 |
Introduction |
Mast cells are specialized secretory cells that belong to
the immune system. Through triggered exocytosis
of their secretory granules (SGs),1 mast cells release biologically active substances, including vasoactive amines, proteases, and preformed cytokines. In addition, after their activation, mast cells produce and release arachidonic acid
metabolites such as leukotrienes, prostaglandins, and multifunctional cytokines. Together, these mediators play central roles in both the immediate and late phase inflammatory reactions (1, 2). Although their physiological role in
the body is less clear, mast cells importantly contribute to
host defense against bacterial and parasite infections (3) as
well as to cellular immune responses through their ability
to present antigens and trigger antigen-specific T cell proliferation (9, 10).
Previous studies of exocytosis in mast cells indicate that
the final trigger to exocytosis involves a late acting GTP-binding protein (11, 12) and Ca2+ (13, 14). The molecular
identity of the mast cell exocytic Ca2+ sensor remains obscure. In the synapse, this role has been ascribed to synaptotagmins (Syts) I and II, abundant Ca2+ and phospholipid
binding proteins localized on synaptic vesicles (SVs) (15-
20). Binding of Ca2+ to Syt results in a conformational (21)
or electrostatic (22) change that, by an as yet unresolved
mechanism, allows exocytosis to occur. The finding that
Syt I and II belong to a larger family of ubiquitously expressed proteins suggests that Syt isoforms may function as
general Ca2+ sensors (23, 24). This hypothesis is supported
by the recent demonstration of a role for a Syt isoform in
controlling insulin secretion (25, 26).
We have recently reported that expression of Syt I in
RBL-2H3 cells (a mucosal mast cell line) resulted in prominent potentiation and acceleration of Ca2+-dependent
exocytosis (27). Therefore, in this study we decided to
identify the Syt isoform which is endogenously expressed
in RBL cells, and explore its role in controlling exocytosis.
We found that rat basophilic leukemia (RBL) cells endogenously express the Syt isoforms II, III, and V. The role of
Syt II, the most abundant isoform in RBL cells, was investigated.
 |
Materials and Methods |
Antibodies.
Antibodies used included rabbit polyclonal serum
directed against the cytoplasmic domain of Syt I (a gift from Dr.
T.C. Sudhof, Howard Hughes Medical Institute, University of
Texas Southwestern Medical School, Dallas, TX), mAbs directed
against the NH2-terminal region of Syt II (a gift from Dr. M. Takahashi, Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan), and polyclonal antibodies against cathepsin D (Calbiochem).
Isolation and Growth of Mast Cells.
Bone marrow-derived mast
cells (BMMCs) were obtained as previously described (28). In
brief, femoral bone marrow cells from 6-wk-old BALB/c mice
were cultured in 50% WEHI-3 cells conditioned medium. Culture medium was changed weekly, and nonadhering cells were
used for further growth. After 3 wk, at least 99% of the cells were
identified as mast cells by toluidine blue staining. Rat peritoneal
mast cells (RPMCs) were obtained from Wistar rats by peritoneal
lavage, and purified as previously described (29). In brief, a suspension of washed peritoneal cells was layered over a cushion of
30% Ficoll 400 (Pharmacia Biotech Inc.) in buffered saline and
0.1% BSA and centrifuged at 150 g for 15 min. The purity of
mast cells recovered from the bottom of the tube was >90%, as
assessed by toluidine blue staining. RBL-2H3 cells (hereafter termed RBL cells) were maintained in adherent cultures in
DMEM supplemented with 10% FCS in a humidified atmosphere
of 6% CO2 at 37°C.
Reverse Transcription and PCR Amplification of Syt cDNA Fragments.
RNA was isolated from trypsinized RBL cells collected
by centrifugation at 400 g for 5 min, and from brains that were
rapidly excised from 150-200-g Sprague-Dawley rats killed by
CO2 suffocation and then exsanguinated. Total RNA was isolated on a guanidine thiocyanate/CsCl gradient, extracted twice
with phenol/chloroform, and then ethanol precipitated. The
RNA was dissolved in 0.1% diethyl pyrocarbonate-treated water,
quantified by measuring absorbance at 260 nm, evaluated for
degradation by agarose-formaldehyde gel electrophoresis, and
frozen until used. The mRNA was isolated from total RNA by
oligo-dT cellulose chromatography [Poly(A)Pure; Ambion], and
2 µg was reverse transcribed by 125 U of Moloney's murine leukemia virus-reverse transcriptase (New England BioLabs) in a 50 µl
reaction containing 2.5 µg each of (dT)18 and random octamers,
1 mM of each dNTP, and 40 U of RNAsin (Promega) at 37°C for 30 min, 42°C for 30 min, and 50°C for 15 min. The first
round of nested PCR was performed with 1 µl of AmpliTaq
(Perkin-Elmer Cetus) in 100 µl reaction buffer supplemented
with 1.5 mM MgCl2, 10% (vol/vol) DMSO, 1 µM of each
primer, 50 µM of each dNTP, and 1 µl of the reverse transcription reaction as template. The four primers correspond to RNA
sequences encoding portions of Syt proteins schematized in Fig.
1 A, and their sequences were: A, TCWGACCCYTAYGTCAARRTCT; B, AGACCCARGTGCACMGGAAGAC; C,
SYCYTTSACRTAGGGRTCTGA; D, GGGGTTSAGSGTGTTCTTCTT. For the first round of PCR, six cycles of 94°C
for 1 min ramping to 49°C in 3 min, 49°C for 1 min, 72°C for
1 min were followed by 24 cycles of 94°C for 1 min, 65°C for
1 min, 72°C for 1 min, with a final extension at 72°C for 6 min. 1 µl
of the PCR product obtained with RBL cell cDNA and the A
and D primers was used for template in a second round of PCR
identical to the first except that the initial six cycles with low annealing temperature were not included. The product of the second reaction using the B and C primers was purified by agarose
gel electrophoresis, then ligated into the pCR-II vector (Invitrogen). DH5
cells were transformed with the ligation mixture and
colonies were selected for sequencing.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 1.
