(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
[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.
Tetanus toxin (TeTx), ()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.
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 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.
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.
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 [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.
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 (,
). 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.
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).
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.