GLYCOPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS IN PARAMECIUM TETRAURELIA : POSSIBLE ROLE IN CHEMORESPONSE
University of Vermont, Department of Biology, Burlington, VT 05405,
USA
*
Author for correspondence (e-mail:
jvanhout{at}zoo.uvm.edu
)
Accepted May 30, 2001
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Summary |
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Antisera against the proteins removed by the salt/ethanol washing procedure include antibodies against large surface antigens, which we confirm in this species to be GPI-anchored, and against an array of proteins of smaller molecular mass. These antisera specifically block the chemoresponse to some stimuli, such as folate, which we suggest are signaled through GPI-anchored receptors. Responses to cyclic AMP, which we believe involve an integral membrane protein receptor, and to NH4Cl, which requires no receptor, are not affected by the antisera. Antiserum against a mammalian GPI-anchored folate-binding protein recognizes a single band among the GPI-anchored salt and ethanol wash proteins. The same antiserum specifically blocks the chemoresponse to folate.
Key words: receptor, chemosensory, Paramecium tetraurelia, GPI anchor, signal transduction, folate
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Introduction |
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GPI-anchored proteins have been well characterized as coat proteins from
parasitic protozoa such as trypanosomes (McConville and Ferguson,
1993), and their function in
these cells may include protection and camouflage from the immune system of
the host. Free-living protozoa, such as Paramecium, also have
GPI-anchored surface proteins, but the functions of these proteins in the life
history of the cells is not yet clear. On the surface of Paramecium
spp. are extremely large and glycosylated proteins (`immobilization' antigens,
referred to here as surface antigens). These make a coating on the cell that
is so prevalent as to appear to be `fuzzy' when seen in the transmission
electron microscope (Ramanathan et al.,
1981
). Such a large amount of
protein could perhaps be a form of metal-ion or pH buffering for cells that
live in extremes of ionic strength in freshwater habitats. Regardless of their
function, they are plentiful surface proteins and have been demonstrated to be
GPI-anchored in P. primaurelia (Capdeville et al.,
1987
).
In our search for chemoreceptor proteins among the surface proteins of
P. tetraurelia, we are exploring the GPI-anchored proteins in part
because of their characteristic insolubility in vertebrate systems (Parton and
Simons, 1995; Brown and
London, 1997
). Mammalian
GPI-anchored proteins, such as folate receptors, are found in caveolae (Wu et
al., 1997
; Ying et al.,
1992
) or membrane domains
(detergent-insoluble domains) (Futerman,
1995
; Schnitzer et al.,
1995
; Parton and Simons,
1995
; Brown and London,
1997
), which are characterized
as enriched in phosphoglycosphingolipids and cholesterol, and in many proteins
that are associated with signal transduction (Nosjean et al.,
1997
). Our experience with
Paramecium tetraurelia chemoreceptors is that some are highly
insoluble in Triton X-100 (Sasner and Van Houten,
1989
), indicating that they
may be GPI-anchored proteins. It was necessary to explore some aspects of the
surface of P. tetraurelia before we could search for putative
GPI-anchored chemoreceptors. As we describe below, we have demonstrated that
the A and B large surface antigens of P. tetraurelia, like those of
P. primaurelia, are GPI-anchored, since they can be released,
together with the cleaved GPI anchor, using an exogenous
phosphoinositol-specific phospholipase C (PI-PLC).
The large surface antigens of Paramecium spp. are known to be
liberated from the surface by washing cells in high concentrations of salt and
ethanol (Preer et al., 1981),
which would provide an efficient method of removing and harvesting
GPI-anchored receptors. We demonstrate here that a salt and ethanol wash of
P. tetraurelia contains an enrichment of the A and B large surface
antigens and an array of other GPI-anchored proteins and that there is an
endogenous, probably constitutive, phospholipase that cleaves the GPI anchor.
However, the salt/ethanol treatment does not seem to require a lipase to
release some proteins from the cell surface because these proteins have no
anchor or an intact one. Triton X-114 phase-partitioning has been useful in
the solubilization of GPI-anchored proteins, and we discuss here the
solubility of cell membrane proteins in Triton X-114. Two known integral
membrane proteins, the plasma membrane Ca2+ pump and the cyclic AMP
receptor, are not found among the salt/ethanol- or PLC-released proteins.
In this study, we also use antisera produced against salt/ethanol wash proteins to block a subset of chemoresponses, which we believe to be receptor-mediated. In addition, antiserum against a mammalian GPI-anchored folate-binding protein recognizes a GPI-anchored protein among the salt/ethanol wash proteins, and the same antiserum specifically blocks the chemoresponse to folate.
We anticipate that the results of these studies will generate interest in GPI-anchored proteins as receptors or partners of receptors in chemical sensing. The methods used for efficiently harvesting the proteins from the cell surface may also be more generally applicable.