PCR amplification of Syt isoforms. (A) Schematic portrayal
of a representative Syt protein and the four primers for the PCR reactions. (B) An agarose gel of electrophoretically separated products of an
initial round of PCR performed as described in Materials and Methods,
then stained with ethidium bromide. Primer pairs and the source of
cDNA for each reaction are shown above the gel, and size markers are
shown on the right side. (C) An agarose gel of the products of a second
round of PCR, using for a template of the PCR product of RBL cell
cDNA with primers A and D. Primer pairs for the second-round reactions are shown above the gel, and size markers are shown on both sides.
|
|
Ribonuclease Protection Assay.
Vectors containing the PCR-cloned Syt fragments were linearized with NotI for SP6-directed
synthesis of riboprobes or with BamHI for T7-directed cRNA
synthesis. cRNA hybridization controls were generated in a 50 µl
reaction containing 3 µg of template DNA, 1.6 U/µl RNAsin,
10 mM dithiothreitol (DTT), 0.1 mg/ml BSA, 1 mM of each
NTP, 2.5 µM [3H]UTP (45 Ci/mmol; Amersham), and 100 U
T7 RNA polymerase (New England Biolabs) in the manufacturer's buffer. Reactions were incubated for 4 h at 37°C, 10 U
DNAse I was added, and the incubation was continued for another
20 min, and then cRNA was purified on a Nick-Spin column
(Ambion). Riboprobes were transcribed in a 20 µl reaction using
1 µg of template DNA, 25 µM
-[32P]CTP (800 Ci/mmol; Amersham), 2 U/µl RNasin, 10 mM DTT, 0.1 mg/ml BSA, 0.5 mM
each of other NTPs, and 10 U SP6 RNA polymerase (New England Biolabs) in the manufacturer's buffer for 1 h at 40°C. After
treatment with 5 U of DNAse I, full-length transcripts were isolated by gel purification in 5% acrylamide/8 M urea gels. RNase protection assays were performed using the RPA II kit (Ambion). Cognate cRNA (0.1 pmol) and yeast RNA were used as positive
and negative controls. Each experiment contained 1 pmol riboprobe and varying amounts of RBL cell RNA supplemented
with yeast RNA to complete a total of 40 µg RNA. Hybridization was carried out overnight at 45°C. Protected probes were
electrophoresed through 5% acrylamide/8 M urea gels and visualized by autoradiography.
Preparation of Mast Cell and Brain Lysates.
Mast cells (106) derived from different sources (RPMCs, BMMCs, and RBL-2H3)
were washed in PBS and resuspended in 30 µl of lysis buffer (50 mM
Hepes, pH 7.4, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA,
1% Triton X-100, 0.1% SDS, 50 mM NaF, 10 mM NaPPi, 2 mM
NaVO4, 1 mM PMSF, and 10 µg/ml leupeptin) and centrifuged
at 12,000 g for 15 min at 4°C. The cleared supernatants were
mixed with 5× Laemmli sample buffer to a final concentration of
1×, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting. For the preparation of brain homogenate, whole brain
from a Wistar rat was homogenized in PBS at 4°C using a Polytron (Kinematica, GmbH, Switzerland; 20 s, setting 7). Aliquots
(5-10 µg protein) were mixed with 5× Laemmli sample buffer,
boiled for 5 min, and subjected to SDS-PAGE and immunoblotting.
Subcellular Fractionation of RBL Cells.
RBL cells (7 × 107) were
washed with PBS and suspended in homogenization buffer (0.25 M
sucrose, 1 mM MgCl2, 800 U/ml DNase I [Sigma Chemical
Co.], 10 mM Hepes, pH 7.4, 1 mM PMSF, and a cocktail of
protease inhibitors [Boehringer Mannheim, Germany]). Cells
were then disrupted by 3 cycles of freezing and thawing followed
by 20 passages through a 21-gauge needle. Unbroken cells and
nuclei were removed by sequential filtering through 5- and 2-µm filters (Poretics Co.). The final filtrate was then centrifuged for 10 min at 500 g and the supernatant loaded onto a continuous, 0.45-2.0 M sucrose gradient (10 ml), which was layered
over a 0.3 ml cushion of 70% (wt/wt) sucrose and centrifuged for
18 h at 100,000 g. Histamine was assayed fluorimetrically after
condensation in alkaline medium with o-phthalaldehyde (30).
LDH activity was assayed using LDH reagent according to the
manufacturer's instructions (Merck Diagnostica, Germany).
Secretion from RBL Cells.
RBL-2H3 cells were seeded in
24-well plates at 2 × 105 cells per well and incubated overnight
in a humidified incubator at 37°C. The cells were then washed
three times in Tyrode's buffer (10 mM Hepes, pH 7.4, 130 mM
NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA) and stimulated in the same buffer with the
indicated concentrations of the calcium ionophore A23187 and
the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA; Calbiochem). Secretion was allowed to proceed for 30 min at 37°C. Aliquots from the supernatants were taken for measurements of released
-hexosaminidase activity. Cells in control wells were lysed by addition of 0.1% Triton X-100 to determine the total enzyme content. For Fc
RI induced secretion, cells were passively sensitized by overnight incubation with DNP specific monoclonal IgE (SPE7, a gift of Dr. Z. Eshhar, the Weizmann Institute
of Science, Rehovot, Israel), washed three times in Tyrode's
buffer, and then stimulated with the indicated concentrations of
the antigen, DNP-BSA. Activity of the released
-hexosaminidase
was determined by incubating aliquots (20 µl) of supernatants and
cell lysates for 90 min at 37°C with 50 µl of the substrate solution
consisting of 1.3 mg/ml p-nitrophenyl-N-acetyl-
-D-glucosaminide
(Sigma Chemical Co.) in 0.1 M citrate pH 4.5. The reaction was
stopped by the addition of 150 µl of 0.2 M glycine, pH 10.7. OD
was read at 405 nm, in an ELISA reader. Results were expressed
as percentage of total
-hexosaminidase activity present in the
cells. To assay the release of cathepsin D, supernatants of cells,
stimulated as above, were concentrated in VivaSpin concentrators
with a 10 kD cut-off (VivaScience, UK). The concentrates were
mixed with 5× Laemmli sample buffer, boiled for 5 min, and
subjected to SDS-PAGE and immunoblotting with anti-cathepsin
D antibodies. For measurement of serotonin release, cells were incubated overnight with 2 µCi of [3H]5-hydroxytryptamine (NEN),
washed, and stimulated as above. Aliquots from the supernatants
were taken for measurement of radioactivity.
