(Received for publication, December 19, 1995; and in revised form, February 16, 1996)
From the
Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover, VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.
Trypanosoma brucei, the parasitic protozoan causative
of Nagana in the African cattle, is transmitted between mammals by the
tsetse fly. Its life cycle includes several distinct nonreplicative
(infective) and proliferative (noninfective) stages in both the
mammalian host and the insect vector. In addition to biochemical and
morphological modifications, the differentiation from the mammalian
bloodstream form to the insect procyclic form is accompanied by
important changes in protein composition of the cell surface, probably
as a protective adaptation to different host defenses. In particular,
the major surface antigen of the bloodstream form, the VSG, ()is replaced in procyclic forms by another predominant
surface glycoprotein, termed procyclin(1) . So far the
mechanisms involved in the induction of these transformations are
unclear. On the basis of observations made in other eukaryotes, it is
probable that activation of cell surface receptors by appropriate
ligands may cause the generation of second messengers which trigger
changes in the programming of gene expression. Typical in this respect
is the generation of cAMP produced as a result of the stimulation of
adenylate cyclase(2, 3, 4) .
In a variety
of trypanosomal species (5, 6, 7, 8) and particularly in T. brucei(9, 10) , changes in cAMP levels
seem to be associated with events triggering cell proliferation and
differentiation. In T. brucei, adenylate cyclase is located in
the plasma membrane(11, 12) . This activity is encoded
by four gene families, two of which contain at least 10
members(13, 14) . ()Genes from one of these
families, termed ESAG 4 (for expression site-associated gene 4) belong
to the transcription units of the VSG genes and are thus only expressed
in the bloodstream form, whereas the genes from the three other
families termed GRESAG 4.1, 4.2, and 4.3 (for gene related to ESAG 4),
are not linked to the VSG genes and are expressed in both bloodstream
and procyclic forms. An adenylate cyclase activity stimulated by
calcium was found to be restricted to the bloodstream form and is
likely to be the product of the ESAG 4 genes(15, 16) .
The different adenylate cyclases appear to be transmembrane
glycoproteins with a divergent external domain at the N terminus and a
conserved catalytic domain located in front of a C-terminal
extension(16) .
In dividing procyclic and bloodstream forms
the activity of adenylate cyclase is down-regulated, since it can be
strongly stimulated upon rupturing of the cells(17) . However,
in both intact cells ()and isolated plasma
membranes(11) , the adenylate cyclase activity of trypanosomes
was found to be insensitive to agents known to activate
G-protein-responsive adenylate cyclases, such as GTP
S, Gpp(NH)p,
forskolin, cholera, and pertussis toxins. These results are in keeping
with the general structure of the kinetoplastid
cyclases(14, 18) , which is very similar to that of
G-protein-independent single transmembrane span
cyclases(19, 20, 21) . In view of the
observations reported for Dictyostelium(20) , it may
be suggested that the trypanosomal adenylate cyclases differ in their
response to specific ligands and are involved in distinct events of
transformation and/or proliferation in the parasite life cycle.
In
the bloodstream form, stress conditions that trigger the release of the
VSG (osmotic shock, Ca, local anesthetics) were also
found to activate adenylate cyclase, whereas zinc inhibited both
processes(22, 23, 24, 25) . In
addition, a transient activation of adenylate cyclase was found to
follow VSG release during the cold shock-induced differentiation from
bloodstream to procyclic forms(26) . These observations
suggested that VSG release and adenylate cyclase activity were
dependent on the same stimuli. In order to evaluate this hypothesis we
submitted trypanosomes to different experimental conditions known to
induce the release of the VSG, and we monitored simultaneously the
release of the VSG and the activity of adenylate cyclase. In wild type
cells, activation of adenylate cyclase was found to correlate with VSG
lipase-dependent VSG release independently of the experimental
conditions used to trigger this release (low pH or trypsin). Moreover,
specific inhibitors of protein kinase C (PKC) were found to stimulate
both processes. Significantly, in a mutant lacking the VSG lipase,
stimulation of adenylate cyclase occurred under conditions where the
VSG was not released, indicating that adenylate cyclase activation is
not simply the result of the disruption of the VSG coat. Moreover, the
differential sensitivity of VSG lipase and adenylate cyclase to pCMPS
allowed the design of experiments where VSG release still occurred
under conditions where adenylate cyclase was totally inhibited. We
conclude that stimulation of VSG lipase and adenylate cyclase occur
independently in response to different stress conditions and that PKC
activity may be involved in the control of these responses.