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Materials and methods |
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Pellicle preparation
Surface membranes (pellicles) of cells were prepared from 61 of culture
using a modification of a protocol published previously (from Bilinski et al.,
1981). The final pellet was
resuspended in 2 ml of homogenization buffer and stored at -70°C for up to
1 week (Van Houten et al.,
1991
).
Phospholipase treatments
Frozen pellicle membranes (9 mg) were thawed on ice and brought to 1 ml
with 5 mmoll-1 KCl buffer (in mmoll-1: 5 KCl, 1.3 Tris
base, 1 Ca(OH)2, 1 citric acid, pH 7.0). This low-ionic-strength
buffer was used in anticipation of treating whole cells with PLC and because
whole cells require a buffer comparable with this one. The sample was divided
in two, one half for bacterial phosphoinositol-specific phospholipase C (PLC;
Sigma) treatment and the other for sham treatments. A 500 µl sample was
used to solubilize 5 units of lyophilized PLC and was incubated for 20 min at
37°C (or just incubated at 37°C for sham treatment) before
centrifugation at 11 300g in a Beckman microcentrifuge for 15
min at 4°C. The supernatants were either collected and concentrated to 200
µl with Amicon Centricon 10 concentrators or precipitated with acetone and
washed twice with acetone before resuspension in sample buffer for sodium
dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).
Salt/ethanol washes
Cultures (3 l) of cells grown in wheat extract medium (Wright et al.,
1990) were filtered,
centrifuged at 350g in an IEC-HNSII centrifuge and resuspended
in 3 ml of bacterium-free culture medium. Salt/ethanol stock solution (1.8
ml), consisting of 10 mmoll-1 Na2HPO4, 150
mmoll-1 NaCl and 30 % ethanol (Preer et al.,
1981
), was added to the
sample, which was kept on ice for 1 h. The sample was then centrifuged at
551g in a Beckman J2-21 centrifuge and JA-17 rotor for 5 min
at 4°C, and the supernatant was collected as described above.
Phospholipase inhibitor treatment
Small-scale salt/ethanol washes were carried out in the presence of
PLC-specific inhibitors. A culture of cells (11) was centrifuged at
350g, and the pellet was resuspended in 1 ml of sterile
culture fluid. To 200 µl samples of the resuspended cells were added 20
µl each of 10 mmoll-1
2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; dissolved in
ethanol) and 10 mmoll-1 phenylmethylsulfonyl fluoride (PMSF;
dissolved in isopropanol), to give a final concentration of 1
mmoll-1 for each inhibitor, or 17 µl of a 100 mmoll-1
stock of p-chloromercuriphenylsulfonic acid (PCMPSA dissolved in
water), to give a final concentration of 1 mmoll-1. Neomycin,
another reported PLC inhibitor, was also used at a final concentration of 1
mmoll-1. (Results are not shown for the neomycin treatment, since
this produced no change in the effects of PLC.) A stock of salt/ethanol (10
mmoll-1 Na2HPO4, 150 mmoll-1 NaCl
and 30 % ethanol) was added to bring the volume to 300 µl and to a
salt/ethanol concentration of 3-4 mmoll-1
Na2HPO4, 50-70 mmoll-1 NaCl and 10-14 %
ethanol). Samples were incubated on ice for 1 h and centrifuged at 11
300g in a Beckman microcentrifuge for 5 min at 4°C. The
supernatants were collected.
Triton X-114 phase extraction
A sample of pellicle membranes (500 µl; 3-17 mg) was incubated with 5
µl of 10 % Triton X-114, which had been preconcentrated with Tris-buffered
saline (TBS, 20 mmol1-1 Tris, 137 mmol1-1 NaCl, pH 7.6)
at 30°C overnight to remove contaminants (Bordier,
1981). The Triton/pellicle
mixture was incubated at 4°C with gentle mixing for 2 h. The sample was
then centrifuged at 11 300 g for 30 min at 4°C. The
supernatant was layered over 100 µl of 6 % sucrose in TBS and incubated at
37°C for 4 min. The samples were then centrifuged for 2 min at 11 300
g at room temperature (21-22°C), and the aqueous phase and
detergent pellet were collected. The separate layers were precipitated with 1
ml of cold acetone (4°C) and washed three times in cold acetone. The
samples were then resuspended in 20 µl of sample buffer for loading onto 8%
acrylamide gels and eventual transfer to nitrocellulose.
SDSPAGE and western blots
Homogeneous and gradient SDS/polyacrylamide gels (for details of the
percentage of acrylamide used, see figure legends) with 3 % stacking gels were
used to separate proteins electrophoretically in a Hoeffer Tall Mighty Small
apparatus (Laemmli, 1970).