SDS-PAGE and Immunoblotting.
Samples (normalized according to protein content or number of cells) were separated by
SDS-PAGE using 10 or 12% polyacrylamide gels. They were
then electrophoretically transferred to nitrocellulose filters. Blots
were blocked for 3 h in TBST (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, and 0.05% Tween 20) containing 5% skim milk followed
by overnight incubation at 4°C with the indicated primary antibodies. Blots were washed three times and incubated for 1 h at
room temperature with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG; Jackson Research Labs.). Immunoreactive bands were visualized by the
enhanced chemiluminescence method according to standard procedures.
Cell Transfection.
A full-length rat Syt II cDNA (provided by
Dr. T.C. Sudhof) was subcloned into the EcoRI site of the
pcDNA3 expression vector (Invitrogen) both in the sense and
antisense orientations. RBL-2H3 cells (8 × 106) were transfected
with 20 µg DNA of pcDNA3-Syt II or pcDNA3 alone, by electroporation (0.25 V, 960 µF). Cells were immediately replated in
tissue culture dishes containing growth medium (supplemented DMEM). G418 (1 mg/ml) was added 24 h after transfection and
stable transfectants were selected within 14 d.
 |
Results |
Expression Analysis of Endogenous Syt Isoforms.
Primers
were chosen from the conserved C2 domains (Fig. 1 A)
that averaged 91% identity with known Syt isoforms. An
initial round of PCR with four different primer pairs did
not yield any visible product in reactions containing RBL
cell cDNA, even though abundant product was obtained
from brain cDNA using two different primer pairs (Fig.
1 B). When the PCR product of RBL cell cDNA with
primers A and D was then used as template in a second
round of PCR, the nested reaction with primers B and C
yielded abundant product of the predicted size of 365 bp
(Fig. 1 C). Sequencing the inserts of 21 colonies of subcloned PCR product yielded 10 colonies encoding a fragment of Syt II, 9 colonies encoding Syt III, and 2 colonies
encoding Syt V. These findings were supported by the results of restriction digestions of the PCR product with multiple frequent cutting enzymes (data not shown) that
were consistent with the presence of these three isoforms
and did not indicate the presence of additional isoforms
based on the known sequences of Syt isoforms.
RNase protection assays were then performed to quantitatively assess the expression of Syt isoforms in RBL cells.
Syt II was the most abundant (Fig. 2), and serial dilution of
mRNA used to protect the Syt II probe indicated that this
isoform was approximately fivefold more concentrated in
RBL cells than in Syt III. The Syt V isoform was not protected even when mRNA was present at a level at least 10-fold higher than that which measurably protected the Syt II
probe (Fig. 2), suggesting that Syt V mRNA is present in RBL cells at a concentration less than one-tenth the level
of Syt II.

View larger version (86K):
[in this window]
[in a new window]
|
Fig. 2.
RNase protection assay of RBL cell transcripts. Autoradiogram of the products of an RNase protection assay after PAGE. The riboprobes used for hybridization are listed in the top row, the amount of
mRNA loaded in each lane is listed in the second row, and a size marker
is shown on the left side. This figure is a representative example of an experiment that was repeated four times.
|
|
Because Syt II, which shares the highest homology with
the predominant neural isoform Syt I (31), was the most
abundant isoform, we chose to focus this study on Syt II.
We next examined the expression of the Syt II protein using specific antibodies (mAb 8G2B, directed against the
NH2 terminus of Syt II). A single immunoreactive protein
was detected in RBL cells (Fig. 3, lane 2). Immunoreactivity in RBL cells (Mr ~80 kD) had less mobility on SDS-PAGE than immunoreactivity in the brain (Fig. 3, lane 1). Nevertheless, an 80-kD Syt II-immunoreactive protein
was also detected in lysates from fully differentiated, connective tissue-type, RPMCs (Fig. 3, lane 3) and primary
murine BMMCs (Fig. 3, lane 4). These size differences in
Syt II may thus arise from tissue-specific posttranslational
modifications.

View larger version (115K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of Syt II
protein in mast cells. Whole lysates
(106 cell equivalents) derived from
RBL-2H3 cells (lane 2), RPMCs
(lane 3), BMMCs (lane 4), and a
crude brain homogenate (lane 1, 10 µg protein), were resolved by
SDS-PAGE and immunoblotted
using the mAb 8G2B directed
against the NH2 terminus of Syt II.
|
|
Effect of Syt II Overexpression on Ca2+-induced Exocytosis.
To study the functional role of Syt II, we stably transfected
RBL cells with neural Syt II cDNA and selected clones
with increased levels (approximately twofold) of Syt II expression (Syt II+; Fig. 4, lanes 1-3) for further studies. Notably, transfection with neural Syt II cDNA resulted in
overexpression of the same 80-kD Syt II-immunoreactive
protein, strengthening the concept that the increased apparent Mr of RBL-Syt II was due to tissue-specific posttranslational modifications.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Overexpression of
Syt II in RBL cells. Whole lysates
derived from G418-resistant RBL
clones (1.5 × 106 cell equivalents), transfected with either the
pcDNA3-Syt II recombinant vector (Syt II+, lanes 1-3) or with the empty
pcDNA3 vector (control, lanes 4-6) were resolved by SDS-PAGE and
immunoblotted using monoclonal 8G2B anti-Syt II antibodies.
|
|
Overexpression of Syt II had no effect on the spontaneous release of the SG-associated enzyme,
-hexosaminidase
(32). In the absence of any stimulus, both control cells
(empty vector-transfected) and cells overexpressing Syt II
released up to 5% of their total
-hexosaminidase (Fig. 5 A).