Figure 3:
Mild acid treatment of cells also
initiates concurrent activation of adenylate cyclase and release of the
VSG. Bloodstream form (AnTat 1.1) trypanosomes (3 10
cells/ml) were incubated in isosmotic phosphate buffer (pH 5.5, 4
°C) for 90 min. At various times, samples (2 ml, 6
10
cells) were removed from the incubation and centrifuged.
Following separation of the pellets and supernatants, the pellets were
resuspended in swell dialysis medium (0.1 ml, 4 °C). Three samples
(20 µl each) were analyzed for adenylate cyclase activity under
conditions of swell dialysis (5 min, 37 °C). The remaining portion
(40 µl) was incubated under the same conditions. The cells were
separated from the assay medium by centrifugation after adjustment of
the osmotic strength of the medium to 340 mOsm by the addition of KCl.
The pellets and supernatants from 10
cells were analyzed
for the presence of the VSG as well as for the presence of the CRD
found on the GPI anchor of the VSG. In addition, trypanosomes that had
been previously surface radioiodinated were subjected to a parallel
incubation that was conducted with the same protocol as that used with
unlabeled cells. In this case the
I-labeled VSG present
in the supernatants of cells that had been removed at various times and
centrifuged was processed and subjected to liquid scintillation
spectrophotometry as described under ``Materials and
Methods.'' A shows the time course of activation of
adenylate cyclase (
) and the release of the
I-labeled VSG (┌). Each value represents the mean
± S.E. of three separate determinations. The measurements of VSG
release are expressed as percentage of the total releasable VSG, i.e. the amount released by hypotonic lysis of an equivalent
number of cells.
, cAMP synthesis in control cells incubated at
neutral pH. B shows that the labeled material released in the
medium essentially contains VSG, as determined by Coomassie Blue
staining (lanes 1-3) and autoradiography (lanes
4-6) of equivalent samples of the supernatants after
hypotonic lysis (lanes 1 and 4) (pH 5.5) treatment
for 20 min (lanes 2 and 5) and in control cells (lanes 3 and 6). The two lower bands present in lanes
1-3 are due to components present in the swell dialysis assay
medium (see legend to Fig. 6). C shows the time course
of release of the VSG and the cross-reacting determinant as assessed by
antibody probing of Western blots of the supernatants that had been
subjected to SDS-PAGE, following withdrawal from the incubation at the
times indicated at the top of each lane. Lanes labeled C0 and C90 contained the pellets and supernatants of cells incubated
at pH 7.5 for 0 and 90 min, respectively. In the upper portion of panel (C) the probe was conducted with polyclonal
antibodies raised against the VSG, and in the lower portion of
panel (C) the probe was conducted with the polyclonal anti-CRD
antibodies.
Figure 6:
VSG release is not required for activation
of adenylate cyclase in bloodstream form trypanosomes. Bloodstream form
trypanosomes of either the wild type (AnTat 1.1) or GPI-PLC null mutant
were incubated at 4 °C for 60 min either in isosmotic TES buffer
(pH 7.5) or in isosmotic phosphate buffer (pH 5.5). At the end of the
incubation a sample (2 ml, 6 10
cells) was removed
and centrifuged. The cells were resuspended in swell dialysis medium
(0.1 ml). Three samples (20 µl each) of the resuspended cells were
analyzed for adenylate cyclase activity under conditions of swell
dialysis (37 °C, 5 min). The remaining portion (40 µl) of the
resuspended cells were incubated under the same conditions, then
centrifuged after adjustment of the osmotic strength to 340 mOsm with
KCl. The supernatants of 10
cells were analyzed by
SDS-PAGE. A shows the relative activity of wild type and
mutant cells. B shows the Coomassie Blue staining of proteins
present in the supernatants of 10
cells incubated at either
pH 7.5 (lanes c) or pH 5.5 (lanes a). The 40- and
35-kDa proteins present in all lanes represent components of the
incubation assay medium and disappeared if creatine kinase was omitted
(data not shown). VSG is indicated by the arrow.
Figure 1:
Exogenous trypsin initiates a
temporally distinct activation of the adenylate cyclase and release of
the N-terminal domain of the VSG in the AnTat 1.1 bloodstream form
variant, but fails to activate adenylate cyclase in the
trypsin-resistant AnTat 1.8 variant. Trypanosomes (3 10
cells/ml) were incubated in an isosmotic buffer (pH 7.5) for 30
min (20 °C) in the presence of cycloheximide (10 µg/ml).