The proteins separated in the SDS gels were electroblotted onto
nitrocellulose membrane (Gelman Sciences) (according to Towbin et al.,
1979) with a modified transfer
buffer (15.6 mmol1-1 Tris-HCl, 120mmol1-1 glycine, 20 %
methanol) (according to Gershoni and Palade,
1982
). Membranes were either
stained for total protein with 0.1% Amido Black in 50 % methanol and 10 %
acetic acid or probed with polyclonal antiserum and anti-rabbit secondary
antibody conjugated with alkaline phosphatase (according to McGadey,
1970
) after blocking with
dried milk (Thean and Toh,
1989
). The antisera used were
anti-A and anti-B developed against salt/ethanol washes of cells expressing
either the A or B surface antigen (kind gifts from J. Forney),
anti-cross-reactive-determinant (CRD) of the GPI anchor (anti-CRD, Oxford
GlycoSystems) or anti-folate-binding-protein (bovine, Biogenesis, Poole,
England).
T-maze assays of chemoresponse behavior
D4-12-144 cells were assayed for chemoresponse using T-mazes, as described
previously (Van Houten et al.,
1982) with the following
modifications. Cells are allowed to distribute between two arms of a glass
stopcock, which is the T-maze. The two arms of the T-stopcock contain buffers
that differ by one component, e.g. acetate in test solution and chloride in
the control arm. After 30 min, the cells are counted, and the index of
chemokinesis, Iche, is calculated as the number of cells
in the test arm divided by the total number of cells in both arms. Indices
above 0.5 indicate attraction to the test solution; indices below 0.5 indicate
repulsion. A sample of cells (100 ml) was centrifuged at 350
g, and the pellet was resuspended in chemokinesis buffer [1
mmol1-1 Ca(OH)2, 1.3 mmol1-1 Tris base, 1
mmol1-1 citric acid, and the salt indicated, usually 5
mmol1-1 KCl or NaCl] and centrifuged again. The resulting pellet of
cells was resuspended in 2 ml of buffer and divided into 1 ml samples. To one
of the samples, 4 or 8 µl of each antiserum against salt/ethanol washes of
A or B cells (anti-A and anti-B sera) was added to one of the cell samples;
pre-immune serum from a different set of rabbits was added to the second
sample as control. (Although this is not the best control, we had no other
pre-immune serum to use. A pre-immune serum from different rabbits was used at
the same dilution in a selection of T-mazes for a different purpose, and there
was no difference between the results with pre-immune treatment and
sham-treated controls; W. E. Bell, personal communication.) After 30 min, the
cells were used in T-maze assays without further washing.
In other T-maze tests, D4-12-144 cells were concentrated as above, resuspended in 2x0.5 ml samples of 5 mmol1-1 NaCl buffer and incubated for 1 h with or without 10 µl of anti-folate-binding-protein antiserum. Cells were then immediately tested in T-mazes for 20 min.
Immobilization tests to verify the serotypes of the cells
Cells were transferred to Dryl's solution (Dryl,
1959) in a glass depression
slide using a micropipette. Ten cells were then transferred to 300 µl of
Dryl's solution with 0.6 µl of anti-A or anti-B serum. The cells were then
observed for motility. Cells rendered immotile by anti-A serum, for example,
were expected to be expressing only anti-A surface antigen (Preer,
1959
). The majority of cells,
which were grown at 28°C, expressed antigen A, as expected for this
condition. The remaining cells appeared to be expressing antigen B.
Antiserum against the C terminus of the plasma membrane Ca2+
pump was prepared in rabbits against a synthetic peptide based on the last 17
amino acid residues of the plasma membrane Ca2+ pump clone (Elwess
and Van Houten, 1997). The
peptide was conjugated to keyhole limpet hemocyanin and used as an immunogen
in rabbits (Quality Controlled Biochemicals, Hopkinton, MA, USA). The
antiserum was affinity-purified. The specificity of binding to western blots
of the bacterially expressed C terminus (10 kDa) of the pump was determined by
comparison of results with and without preabsorption of the antiserum with
excess peptide (Yano et al.,
1997
). The antiserum is
referred to as anti-cbd in Fig.
6 because the C terminus of the pump, used in the fusion protein
as antigen, is the calmodulin-binding domain
(CBD).
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Antiserum against 15 amino acid residues of the N terminus of the cyclic
AMP receptor (Van Houten et al.,
1991) was prepared by Lampire
Biological Laboratories (Pipersville, PA, USA) in rabbits using a
glutathione-S-transferase (GST) fusion protein expressed from pGEX
(Strategene) vector in BL21 cells. To control for recognition of GST, we
preabsorbed the antiserum with expressed bacterial GST both on nitrocellulose
strips and in solution. There still remained some recognition of GST on
western blots, as can be see in Fig.
6.
Cilia for phase contrast microscopy were prepared as described by Eisenbach
et al. (Eisenbach et al.,
1983).
