx and IgA protease selectively cleaved synaptobrevin II from both sources. Additionally, LC-TeTx (Yamasaki et al., 1994) (but not IgA protease) split recombinant cellubrevin, which appeared like synaptobrevin as an 18-kDa band in the gels and blots representing recombinant proteins and nontreated synaptic vesicles, respectively. Synaptotagmin, synaptophysin, Rab3A, and synaptobrevin I were not hydrolyzed by TeTx and IgA protease (Fig. 6).


Figure 6: Cleavage of vesicular proteins by LC-TeTx and IgA protease. a, rat brain vesicles were prepared and incubated with 200 nM TeTx and 275 nM IgA protease as described under ``Experimental Procedures.'' Aliquots were separated on 15% SDS-gels followed by Western blotting using monoclonal antibodies as indicated from top to bottom on the left. b, [S]methionine-labeled recombinant cellubrevin, synaptobrevin I, and synaptobrevin II, respectively, were incubated in the absence or presence of 275 nM IgA protease, and aliquots of the reaction mixture were separated on 15% SDS-gels. Molecular weights given on the left correspond to a molecular weight marker (first line).




DISCUSSION

The light chain of tetanus toxin is an endoprotease with high substrate specificity for synaptobrevin II and cellubrevin. Both substrates have an identical amino acid sequence around the cleavage site (Link et al., 1992). Synaptobrevin II is a vesicle-associated protein that is part of the core of the fusion complex (Niemann et al., 1994), and its degradation by LC-TeTx specifically blocks neuronal transmission. BoNt/A inhibits release of transmitters by cleavage of SNAP 25, a protein that is loosely attached to the plasma membrane and that interacts with synaptotagmin and syntaxin (Niemann et al., 1994). The block caused by BoNt/A, even if almost complete, can be reduced by enforced stimulation with calcium at early stages, which was also demonstrated in short-lived synaptosomes (Ashton and Dolly, 1991). At later stages, however, it becomes firmly established (see also Marxen et al., 1991). It was suggested that an unbound pool of SNAP 25 exists in the cytoplasm that is not accessible to BoNt/A (Niemann et al., 1994). Membrane-bound SNAP 25 cleaved by the toxin could be replaced in a calcium-dependent manner by intact molecules from this pool before they were also cleaved by BoNt/A. Only when all molecules of the cytosolic pool are bound to the plasma membrane and cleaved by BoNt/A does the block of exocytosis resist enforced Ca stimulation. Since, however, SNAP 25 is continuously resynthesized, a complete block of exocytosis cannot be achieved by BoNt/A. Alternatively, the toxin might split an as yet unknown second protein at a different rate. Only when both SNAP 25 and the putative protein are cleaved is exocytosis irreversibly inhibited. Which one of the proteins is more crucial or is cleaved first is unclear. The cleavage of synaptobrevin II by LC-TeTx is assumed to be its essential action. However, there is no proof for an association of synaptobrevin II with the small vesicles of chromaffin cells. To investigate whether cleavage of cellubrevin contributes to the toxin action in chromaffin cells, experiments with IgA protease were made. This bacterial protease splits only synaptobrevin II and not cellubrevin, which lacks the motif PPXP (Fig. 7) (McMahon et al., 1993). IgA protease has the same efficacy as TeTx, although it seems to possess a lower potency. However, we have to keep in mind that IgA protease is inactivated inside the cell much faster than TeTx. Release experiments for the construction of dose-response curves were performed 48 h after the incorporation of toxins by electroporation. During this time a partial recovery might have occurred (see below). The substrate of IgA protease may play a part in the exocytosis that is independent of the intracellular calcium concentration, because, as in the case of TeTx poisoning, the block cannot be circumvented by elevated calcium concentrations. Moreover, the restoration of exocytosis in TeTx- and IgA protease-treated cells follows an identical time course, if TeTx is neutralized intracellularly. Control experiments designed to assess the restoration of exocytosis blocked by IgA protease and LC-TeTx indicated that IgA protease lost its activity severalfold faster than LC-TeTx. We assume that IgA protease is inactivated within the cell much faster than TeTx. After the inactivation of IgA protease and the neutralization of TeTx by antibody, the substrate, i.e. synaptobrevin II, is resynthesized, leading to the reconstitution of cellular function. Other vesicular proteins involved in the late steps of neuronal exocytosis (synaptophysin, synaptotagmin, and Rab3A) are not cleaved by IgA protease, and more unspecific proteases (trypsin, endoprotease Glu-C) cannot mimic its effects. The cleavage site of IgA protease is found in rat and bovine synaptobrevin II and is located in the N-terminal highly heterologous region of synaptobrevin II (Fig. 7). Thus, the first 20 amino acid residues probably play an essential role in the proper function of synaptobrevin II late in the course of exocytosis.


Figure 7: Sequences of putative toxin substrates. Complete sequences of rat synaptobrevin II (a) and rat cellubrevin (c) are shown. The first 27 amino acids of bovine synaptobrevin II (b) contain a cleavage site for IgA protease.



Like the clostridial light chains the IgA protease is nontoxic for nerve cells under physiological conditions, because the enzymes cannot pass through the plasma membrane. Bacteria of the genus Clostridium have overcome the barrier by conjugating a transport protein with the protease, thereby giving them access to the intracellular compartment where the substrates are located. With respect to this it should be of interest to elucidate whether the intracellular occurrence of Neisseria is of any pathogenetic significance because, once inside the cell, these bacteria could release their protease directly into the substrate-containing compartment. A conjugation with a transporter would be unnecessary.


FOOTNOTES

*
This research was supported by the Deutsche Forschungsgemeinschaft (Bi 274/4-4). 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.

§
This work is part of the Ph.D. thesis of this author.

To whom correspondence should be addressed: Medical School of Hannover, OE 5340, 30625 Hannover, Federal Republic of Germany. Tel.: 49-511-532-2815; Fax: 49-511-532-2879.

(^1)
The abbreviations used are: TeTx, tetanus toxin; BoNt/A, botulinum A neurotoxin; DC, di-chain; LC, light chain; F, farad; SNAP 25, synaptosomal associated protein of 25 kDa.


ACKNOWLEDGEMENTS

We thank Ulrike Fuhrmann for excellent assistance. We are grateful to U. Weller (Mainz, Germany) for DC-TeTx and LC-TeTx, J. Frevert (Flörsheim, Germany) for DC-BoNt/A, and R. Jahn (New Haven, CT) for the monoclonal antibodies.


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