Trypsin (50 µg/ml final concentration) was then added (arrow labeled T) and the incubation continued for a further 2
h. At various times duplicate samples (0.5 and 1.0 ml) were withdrawn
and mixed with soybean trypsin inhibitor (250 µg/ml final
concentration). The cells were then centrifuged and the pellet and
supernatant of the first sample analyzed for their content of VSG,
while the cells in the pellet of the second sample were analyzed for
their adenylate cyclase activity by measuring cAMP production during a
short (5 min) assay period (37 °C) as described under
``Materials and Methods.'' (Zero time: both trypsin and
trypsin inhibitor were added simultaneously.) A shows the time
course of activation of adenylate cyclase in the AnTat 1.1 (
) and
AnTat 1.8 (
) clones. Each value represents the mean ±
S.E. of three separate determinations. Where no error bar is shown, the
S.E. was less than the representation of the point. B shows
the time course of VSG release assessed by Western blot analysis of
pellets (lanes labeled P) and supernatants (lanes
labeled S) from 10
cells taken at various times
during the incubation. One sample (C30) represents a parallel control
incubation of 30 min without trypsin. Each blot was probed with
polyclonal antibodies raised against either AnTat 1.1 (upper
portion of B) or AnTat 1.8 (lower portion of B). The full-sized VSGs are denoted by arrowheads.
The 43-kDa AnTat 1.1-specific band represents the N-terminal domain of
the VSG, liberated by trypsin cleavage in the hinge region of the
protein.
We examined whether trypsin was required throughout the time course of cyclase activation. AnTat 1.1 trypanosomes were treated with trypsin for 15 min then soybean trypsin inhibitor was added, the cells were centrifuged and resuspended in a buffer supplemented with trypsin inhibitor before incubation at 4 or 20 °C for different periods. Under these conditions adenylate cyclase activation occurred as in the case of a continuous presence of trypsin, in a process dependent on temperature and requiring a lag phase of at least 15 min (Fig. 2A). In accordance with the data of Fig. 1, the 15-min preincubation with trypsin led to a virtually complete removal of the N-terminal domain of the VSG, although some VSG remained refractory to cleavage (data not shown). As shown in Fig. 2B, two distinct components were found to be released after the preincubation period, despite the inactivation of trypsin. In addition to the VSG still uncleaved by trypsin, anti-CRD antibodies, which only allow the detection of the C-terminal domain of the VSG after cleavage of the GPI anchor by the GPI-PLC(41) , revealed the presence of a diffuse band with an apparent molecular mass of 30 kDa (Fig. 2B). This component could not be detected in case of a continuous presence of trypsin (conditions used in Fig. 1B), probably due to uninterrupted proteolysis. In contrast with the observations concerning the N-terminal domain of the VSG, the 30-kDa component was found to be released with a kinetics similar to that of the cyclase stimulation, with a maximum after 90 min (Fig. 2, A and B). Under conditions where the surface coat was removed (15-min incubation with trypsin then 60-min incubation without trypsin), the majority of the cells remained physiologically intact as judged by the low percentage of a cytoplasmic enzyme activity found in the supernatant of treated cells (Table 2A). These results are consistent with the view that the removal of the N-terminal domain of the VSG by trypsin is followed by an active process which requires physiological temperatures but not trypsin and which induces the progressive release of the CRD positive C-terminal domain. The kinetics of this process was similar to that of the activation of adenylate cyclase, suggesting a relationship between cyclase stimulation and release of the C-terminal domain of the VSG.
Figure 2:
Exogenous trypsin initiates concurrent
activation of the adenylate cyclase and VSG lipase-mediated release of
the C-terminal domain of the VSG, even when present for only a short
period. Trypanosomes (3 10
cells/ml) were incubated
in isosmotic buffer (pH 7.5) for 30 min (20 °C) in the presence of
cycloheximide (10 µg/ml). Trypsin (50 µg/ml final
concentration) was then added (arrow labeled T) and
the incubation continued for only 15 min before adding soybean
inhibitor (250 µg/ml final concentration). The cells were
immediately centrifuged and resuspended in the same buffer supplemented
with trypsin inhibitor and leupeptin (20 µg/ml) (arrow labeled R) and the incubation continued for a further 2 h
at 20 or 4 °C. At various times samples were removed from the
incubation, centrifuged, and the supernatants analyzed for their
content of VSG, while the cells in the pellet were analyzed for their
adenylate cyclase activity under conditions of swell dialysis (5 min at
37 °C). A shows the time course of cAMP production in
AnTat 1.1 clone incubated at 20 °C (
) or 4 °C (
).
