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(Received for publication, August 3,
1994) From the
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
[ Tetanus toxin (TeTx), ( 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
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
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
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
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
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 [
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 (
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,
[
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
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.
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1770-1774
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]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.
)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).
-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.
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-H])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 
10
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
. 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 10
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).
[
Chromaffin cells were preloaded with 0.125
µCi/ml of L-[H]Noradrenaline Release
ExperimentsH]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, 1.2
MgSO
, 1.2 KHPO, 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
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 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, 0.8 MgCl, 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
, 3.04
MgCl, 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
, 2.6 MgCl, 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
) and the capacitance (C)
of the cell membrane. It provided two output voltages, which were
proportional to changes in G and C, 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 and C 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.
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)).
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.
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.
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
[H]noradrenaline, and the release experiment was
performed (Fig. 4). Both DC-TeTx and IgA protease suppressed the
release of [H]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.
H] noradrenaline. Exocytosis was
induced by stimulation with carbachol. The inhibition of exocytosis was
calculated from [H]noradrenaline release by
control cells and cells treated with the respective toxin. Each toxin
concentration was tested in triplicate.
, 
). 24 h later the cells were
electroporated in the presence of 166 nM IgA protease
(
), 50 units/ml anti-TeTx antibodies (), or plain
poration medium () 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
[H]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).
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).
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.
)
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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