The Pierce protein microassay was used for photometric analysis of protein using bovine serum albumin (BSA) as standard.
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Results |
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GPI-anchored proteins are recognized by an anti-CRD antibody only when the
anchor is cleaved because only then is the epitope revealed. The anti-A and
anti-B antisera were originally used to identify the serotype of P.
tetraurelia, i.e. the surface antigen that is expressed on the cells.
There are 12 surface antigens, and cells express exclusively only one of the
genes (Preer et al., 1981;
Schmidt, 1988
). Historically,
a simple wash of the whole cells with salt and ethanol (Preer et al.,
1981
) was used to prepare
antisera to surface antigens and, since these surface proteins are very
immunogenic, the antisera contained a high titer of anti-surface-antigen
antibodies. Hence, the antisera could immobilize cells expressing that
specific antigen. We will refer to these antisera as anti-A and anti-B because
of the cell types that were washed with salt/ethanol to produce them, but they
also contain antibodies to proteins other than the surface antigens.
Cells exclusively express only one surface antigen at a time, and we
expected that most of the cells grown at 28°C would express antigen A, the
balance expressing antigen B (Beale,
1954; for a review, see
Schmidt, 1988
). Cells become
immobilized when exposed to antiserum prepared against the salt/ethanol washes
containing the antigen they are expressing and, using this immobilization
test, we verified that most of the cells were expressing antigen A and the
balance antigen B (results not shown).
In Fig. 1A, proteins from the pellet or supernatant of PLC-treated or sham-treated pellicle are visualized with a mixture of anti-A and anti-B antisera; in Fig. 1B, the same proteins are visualized with anti-CRD antiserum. In sham-treated pellicles, the large surface antigen remained in the pellet, as did many proteins in the 40-60 kDa range (Fig. 1A, lanes 1 and 2). Treatment with PLC shifted the surface antigen from the pellet to the supernatant, together with other proteins of lower molecular mass (Fig. 1A, lanes 3 and 4). In Fig. 1B, we see that only after PLC treatment is the GPI anchor recognized on proteins and that these reactive proteins are almost exclusively in the supernatant from the PLC treatment. Note that the filled arrows show the surface antigens, which are among the proteins in the supernatant and have a cleaved GPI anchor after PLC treatment. Thus, exogenous PLC does release GPI-anchored proteins from the pellicle. (To show the large surface antigen clearly, we used 8 % gels, which did not provide good resolution of the lower-molecular-mass proteins. These are better displayed in other figures.)
To demonstrate in a different way that PLC was responsible for the protein release shown in Fig. 1, we incubated pellicles with and without lipase at 0°C to inhibit any endogenous and exogenous enzymes, and compared this with incubations at 37°C (Fig. 2). Note in Fig. 2A (lanes 1-3) that proteins are released from the pellicle at both 0 and 37°C, but that only at 37°C with PLC (lane 4) are the surface antigens and lower-molecular-mass proteins enriched. Also, only the PLC-cleaved proteins (Fig. 2B, lane 4) show a GPI anchor epitope recognized by the anti-CRD antibody.
|
Many of the proteins released by salt/ethanol washes are
GPI-anchored
Whole cells in sterile culture fluid were washed in a salt/ethanol mixture,
as described in Materials and methods. The proteins from the wash were
compared with the profiles of proteins released from the cell surface
pellicles with PLC using immunoblots with anti-A and anti-B sera.
We have focused on two sets of proteins among those released by PLC and salt/ethanol from the surface of P. tetraurelia, i.e. the surface antigens and the cluster of proteins ranging in mass from 40 to 60 kDa. PLC and salt/ethanol treatment release proteins of similar molecular masses and, like the proteins in the salt/ethanol washes, the proteins released by PLC are recognized by anti-A and anti-B antisera (Fig. 3A, lanes 2 and 5) and many are detected with the anti-CRD antiserum that identifies GPI-anchored proteins (Fig. 3B, lanes 2 and 5).
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Because some GPI-anchored proteins are liberated with a cleaved anchor epitope, a lipase rather than a protease seems to be active during the release of proteins with the salt/ethanol treatment. Furthermore, inhibition of PLC and the lipases that are active in salt/ethanol reduces the quantities of proteins recognized by anti-CRD antiserum (see below). Therefore, GPI-anchored proteins are among those released by PLC and salt/ethanol treatment, including the surface antigens of P. tetraurelia.
To examine whether salt/ethanol treatment solubilized proteins had been
cleaved by an endogenous lipase, we included PLC inhibitors in both the
exogenous PLC treatment (as control) (Fig.