Each value represents the mean ± S.E. of three separate
determinations. The S.E. where no error bar is shown was less than the
representation of the point. B shows the time course of VSG
release as assessed by Western blot analysis of supernatants from
10
cells taken at various times during the incubation. Each
blot was probed with polyclonal antibodies raised against the
cross-reacting determinant on the GPI anchor of the VSG
(anti-CRD).
The acid-induced adenylate cyclase activation was characterized further by varying the experimental conditions (Fig. 4). First, this stimulation was only transient and decreased with increasing incubation time in the swell medium used for the assay of activity (Fig. 4A). Second, the stimulation was reversed by a subsequent incubation at neutral pH only at physiological temperatures (compare the effect of 30 and 4 °C in Fig. 4B). Third, the addition of the protonophore FCCP, which causes an acidification of the cytosol(43) , allowed adenylate cyclase activation at neutral pH (Fig. 4C). Taken together, these data indicated that the stimulation of adenylate cyclase by acid treatment is a transient process which occurs in response to a slight acidification of the cytosol. Moreover this process is reversed when cytoplasmic pH is returned to physiological values. These results strongly suggest that the stimulation of the cyclase does not result from cell breakage and are consistent with the observation that only 3.7% of a cytoplasmic marker is found in the supernatant of cells incubated for 60 min at 4 °C in pH 5.5 buffer (Table 2A).
Figure 4:
Characterization of the acid-induced
activation of adenylate cyclase in AnTat 1.1 bloodstream forms. A shows the inhibition of adenylate cyclase stimulation as a
function of the incubation time in swell dialysis medium prior to assay
of the activity. Adenylate cyclase activity was first stimulated by a
30-min preincubation in pH 5.5 buffer at 4 °C. The cells were then
centrifuged, resuspended in swell dialysis medium, and incubated at 37
°C. Samples taken at various times were assayed as described under
``Materials and Methods.'' The curves show the
results obtained from two separate experiments. B presents the
reversibility of acid-induced adenylate cyclase activation. The cells
were treated at 4 °C in pH 5.5 buffer (). After 30 min, they
were centrifuged, resuspended in a pH 7.5 buffer and incubated at
either 30 °C (
) or 4 °C (
) for various periods
prior to assay of adenylate cyclase activity.
, cells incubated
at 4 °C in pH 5.5 buffer for 60 min. C shows the effect of
H
concentration and of the presence of a protonophore
(FCCP) on the activity of adenylate cyclase. Cells were incubated at 4
°C at various concentrations of H
as indicated, in
the presence (
) or absence (
) of the ionophore FCCP (1.2
µM). After 60 min, the cells were centrifuged and assayed
for adenylate cyclase activity. Each value represents the mean ±
S.E. of three separate determinations. Where no error bar is
shown, the S.E. was less than the representation of the point. The
strong stimulation at pH 5.0 is probably due to cellular
toxicity.
Figure 5:
Inhibitors of PKC induce adenylate cyclase
activation and VSG release in AnTat 1.1 bloodstream forms. Bloodstream
form trypanosomes were incubated for 5 min at 37 °C under swell
dialysis conditions in the presence or absence of the indicated
compounds and assayed for cAMP production (A) and for VSG
release (B and C). A shows the dose response
curve of adenylate cyclase assayed in the presence of the indicated
concentrations of Myr- PKC-
(
), Myr-
PKC-
(
), or Myr-random peptide (
). Each value represents the
mean of three separate determinations and in all cases the S.E. was
less than the representation of the point. B shows a Western
blot analysis of supernatants of cells incubated under the same
experimental conditions with the indicated inhibitor concentrations and
probed with either anti-AnTat 1.1 VSG (top panels) or anti-CRD (bottom panels) antibodies. C shows Western blots of
proteins present in the supernatant of cells incubated as indicated
above without peptide (lane 1) or with Myr-
PKC-
(lane 2), Myr-random peptide (lane 3), calphostin C (lane 4), and Myr-
PKA (lane 5), and probed
with either anti-AnTat 1.1 VSG (left panel) or anti-CRD (right panel) antibodies.