However, in contrast to transfection with Syt I (27), overexpression of Syt II failed to potentiate Ca2+-dependent
exocytosis evoked by a Ca2+ ionophore alone (Fig. 5 A), or
in the presence of phorbol ester (Fig. 5 B). Instead, a mild
inhibition could be observed when the cells were triggered
with low (<10 µM) concentrations of the Ca2+ ionophore.

View larger version (27K):
[in this window]
[in a new window]

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Modulation of exocytosis by Syt II. Control ( ), Syt II+
( ), and Syt II ( ) cells were incubated for 30 min at 37°C with
the indicated concentrations of
the Ca2+ ionophore A23187,
alone (A) or together with 50 nM
TPA (B). The extent of release is
presented as percentage of total
-hexosaminidase activity. The results presented in A are from one
experiment, which included single clones stably transfected with
the empty pcDNA3 vector, the
pcDNA3-Syt II recombinant vector, or the pcDNA3-Syt II recombinant vector in the antisense orientation. Similar results were
obtained on five occasions and using two additional clones. The data points presented in B are means ± SEM of four determinations and include two independent clones stably transfected with
the empty pcDNA3 vector, one clone stably transfected with the pcDNA3-Syt II recombinant vector, and two independent clones stably transfected with
pcDNA3-Syt II in the antisense orientation. Similar results were obtained on five occasions. Inset: -hexosaminidase release of individual clones (1-3 stably
transfected with the empty pcDNA3 vector and 4-6 stably transfected with pcDNA3-Syt II in the antisense orientation) at a representative concentration of
agonist: A, 10 µM A23187; B, 1 µM A23187 and 50 nM TPA.
|
|
Effect of the Suppression of Syt II Expression on Ca2+-induced
Exocytosis.
The fact that Syt II, unlike Syt I, is endogenously expressed in RBL cells enabled us to extend these
results by investigating the effect of reducing the level of
Syt II expression on exocytosis. To this end, cells were stably transfected with Syt II cDNA subcloned in the antisense orientation, resulting in substantially reduced levels of
Syt II expression (15, 6, 47, and 6% of control levels) (Fig.
6). Clones expressing the lowest levels (6-15%) were chosen for further analyses. In these cells (Syt II
), the basal,
spontaneous release of
-hexosaminidase was not affected,
revealing that Syt II was not acting as a limiting fusion clamp. However,
-hexosaminidase release triggered by a
Ca2+ ionophore alone (Fig. 5 A) or with phorbol ester
(Fig. 5 B) was markedly (by up to fivefold) potentiated.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Suppression of Syt
II expression. Whole lysates
derived from G418-resistant
RBL clones (1.5 × 106 cell
equivalents), transfected with
either the pcDNA3-Syt II antisense orientation (Syt II , lanes 1-4) or
with the empty pcDNA3 vector (control, lanes 5-7) were resolved by
SDS-PAGE and immunoblotted as in Fig. 3.
|
|
The Role of Syt II in Fc
RI-induced Exocytosis.
Physiologically, exocytosis in mast cells can be triggered by antigen-
induced aggregation of the high-affinity receptors (Fc
RI) for
IgE (33). To investigate the involvement of Syt II in controlling Fc
RI-mediated exocytosis, antigen-induced secretion was studied in the Syt II
and Syt II+ cells. Secretion of
-hexosaminidase was unaffected in the Syt II+ cells but was
significantly elevated (by fourfold) in the Syt II
cells (Fig.
7). Taken together, these results suggest that Syt II negatively
regulates release of
-hexosaminidase, whether triggered by
Ca2+ ionophore or by an immunological trigger.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
Modulation of Fc RI-dependent release by Syt II. Passively
sensitized control ( ), Syt II+ ( ), and Syt II ( ) cells were incubated
for 30 min at 37°C with the indicated concentrations of the corresponding antigen, DNP-BSA. The extent of release is presented as percentage
of total -hexosaminidase activity. The results presented are of a representative experiment, that included three independent clones, stably
transfected with the empty pcDNA3 vector, three independent clones
stably transfected with the pcDNA3-Syt II recombinant vector and three
independent clones stably transfected with pcDNA3-Syt II in the antisense orientation. The data points are means ± SEM of six determinations. Similar results were obtained on five occasions. Inset: -hexosaminidase release of individual clones (1-3 stably transfected with the
empty pcDNA3 vector and 4-6 stably transfected with pcDNA3-Syt II in
the antisense orientation) at a representative concentration of antigen (10 ng/ml DNP-BSA).
|
|
Subcellular Distribution of Syt II in RBL Cells.
To understand the opposite effects exerted by the transfected Syt I
and Syt II proteins, we investigated their distribution in RBL
cells using a continuous sucrose gradient. All of the Syt II
immunoreactivity present in either the control or the Syt II- transfected cells comigrated with 60% of the
-hexosaminidase activity, present in fractions 6-13 at ~0.75 M sucrose
(Fig. 8, A, B, E, and F). These fractions did not include the
histamine-containing SGs, which migrated at fractions 16-23
at 1.3 M sucrose and included the remaining
-hexosaminidase activity (Fig. 8, E and F). Histamine was also found at
the top of the gradient (Fig. 8 E), but this probably reflected the contents of SGs that were released during cell
disruption. Therefore, these results indicate that, unlike transfected Syt I (Fig. 8 C), the endogenous and transfected
Syt II proteins were not targeted to the histamine-containing SGs, but to a different intracellular compartment.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 8.
Subcellular fractionation of control and Syt II-transfected RBL cell lysates.