3A, lanes 3 and 4) and salt/ethanol washes
(Fig. 3A, lanes 6 and 7). The
PLC inhibitors neomycin (Lipsky and Lietman,
1982), PMSF (Ohia and
Jumblatt, 1993
), NCDC (Jino et
al., 1994
; Clark and Garland,
1993
) and PCMPSA (Capdeville
and Benwakrim, 1996
; Low,
1987
) were used, but the
results with neomycin are not shown because it had no inhibitory effect on
protein release. These results are not unexpected since it has been reported
that neomycin can actually activate PI-PLC in other cell types (Morris et al.,
1996
) and has unpredictable
effects on the Tetrahymena GPI system
(Kovács and Csaba,
1996
).
Individually, PMSF and NCDC produce no inhibition of cleavage by exogenous PLC (results of individual tests are not shown), but NCDC and PMSF in combination reduce levels of the surface antigen and other proteins released by bacterial PLC treatment of Paramecium pellicle (Fig. 3A, lane 2 versus lane 3; also see Fig. 5A, lane 1 versus lane 2). The effect of inhibitor on this exogenous PLC is more evident when examining the proteins released with cleaved GPI anchors and, therefore, recognized by the anti-CRD antibodies (Fig. 3B, lane 2 versus lane 3). The combination of NCDC and PMSF has little effect on salt/ethanol wash profiles (Fig. 3A,B, lane 5 versus lane 6). Thus, the conventional PLC inhibitors affect the exogenously added PLC but not the endogenous lipase of P. tetraurelia.
|
The opposite effect is shown by PCMPSA, which inhibits the endogenous but not the exogenous lipase. This inhibitor affects exogenous PLC treatment profiles of pellicle proteins only slightly (Fig. 3, lane 2 versus lane 4). Pellicle proteins released by PLC even in the presence of this inhibitor retain the epitope recognized by anti-CRD antibody, i.e. they have a cleaved GPI anchor (Fig. 3A,B, lanes 4). The inhibitor PCMPSA more effectively reduces, but does not eliminate, the release of proteins from whole cells with a salt/ethanol wash (Fig. 3A, lane 5 versus lane 7). Some proteins that are released by a salt/ethanol treatment are recognized by the anti-CRD antiserum, but when the inhibitor PCMPSA is included in the salt/ethanol wash fewer proteins are released (Fig. 3A, lane 5 versus lane 7) and they are not recognized by the anti-CRD antibody, i.e. they either have intact GPI anchors or never had one (Fig. 3B, lane 5 versus lane 7). Thus, PCMPSA inhibits the endogenous lipase of P. tetraurelia, but has little effect on exogenously added bacterial PLC.
Ethanol treatment is known to affect membrane protein function (Schultz et
al., 1997), perhaps by
solubilization of some membrane proteins. We do not believe solubilization to
be the major contributor to the appearance of proteins in the salt/ethanol
wash because of the differences between the proteins in
Fig. 3A (lanes 6 and 7) and
Fig. 3B (lanes 6 and 7). If
solubilization and not lipase activity were responsible for the liberation of
the membrane proteins, the proteins in lanes 6 and 7 should be the same in
quantity and pattern, but they are not. Solubilization would not provide an
explanation for the loss of the anti-CRD epitope in
Fig. 3B (lane 7). We consider
the proteins in Fig. 3A (lane
7) to be those solubilized by the salt/ethanol treatment and to consist of
GPI-anchored proteins with the intact anchor attached and peripheral proteins
that never had a GPI anchor (Fig.
3B, lane 7). These same proteins will probably be included in
lanes 5 and 6 of Fig. 3 as
well.
A trivial explanation for the appearance of the surface antigens and 40-60
kDa proteins in the salt/ethanol wash could be the inadvertent deciliation and
collection of cilia instead of cleaved proteins in the wash. Ethanol (5 %) is
commonly used to deciliate cells, making this a concern. However, the high
salt concentration and significantly higher ethanol concentration (15-20 %)
used in the salt/ethanol appears to fix the cells, and cilia remain attached
(Fig. 4). We have also examined
our pellicle preparation for contamination with cilia using phasecontrast
microscopy. Although we can identify cilia in preparations designed to harvest
cilia (Eisenbach et al.,
1983), we see only an
occasional structure that could be a cilium in the pellicle preparation (data
not shown). This result is expected from the low speeds used to pellet sheets
of pellicle surface membranes (3000g).
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Triton X-114 treatment to solubilize GPI-anchored proteins
Glycophosphatidylinositol (GPI)-anchored proteins are notoriously insoluble
in Triton X-100, but can be solubilized and analyzed by phase partitioning in
Triton X-114 (Ko and Thompson,
1995). Proteins with an intact
GPI anchor would be enriched in the detergent phase (approximately 11 %),
while those with no anchor or a cleaved anchor would be enriched in the
aqueous phase, which still retains some detergent (approximately 0.06 %;
Bordier, 1981
). P.
tetraurelia pellicles were extracted with Triton X-114, the extracts were
separated into detergent and relative aqueous phases, and the proteins were
analyzed by SDSPAGE and western blotting using anti-A and anti-B
antisera (Fig. 5A) and anti-CRD
antiserum (Fig. 5B).