The stimulation of adenylate cyclase by the PKC inhibitors prompted
us to examine the effects of these compounds on the release of VSG. Table 2B shows that a short incubation with 100 µM Myr- PKC-
induced the release of almost 80% of the
total releasable VSG. Significantly, the release of VSG was dependent
on the concentration of Myr-
PKC-
and Myr-
PKC-
in a similar manner to that observed for cyclase activation (compare Fig. 5, A and B, upper panels). Moreover,
neither treatment with these inhibitors of PKC nor the concomitant
activation of adenylate cyclase and release of VSG had a deleterious
effect on cellular integrity as indicated by the very low release of a
cytoplasmic marker enzyme under these conditions (Table 2A).
Another specific PKC inhibitor, calphostin C, also induced VSG release (Fig. 5C, lane 4, left panel), whereas control peptides
were ineffective (Fig. 5C, lanes 3 and 5, left
panel). Finally, the released VSG was CRD-positive in all cases,
demonstrating the involvement of VSG lipase in VSG release (Fig. 5B, lower panel, and C, right panel).
Taken together, these results suggested that inhibition of PKC induces the activation of both adenylate cyclase and VSG lipase, without damaging the cells.
Thus, while it was clear that activation of adenylate cyclase does not require VSG release, an alternative model involving cyclase-dependent activation of GPI-PLC remained possible. To test this idea, we exploited the observation that 100 µM pCMPS prevents adenylate cyclase activation under all treatments tested (acid, trypsin, and PKC inhibitors, Fig. 7A). This concentration of pCMPS is 50-fold lower than that reported to be necessary for inhibition of the trypanosomal GPI-PLC(50, 51) . Significantly, we found that over the concentration range of pCMPS required to inhibit totally adenylate cyclase (15-100 µM, Fig. 7B), VSG release was not affected (Table 2B and Fig. 7C). We verified that pCMPS does not inhibit adenylate cyclase indirectly through inactivation of the creatine kinase present in the assay medium, since inhibition was observed in the absence of creatine kinase (data not shown). Therefore, VSG release was observed in the absence of adenylate cyclase activation. We conclude that the stimulation of adenylate cyclase is not required to induce the release of VSG.
Figure 7:
VSG release can occur in the absence of
activation of adenylate cyclase in the AnTat 1.1 bloodstream variant. A shows the effect of 100 µM pCMPS on adenylate
cyclase activity. Bloodstream form trypanosomes (3 10
cells/ml) were preincubated either in isosmotic TES buffer with
or without trypsin (30 min, 20 °C) or in isosmotic pH 5.5 phosphate
buffer (60 min, 4 °C) as described in the legends to Fig. 1and Fig. 3and under ``Materials and
Methods.'' The cells were then centrifuged and assayed for
adenylate cyclase activity under swell dialysis conditions (5 min, 37
°C) in the absence or presence of 100 µM pCMPS. The
activity was also assayed in the presence of 100 µM Myr-
PKC-
with or without pCMPS. The results are
expressed as relative activity (control = 1). B shows
the effect of the concentration of pCMPS on Myr-
PKC-
-induced adenylate cyclase activation. Trypanosomes (3
10
cells/ml) were assayed under swell dialysis conditions
in the presence of 100 µM Myr-
PKC-
at various
concentrations of pCMPS. Each value represents the mean ± S.E.
of three separate determinations. Where no error bar is shown
the S.E. was less than the representation of the point. C shows the effect of pCMPS on PKC inhibitor-induced VSG release.
Bloodstream form trypanosomes were incubated under swell dialysis
conditions (37 °C, 5 min) in the presence or absence of 100
µM Myr-
PKC-
and with or without 100 µM pCMPS as indicated. The cells were separated from the incubation
medium by centrifugation after the osmotic strength was adjusted to 340
mOsm with KCl and the supernatants from 10
cells were
subjected to Western blot analysis using either anti-AnTat 1.1 VSG (left panel) or anti-CRD (right panel)
antibodies.
The biological
significance of the relationship between VSG release and activation of
the cyclase is unclear, but may relate to the triggering of a cell
signaling process under stress conditions. In this regard it is
interesting to note that trypanosomes from highly infected mice (around
10 parasites/ml of blood), thus presumably subjected to
growth limiting stress, reproducibly exhibit higher basal levels of
adenylate cyclase activity than cells isolated during lower
parasitaemia.
Stress-dependent VSG release and activation
of adenylate cyclase may initiate a cascade that results in either a
metabolic change or an altered pattern of gene expression that enables
bloodstream forms of the parasite to adapt to environmental conditions.