Fractions from a continuous sucrose gradient were collected from the top, and assayed
for: A, Syt II immunoreactivity in control
cells; B, Syt II immunoreactivity in Syt II+
cells; C, Syt I immunoreactivity in Syt I+ cells;
D, pro-cathepsin D immunoreactivity; E,
-hexosaminidase activity (presented as OD
read at 405 nm) ( ); histamine content ( )
and LDH activity ( ); (F) protein ( ) and
sucrose density ( ). The data presented in
panels E and F is the average of three
sucrose gradients performed on control, Syt
II+, and Syt I+ cells.
|
|
The presence of
-hexosaminidase in fractions 6-13 of the
sucrose gradient suggested the presence of a lysosomal organelle distinct from the histamine-containing SGs. Indeed,
fractions 6-13 also contained procathepsin D, Mr 53 kD, the
precursor of the lysosomal protease cathepsin D (Fig. 8 D).
Effects of Syt II on the Release of Cathepsin D.
Lysosomes
were recently shown to behave as Ca2+-regulated exocytic
vesicles (34). Since
-hexosaminidase is distributed between histamine-containing SGs and procathepsin D-containing
lysosomes in RBL cells, it was important to determine
whether secretion of the content of the latter compartment
is negatively regulated by Syt II. To address this question,
we examined whether Syt II could modulate release of the
lysosomally processed form of cathepsin D (mature cathepsin D, Mr ~43 kD) (35). Concentrating the cell supernatants by 20-fold allowed the detection of cathepsin D in supernatants from Ca2+ ionophore- or antigen-triggered cells
(Fig. 9 A, lanes 1-3). The amount of secreted mature
cathepsin D was significantly inhibited or increased in the
Syt II+ or Syt II
cells, respectively (Fig. 9). Ca2+ ionophore was more effective than the immunological stimulus in the Syt II+ cells, but did not differ significantly in Syt II
cells. The precursor form of cathepsin D (53 kD) was detected in supernatants of both triggered and nontriggered
cells (data not shown), reflecting the constitutive release of
unprocessed cathepsin D (34). These results demonstrate
that mast cell activation triggers exocytosis of a lysosomal
fraction distinct from histamine-containing SGs, and that
mobilization of this compartment depends substantially on
the expression level of Syt II.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 9.
Release of Cathepsin D. (A) Control RBL cells (lanes 1-3),
Syt II+ cells (lanes 4-6), and Syt II cells (lanes 7-9) were incubated for
30 min at 37°C with buffer (lanes 1, 4, and 7), 50 ng/ml of the DNP-BSA antigen (lanes 2, 5, and 8), or 10 µM of the Ca2+ ionophore A23187
(lanes 3, 6, and 9). The concentrated cell supernatants were resolved by
SDS-PAGE and immunoblotted using anti-cathepsin D (Cat D) antibodies. (B) The intensity of the band corresponding to mature cathepsin D
was quantitated by densitometry (using a B.I.S. 202D densitometer,
Dinco & Rhenium, Israel) and is presented as fold stimulation of the level
in control, nonstimulated cells.
|
|
Effects of Syt II on the Release of Serotonin.
We have also
evaluated the effects of Syt II on the triggered release of serotonin, to exclusively monitor exocytosis of SGs (36). Overexpression of Syt II had no significant effect on serotonin
release triggered by either secretagogue (Fig. 10, A-C). However, reducing its level of expression in the Syt II
cells
had a small but significant stimulatory effect (Fig. 10, A-C).

View larger version (23K):
[in this window]
[in a new window]

View larger version (27K):
[in this window]
[in a new window]

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 10.
Release of serotonin. Control ( ), Syt II+ ( ) and Syt II ( ) cells, loaded with [3H]5-hydroxytryptamine (serotonin), were stimulated for
30 min at 37°C with the indicated concentrations of the Ca2+ ionophore A23187 alone (A), together with 50 nM TPA (B), or with the antigen DNP-BSA (C). The extent of serotonin release is presented as percentage of the total radioactivity in the cells. The data points presented are means ± SEM of
8-12 determinations and include three independent clones stably transfected with the empty pcDNA3 vector, three independent clones stably transfected
with the pcDNA3-Syt II recombinant vector, and three independent clones stably transfected with pcDNA3-Syt II in the antisense orientation. Statistical
analysis was performed using two-tailed student's t test. *P < 0.05; **P < 0.01. Inset: serotonin release of individual clones (1-3 stably transfected with
the empty pcDNA3 vector and 4-6 stably transfected with pcDNA3-Syt II in the antisense orientation) at a representative concentration of agonist: A,
10 µM A23187; B, 1 µM A23187 and 50 nM TPA; and C, 10 ng/ml DNP-BSA.
|
|
 |
Discussion |
Previous studies have already alluded to the possibility
that Syt isoforms may serve the role of general Ca2+ sensors, controlling regulated exocytosis also in nonneuronal secretory cells (23, 25, 26). We and others have previously shown that mast cells express Syt and SNAREs that probably function to control mediators released from these cells
(27, 37). Here, we demonstrate that RBL cells endogenously express at least three distinct isoforms of Syt, including Syt II, III, and V. Syt II was identified both by
RNAase protection assays (Fig. 2) and at the protein level,
on the basis of its immunoreactivity with specific antibodies
(Fig. 3). However, in contrast to its location on SVs or SGs
in neurons or endocrine cells, in the RBL cells, Syt II cofractionates with the lysosomal fraction rather than with the histamine-containing SGs (Fig. 8). Furthermore, transfection of the RBL cells with neural Syt II cDNA resulted in
overexpression of Syt II (Fig. 4) and its targeting to the
same fraction (Fig. 8).