Fig. 5 shows the proteins
released by PLC treatment of pellicle membranes without
(Fig. 5A,B, lanes 1) and with
(Fig. 5A,B, lanes 2) PLC
inhibitors for comparison with the aqueous and detergent phases of the Triton
X-114 extraction and phase separation without PLC treatment
(Fig. 5A,B, lanes 3 aqueous,
lanes 4 detergent phase, respectively). Note that the profile of proteins
released by PLC is similar to that (focusing on surface antigens and the 40-60
kDa range) of the relative aqueous phase of the Triton X-114 extract whether
probed with anti-A and anti-B or anti-CRD antiserum
(Fig. 5A,B, lanes 1
versus lanes 3). Much less protein is typically found in the
detergent phase than in the aqueous phase
(Fig. 5A, lane 3
versus lane 4). Notably, there is less surface antigen in the
detergent phase.
Anti-CRD antiserum recognizes only the cleaved GPI-anchored proteins and, therefore, it was expected that hydrophobic proteins with intact GPI anchors would be found in the detergent phase and would not react with the anti-CRD antiserum. However, we found little of the surface antigen in the detergent phase as detected by the anti-A and anti-B antisera (Fig. 5A, lane 4), and none of it appeared to react with the anti-CRD antibody (Fig. 5B, lane 4). It was unexpected that so many of the GPI-anchored proteins should be found in the aqueous phase, with GPI anchors cleaved (Fig. 5A,B, lanes 3). These Triton X-114 extraction studies provide new information about the surface proteins of Paramecium, and of P. tetraurelia in particular.
Integral membrane proteins are not among the GPI-anchored
proteins
A surface membrane protein, the Ca2+ pump, should be an integral
membrane protein (Wright and Van Houten,
1990; Elwess and Van Houten,
1997
) and, therefore, not
among the GPI-anchored proteins liberated by PLC, by a salt/ethanol wash or in
the Triton X-114 aqueous phase. The cyclic AMP chemoreceptor is readily
soluble in Triton X-100 and is also likely to be an integral membrane protein
(Van Houten et al., 1991
). We
examined the supernatant from PLC treatment of pellicle and the salt/ethanol
wash for these proteins using antibodies to the C terminus of the
Ca2+ pump and the N-terminal sequence of the cyclic AMP receptor
(see Materials and methods). A bacterially expressed protein of approximately
10 kDa of the C terminus of the Ca2+ pump fused with GST and the
GST fusion protein from the N terminus of the cyclic AMP receptor were used as
positive controls for the antisera in western blots
(Fig. 6). The anti-A and anti-B
antisera reacted with an array of bands in the PLC supernatant, but not with
the fusion proteins (Fig. 6A,
lane 1 versus lanes 2 and 3).
The antiserum against the Ca2+ pump peptide and cyclic AMP receptor fusion protein do not react with any proteins from PLC- or salt/ethanol-treated cells (Fig. 6B, lanes 1 and 2; Fig. 6C, lanes 1 and 3), indicating that these integral membrane proteins are probably not present in the PLC supernatant or salt/ethanol wash. Similarly, proteins in the supernatants from the sham- and PLC-treated pellicles do not react with anti-Ca2+-pump (Fig. 6B, lanes 1 and 2) or anti-cyclic-AMP receptor (Fig. 6B, lanes 3 and 4) antiserum. Even though sufficient protein is present to produce a strong reaction with anti-CRD antibodies (Fig. 6B, lane 6).
Proteins from salt/ethanol washes also fail to react with anti-Ca2+-pump and anti-cyclic-AMP receptor antisera (Fig. 6C, lanes 1 and 3) on western blots, even though sufficient protein is present to produce a reaction with anti-A and anti-B antisera (Fig. 6C, lane 2).
Positive and negative controls for the antiserum against the Ca2+ pump and cyclic AMP receptor are shown in Fig. 6A. The anti-A and anti-B antisera recognize proteins in the supernatant of PLC-treated cells (Fig. 6A, lane 1), but the anti-Ca2+-pump and anti-cyclic-AMP-receptor antisera do not recognize proteins from the supernatant (Fig. 6A, lanes 2 and 3). In contrast, the anti-Ca2+-pump and anti-cyclic-AMP-receptor antisera recognize their antigens because the anti-Ca2+-pump polyclonal antiserum reacts with the Ca2+ pump C terminus fusion protein (filled arrow, Fig. 6A, lane 4), and the anti-cyclic-AMP-receptor polyclonal antiserum reacts with the receptor fusion protein (open arrow, Fig. 6A, lane 5). In addition, Fig. 6B (lane 6) demonstrates that there is sufficient protein in the PLC supernatant for recognition by anti-CRD antibody. Likewise, Fig. 6C (lane 2) shows that there is sufficient protein in the salt/ethanol wash to be recognized by the anti-A and anti-B antisera. Therefore, low antibody titers or insufficient protein loaded on the gels cannot account for the lack of recognition of the Ca2+ pump (133 kDa) or the cyclic AMP receptor (48 kDa) among the proteins of the PLC supernatant and salt/ethanol wash by the anti-Ca2+-pump and anti-cyclic-AMP-receptor antisera in Fig. 6B (lanes 2 and 4) and Fig. 6C (lanes 1 and 3).