Mast cells belong to immune cells of the hemopoietic lineage, where an intimate connection exists between lysosomes
and SGs (38). The SGs of mast cells include, in addition to
their secretory cargo of vasoactive amines (e.g., histamine and
serotonin), lysosomal enzymes such as
-hexosaminidase,
-glucuronidase, arylsulfatase, and carboxypeptidases (32),
as well as lysosomal integral membrane proteins (LIMPs)
(39). Therefore, mast cell SGs can be defined as secretory
lysosomes. Nevertheless, in consistence with previous data
(40, 41), our data indicate that in addition to the lysosomal, amine-containing SGs, mast cells also contain lysosomes,
which lack biogenic amines and with which Syt II is associated (Fig. 8). Such amine-free lysosomes were previously
reported to resist cell triggering by immunologic or Ca2+
ionophore stimulation (40, 41). Whether the two populations of granules are sequentially formed and by what
mechanism selective retention of the nonsecretory lysosomes is achieved, remained unknown. We now demonstrate
that mast cells can, to some extent, release also their lysosomal pool of hydrolases, upon both an immunologic and a
Ca2+ ionophore trigger. In this process both lysosomal enzymes, which are distributed between both SGs and lysosomes, such as
-hexosaminidase, as well as hydrolases localized exclusively to the amine-free lysosomal fraction, such as
cathepsin D (Fig. 9), are released. However, this release is
inhibited by overexpression of Syt II and markedly potentiated by reducing the level of Syt II expression (Fig. 9). Recently, three types of granules were ultrastructurally distinguished in IFN-
-treated mast cells (Table I). Type I and type II granules were both labeled by a fluid phase endocytic marker and both contained MHC class II as well as
lysosomal markers (42). These results have therefore suggested their position in the endocytic pathway, similarly to
lysosomal compartments (42). Serotonin was localized to
type II and type III granules, of which the latter type did
not internalize the fluid phase endocytic marker, nor did it
contain MHC class II (42). Based on these results, it was
suggested that a fusion event between type I (amine-free lysosomes) and III (e.g., SGs) granules may account for the
formation of type II granules (42). Our results are compatible with this model and define Syt II as the molecular entity, which may control this fusion event and effect selective retention of the nonsecretory lysosomes during cell
activation (see model shown in Fig. 11). Furthermore, this
model predicts that downregulation of Syt II should also
indirectly affect SG exocytosis by facilitating the fusion
event between the amine-free lysosomes and SGs. Indeed, we found that suppression of Syt II level of expression also
moderately potentiates serotonin release (Fig. 10).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 11.
Model of regulation of lysosomal exocytosis by Syt II. According to this model, Syt II is localized to lysosomes (L), where it acts to
inhibit fusion with SGs and the plasma membrane, at Ca2+ concentrations
that already support SG exocytosis. External signals are predicted to
downregulate Syt II and thereby remove this inhibition and facilitate
lysosomal exocytosis as well as fusion with SGs.
|
|
The molecular mechanism by which Syt II inhibits lysosome exocytosis is currently unknown. Syts fall into three
distinct classes that for syntaxin binding require either high
Ca2+ concentrations (200 µM) (class A) or low Ca2+ concentrations (<1 µM) (class B), or do not bind syntaxin in a
Ca2+-dependent manner (class C) (20). Syt II, the major
RBL isoform, and Syt V, the least abundant isoform, are
class A proteins. However, although Ca2+ concentrations
measured in neurons during an action potential are high
enough to support Ca2+-dependent interaction of class A
Syts with syntaxins, the rise of intracellular Ca2+ concentration in mast cells induced by Fc
RI cross-linking rarely exceeds 1 µM and would not be predicted to support such
interaction (43). Calcium-dependent Syts negatively regulate neuronal exocytosis at basal Ca2+ concentrations (44),
whereas positive effects on exocytosis are observed only at
elevated Ca2+ concentrations and are thought to depend on
interaction with syntaxin (20). In the mast cells Syt II seems
to increase the Ca2+ requirements for lysosomal exocytosis,
since Ca2+ ionophore is far more effective than immunologic stimulation in triggering cathepsin D release from
control cells, but both are equally potent in Syt II
cells
(Fig. 9). It is of great interest that Syt II appears to be used
in mast cells as a negative regulator of Ca2+-dependent
exocytosis and of a subclass of secretory vesicles, and is the
first example to our knowledge. Syt II inhibitory function appears to be linked to its lysosomal association since Syt I, although highly homologous to Syt II, potentiated Ca2+-dependent exocytosis of SG when transfected into the RBL
cells, alongside its SG targeting (27). The reasons for this
differential targeting of Syt I and Syt II remain unknown.
Although not proven here, the remaining Syt isoform
expressed in RBL cells, Syt III, which is a class B protein,
would be an adequate candidate to serve as the positive
regulator of SG exocytosis, whose action is mimicked by
transfected Syt I.
In conclusion, our findings provide unequivocal evidence for an active role of Syt II in negatively controlling
Ca2+-regulated lysosomal exocytosis. This observation extends the function of Syt II to regulation of exocytosis of
secretory organelles exclusive to SVs or SGs. Specifically, in
mast cells regulation by Syt II may have important implications on their function as APCs in host defense mechanisms, as this process requires uptake and lysosomal processing of antigens, followed by presentation of MHC class
II-peptide complexes on the mast cell surface (10, 45). Our
model predicts that the cellular level of Syt II could be up-
or downregulated to determine the balance of mast cell effector function between the secretion of inflammatory mediators from SG exocytosis and the presentation of antigen
by externalization of MHC II-containing lysosomes. Syt II
may thus play a central role in controlling the physiological
functions of mast cells.
Address correspondence to Ronit Sagi-Eisenberg, Department of Cell Biology and Histology, Sackler
School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel. Phone: 972-3-640-9500; Fax: 972-3-640-7432; E-mail:
We thank Drs. Y. Zick and D. Neumann for helpful discussions and a critical reading of this manuscript;
and Drs. T.C. Sudhof, M. Takahashi, and Z. Eshhar for their generous gifts of cDNA and antibodies.
This work was supported by grants from the Israel Science Foundation, founded by the Israel Academy for
Sciences and Humanities, and by the Thyssen Stiftung (to R. Sagi-Eisenberg).
1.
|
Stevens, R.L., and
K.F. Austen.
1989.
Recent advance in the
cellular and molecular biology of mast cells.
Immunol. Today.
10:
381-385
[Medline].
|
2.
|
Galli, S.J.,
J.R. Gordon, and
B.K. Wershil.
1991.
Cytokine
production by mast cells and basophils.
Curr. Opin. Immunol.
3:
865-872
[Medline].
|
3.
|
Metcalfe, D.D.,
M. Kaliner, and
M.A. Donlon.
1981.
The
mast cell.
Crit. Rev. Immunol.
3:
23-74
[Medline].
|
4.
|
Malaviya, R.,
T. Ikeda,
E. Ross, and
S.N. Abraham.
1996.
Mast
cell modulation of neutrophil influx and bacterial clearance at
sites of infection through TNF-alpha.
Nature.
381:
77-80
[Medline].
|
5.
|
Prodeus, A.P.,
X. Zhou,
M. Maurer,
S.J. Galli, and
M.C. Carroll.
1997.
Impaired mast cell-dependent natural immunity in complement C3-deficient mice.
Nature.
390:
172-175
[Medline].
|
6.
|
Echtenacher, B.,
D.N. Mannel, and
L. Hultner.
1996.
Critical protective role of mast cells in a model of acute septic
peritonitis.
Nature.
381:
75-77
[Medline].
|
7.
|
Abraham, S.N., and
R. Malaviya.
1997.
Mast cells in infection and immunity.
Infect. Immun.
65:
3501-3508
[Free Full Text].
|
8.
|
Galli, S.J..
1997.
The Paul Kallos memorial lecture. The mast
cell: a versatile effector cell for a challenging world.
Int. Arch.
Allergy Immunol.
113:
14-22
[Medline].
|
9.
|
Malaviya, R.,
N.J. Twesten,
E.A. Ross,
S.N. Abraham, and
J.D. Pfeifer.
1996.
Mast cells process bacterial Ags through a
phagocytic route for class I MHC presentation to T cells.
J.
Immunol.
156:
1490-1496
[Abstract].
|
10.
|
Frandji, P.,
C. Oskeritzian,
F. Cacaraci,
J. Lapeyre,
R. Peronet,
B. David,
J.G. Guillet, and
S. Mecheri.
1993.
Antigen-dependent stimulation by bone marrow-derived mast cells of MHC
class II-restricted T cell hybridoma.
J. Immunol.
151:
6318-6328
[Abstract/Free Full Text].
|
11.
|
Gomperts, B.D..
1990.
Ge: a GTP-binding protein mediating
exocytosis.
Annu. Rev. Physiol.
52:
591-606
[Medline].
|
12.
|
Aridor, M.,
G. Rajmilevich,
M.A. Beaven, and
R. Sagi-Eisenberg.
1993.
Activation of exocytosis by the heterotrimeric G protein Gi3.
Science.
262:
1569-1572
[Medline].
|
13.
|
Foreman, J.C.,
J.L. Mongar, and
B.D. Gomperts.
1973.
Calcium
ionophores and movement of calcium ions following the physiological stimulus to a secretory process.
Nature.
245:
249-251
[Medline].
|
14.
|
Howell, T.W.,
S. Cockcroft, and
B.D. Gomperts.
1987.
Essential synergy between calcium and guanine nucleotides in
exocytotic secretion from permeabilized rat mast cells.
J. Cell
Biol.
105:
191-198
[Abstract].
|
15.
|
Bommert, K.,
M.P. Charlton,
W.M. DeBello,
G.J. Chin,
H. Betz, and
G.J. Augustine.
1993.
Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin
in exocytosis.
Nature.
363:
163-165
[Medline].
|
16.
|
Brose, N.,
A.G. Petrenko,
T.C. Sudhof, and
R. Jahn.
1992.
Synaptotagmin: a calcium sensor on the synaptic vesicle surface.
Science.
256:
1021-1025
[Medline].
|
17.
|
DeBello, W.M.,
H. Betz, and
G.J. Augustine.
1993.
Synaptotagmin and neurotransmitter release.
Cell.
74:
947-950
[Medline].
|
18.
|
Elfernik, L.A.,
M.R. Peterson, and
R.H. Scheller.
1993.
A
role for synaptotagmin (p65) in regulated exocytosis.
Cell.
72:
153-159
[Medline].
|
19.
|
Geppert, M.,
Y. Goda,
R.E. Hammer,
C. Li,
T.W. Rosahl,
C.F. Stevens, and
T.C. Sudhof.
1994.
Synaptotagmin I: a
major Ca2+ sensor for transmitter release at a central synapse.
Cell.
79:
717-727
[Medline].
|
20.
|
Sudhof, T.C., and
J. Rizo.
1996.
Synaptotagmins: C2-domain
proteins that regulate membrane traffic.
Neuron.
17:
379-388
[Medline].
|
21.
|
Chapman, E.R.,
P.I. Hanson,
S. An, and
R. Jahn.
1995.
Ca2+ regulates the interaction between synaptotagmin and
syntaxin 1.
J. Biol. Chem.
270:
23667-23671
[Abstract/Free Full Text].
|
22.
|
Shao, X.,
C. Li,
I. Fernandez,
X. Zhang,
T.C. Sudhof, and
J. Rizo.
1997.
Synaptotagmin-syntaxin interaction: the C2 domain
as a Ca2+-dependent electrostatic switch.
Neuron.
18:
133-142
[Medline].
|
23.
|
Li, C.,
B. Ullrich,
J.Z. Zhang,
R.G. Anderson,
N. Brose, and
T.C. Sudhof.
1995.
Ca2+-dependent and -independent activities
of neural and non-neural synaptotagmins.
Nature.
375:
594-599
[Medline].
|
24.
|
von Poser, C.,
K. Ichtchenko,
X. Shao,
J. Rizo, and
T.C. Sudhof.