In another set of positive controls (not shown), we demonstrated that the antisera can detect the Ca2+ pump and cyclic AMP receptor proteins in the whole pellicle at the dilutions used in the experiments shown in Fig. 6. We found that the antiserum against the Ca2+ pump reacts specifically with a protein with a molecular mass of approximately 133 kDa, while the antiserum against the cyclic AMP receptor reacts specifically with a protein of molecular mass 48 kDa in pellicle. We determined specificity by observing the disappearance of bands when the antiserum was preincubated with the antigens against which they had been made.
T-maze tests of cells treated with anti-A and anti-B antisera
The salt/ethanol wash of cells clearly contains many GPI-anchored proteins
in addition to the surface antigen, because many proteins that react with the
anti-A and anti-B antisera also react with the anti-CRD antiserum. It is
unlikely that integral proteins are found among the salt/ethanol wash
proteins, except as a result of proteolytic cleavage. Therefore, we
hypothesized that the anti-A and anti-B antisera could contain blocking
antibodies to the surface chemoreceptors that govern attraction to stimuli
such as acetate, folate, glutamate, cyclic AMP and biotin if these receptors
were peripheral or GPI-anchored. All these stimuli appear to bind to specific
sites, probably receptors, at the cell surface to mediate attraction, while
ammonium does not (Van Houten,
1994). We therefore tested
whether anti-A and anti-B antisera mixtures could block the chemoresponse in
mutants lacking the A and B antigens (courtesy of J. Forney). These cells
would not be immobilized by the antisera, but might show behavioral defects if
their chemoreceptors to specific stimuli were blocked by the antisera. As
shown in Table 1, the antisera
produce significant inhibition of the response to folate, while the responses
to acetate and ammonia were unaffected, showing that this is not a general
effect on motility in the T-mazes. The A-B- cells
show a definite attraction, but a more variable response, to glutamate than
wild-type cells (index of chemokinesis, Iche,
0.70±0.07, mean ± S.D., N=8-12). This variability makes
it difficult to determine whether the decrease in glutamate
Iche with antibody treatment indicates that a GPI-anchored
protein is involved in the glutamate chemoresponse. Responses to cyclic AMP
and biotin (not shown) are affected, but only at higher concentrations of the
antisera than are required to block the response to folate or glutamate, and
the response to biotin is only partially reduced.
|
Antisera to a mammalian GPI-anchored folate-binding protein
Mammalian folate-binding proteins are GPI-anchored and mediate the uptake
of folate into cells (Antony,
1996). An antiserum to bovine
folate-binding protein recognized one protein band of approximately 37 kDa
among salt/ethanol wash proteins (Fig.
7, lane 1). Probing of blots with anti-CRD antiserum shows a band
of similar size among the GPI-anchored proteins on the blot
(Fig. 7, lane 2). (Repeated use
of anti-folate-binding-protein antiserum with salt/ethanol washes has
confirmed that one protein of approximately 37 kDa is recognized.)
|
Subsequent use of this antiserum in a pre-incubation of the cells before T-maze tests blocks the chemoresponse to folate (Table 2). Chemoresponses to other stimuli do not appear to be affected by the antiserum treatment.
|
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Discussion |
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As anticipated from work on P. primaurelia (Capdeville et al.,
1987), the traditional method
of harvesting the surface antigens for making antisera using a salt/ethanol
wash of the cells also releases an array of proteins (particularly proteins of
40-60 kDa) that react with the anti-CRD antiserum
(Fig. 3). These proteins can be
slightly reduced in quantity using the same lipase inhibitors that inhibit the
exogenous PLC used in these experiments
(Fig. 3,
Fig. 4), and many of these
proteins show reactivity with the anti-CRD antiserum. However, another PLC
inhibitor (PCMPSA), which does not greatly affect exogenous PLC, reduces the
amount of protein in the wash and inhibits cleavage of the GPI anchor. These
proteins of 40-60 kDa are not simply degradation products of the surface
antigens; they cross-react with antisera from cells with surface antigens
other than A and B (Eisenbach et al.,
1983
) and, in addition,
salt/ethanol washes of A-B- cells show this same array
of proteins when visualized with anti-A and anti-B antisera (C. A. Paquette,
V. Rakochy, A. Bush, and J. L. Van Houten, unpublished observations).
We demonstrated by examining cells sham-treated in Dryl's solution and treated with salt/ethanol mixtures that the appearance of the surface antigen in the salt/ethanol washes of cells was not the result of accidentally harvesting cilia, which are enriched in membrane and the surface antigen proteins. We found that the cells from both treatments appeared intact and there was no measurable loss of cilia, as determined by visual inspection of fixed cells using phase microscopy (Fig. 4).