1997.
The evolutionary pressure to inactivate. A
subclass of synaptotagmins with an amino acid substitution
that abolishes Ca2+ binding.
J. Biol. Chem.
272:
14314-14319
[Abstract/Free Full Text].
|
25.
|
Lang, J.,
M. Fukuda,
H. Zhang,
K. Mikoshiba, and
C.B. Wollheim.
1997.
The first C2 domain of synaptotagmin is
required for exocytosis of insulin from pancreatic beta-cells:
action of synaptotagmin at low micromolar calcium.
EMBO
(Eur. Mol. Biol. Organ.) J.
16:
5837-5846
[Abstract/Free Full Text].
|
26.
|
Mizuta, M.,
T. Kurose,
T. Miki,
K.Y. Shoji,
M. Takahashi,
S. Seino, and
S. Matsukura.
1997.
Localization and functional
role of synaptotagmin III in insulin secretory vesicles in pancreatic beta-cells.
Diabetes.
46:
2002-2006
[Abstract].
|
27.
|
Baram, D.,
M. Linial,
Y.A. Mekori, and
R. Sagi-Eisenberg.
1998.
Ca2+-dependent exocytosis in mast cells is stimulated by
the Ca2+ sensor, synaptotagmin I.
J. Immunol.
161:
5120-5123
[Abstract/Free Full Text].
|
28.
|
Katz, H.R.,
E.T. Dayton,
S.F. Levi,
A.C. Benson,
K.F. Austen, and
R.L. Stevens.
1988.
Coculture of mouse IL-3-dependent
mast cells with 3T3 fibroblasts stimulates synthesis of globopentaosylceramide (Forssman glycolipid) by fibroblasts and surface
expression on both populations.
J. Immunol.
140:
3090-3097
[Abstract/Free Full Text].
|
29.
|
Aridor, M.,
L.M. Traub, and
R. Sagi-Eisenberg.
1990.
Exocytosis in mast cells by basic secretagogues: evidence for direct
activation of GTP-binding proteins.
J. Cell Biol.
111:
909-917
[Abstract].
|
30.
|
Shore, P.A.,
A. Burkhalter, and
V.H. Cohn.
1959.
A method
for the fluorimetric assay of histamine in tissues.
J. Pharmacol.
Exp. Ther.
127:
182-185
.
|
31.
|
Geppert, M.,
B.T. Archer III, and
T.C. Sudhof.
1991.
Synaptotagmin II. A novel differentially distributed form of synaptotagmin.
J. Biol. Chem.
266:
13548-13552
[Abstract/Free Full Text].
|
32.
|
Schwartz, L.B., and
K.F. Austen.
1980.
Enzymes of the mast
cell granule.
J. Invest. Dermatol.
74:
349-353
[Abstract].
|
33.
|
Segal, D.M.,
J. Taurog, and
H. Metzger.
1977.
Dimeric immunoglobulin E serves as a unit signal for mast cell degranulation.
Proc. Natl. Acad. Sci. USA.
74:
2993-2997
[Abstract].
|
34.
|
Rodriguez, A.,
P. Webster,
J. Ortego, and
N.W. Andrews.
1997.
Lysosomes behave as Ca2+-regulated exocytic vesicles
in fibroblasts and epithelial cells.
J. Cell Biol.
137:
93-104
[Abstract/Free Full Text].
|
35.
|
Nishimura, Y.,
K. Kato,
K. Furuno, and
M. Himeno.
1995.
Biosynthesis and processing of lysosomal cathepsin D in primary
cultures of rat hepatocytes.
Biol. Pharmacol. Bull.
18:
825-828
.
|
36.
|
Mazingue, C.,
J.P. Dessaint, and
A. Capron.
1978.
[3H]serotonin release: an improved method to measure mast cell degranulation.
J. Immunol. Methods.
21:
65-77
[Medline].
|
37.
|
Guo, Z.,
C. Turner, and
D. Castle.
1998.
Relocation of the
t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells.
Cell.
94:
537-548
[Medline].
|
38.
|
Griffiths, G.M..
1996.
Secretory lysosomes-a special mechanism of regulated secretion in haemopoietic cells.
Trends Cell
Biol.
6:
329-332
.
|
39.
|
Suarez, Q.C..
1987.
The distribution of four lysosomal integral membrane proteins (LIMPs) in rat basophilic leukemia
cells.
Tissue Cell.
19:
495-504
[Medline].
|
40.
|
Sannes, P.L., and
S.S. Spicer.
1979.
The heterophagic granules of mast cells: dipeptidyl aminopeptidase II activity and
resistance to exocytosis.
Am. J. Pathol.
94:
447-457
[Abstract].
|
41.
|
Jamur, M.C.,
I. Vugman, and
A.R. Hand.
1986.
Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in
vivo.
Cell Tissue Res.
244:
557-563
[Medline].
|
42.
|
Raposo, G.,
D. Tenza,
S. Mecheri,
R. Peronet,
C. Bonnerot, and
C. Desaymard.
1997.
Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.
Mol. Biol.
Cell.
8:
2631-2645
[Abstract/Free Full Text].
|
43.
|
Kim, T.D.,
G.T. Eddlestone,
S.F. Mahmoud,
J. Kuchtey, and
C. Fewtrell.
1997.
Correlating Ca2+ responses and secretion in individual RBL-2H3 mucosal mast cells.
J. Biol.
Chem.
272:
31225-31229
[Abstract/Free Full Text].
|
44.
|
Littleton, J.T.,
M. Stern,
M. Perin, and
H.J. Bellen.
1994.
Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin
mutants.
Proc. Natl. Acad. Sci. USA.
91:
10888-10892
[Abstract/Free Full Text].
|
45.
|
Fox, C.C.,
S.D. Jewell, and
C.C. Whitacre.
1994.
Rat peritoneal mast cells present antigen to a PPD-specific T cell line.
Cell. Immunol.
158:
253-264
[Medline].
|