It was anticipated from the work of Capdeville et al. (Capdeville et al.,
1993) on ciliary proteins of
P. primaurelia that an endogenous lipase could be activated by
salt/ethanol treatment. However, we have demonstrated here that, for P.
tetraurelia, not only are more proteins with cleaved GPI anchors found in
salt/ethanol washes than in buffer washes but that, upon Triton X-114
extraction and phase separation, the GPI-anchored proteins are found primarily
in the aqueous phase with their anchors cleaved. It is interesting that
constitutive endogenous lipases are found in other systems, such as in
trypanosomes (McConville and Ferguson,
1993
). It appears, therefore,
either that both salt/ethanol and Triton X-114 activate a surface lipase or
that P. tetraurelia also has a constitutive lipase. At present, we
are unable to distinguish between these possibilities.
The anti-A and anti-B antisera produced against salt/ethanol washes of
cells react with proteins that are liberated from the cell surface with
exogenous PLC or with salt/ethanol washes
(Fig. 3). Subsets of these
proteins (particularly the surface antigens and 40-60 kDa proteins) react with
the anti-CRD antiserum (Fig.
3). While this is far from proof of congruence between the two
protein profiles, it is at least suggestive that the anti-A and anti-B
antisera react with many GPI-anchored proteins. If there are other proteins
present in the salt/ethanol wash of whole cells, and hence available as
antigens for the antisera, they are likely to be peripheral proteins and not
integral membrane proteins. We reasoned that the anti-A and anti-B antisera
could contain blocking antibodies for chemoreceptors and found that these
antisera block chemoresponse behavior in T-mazes, but not responses to
all stimuli. The response to ammonium, which we believe crosses the membrane
without the assistance of a receptor (Davis et al.,
1998), is not affected by
antiserum treatment, while the responses to folate and glutamate are always
reduced to neutral or near neutral (Table
1). Other responses that we believe are membrane-receptor-mediated
either are not affected (as in the case of acetate) or are affected only at
high antiserum concentrations (cyclic AMP) and to a lesser extent (biotin)
(data not shown). It is possible that these last cases are indicative of
receptors that are not GPI-anchored and that their receptors are not among the
proteins found in the salt/ethanol wash. Alternatively, the antibody titers
may be too low to have a blocking effect on these specific receptors. These
possibilities cannot be resolved until we purify the receptors. The cyclic AMP
receptor, a Triton-X-100-soluble protein, has been purified to homogeneity,
and antibodies against it do not react with any of the proteins in blots of
salt/ethanol washes, supporting the view that this receptor is not a
GPI-anchored receptor (Fig.
6).
The antiserum against a bovine GPI-anchored, folate-binding protein is better defined than the anti-A and anti-B sera and provides better evidence for the participation of a GPI-anchored protein in chemoresponses. A single protein from salt/ethanol washes is the primary protein recognized by this antiserum. Moreover, a protein that shows similar migration on gels is reactive with the anti-CRD antibody, indicating that it is GPI-anchored. The same antiserum very specifically and thoroughly blocks the chemoresponse to folate in T-mazes. While these data are not conclusive, they nonetheless suggest that the P. tetraurelia chemoreceptor for folate is a GPI-anchored protein.
Previous observations have been made (Ramanathan et al.,
1981; Ramanathan et al.,
1983
; Eisenbach et al.,
1983
) about the effects of
antisera against P. tetraurelia ciliary surface proteins, including
the surface antigen and a cluster of proteins of lower molecular mass. There
are some distinct differences between these earlier observations and those
presented here. We have compared proteins from the pellicle (cell body surface
membranes) and whole-cell salt/ethanol washes, while the previous studies
focused on ciliary membrane proteins. Antisera in the previous studies were
prepared against these ciliary proteins using membrane preparations as
antigens rather than salt/ethanol washes (Ramanathan et al.,
1983
; Eisenbach et al.,
1983
). Nevertheless, there are
distinct similarities between the pattern of proteins cleaved by PLC treatment
of pellicle and salt/ethanol washes of whole cells and these earlier protein
profiles. In short, we are probably observing many of the same proteins using
different antisera. Insight into GPI-anchored proteins also helps to explain
the effects of the antisera against ciliary surface proteins on a membrane
Ca2+ conductance (see below).
The recent cloning by complementation of a subunit of the Ca2+
channel of P. tetraurelia introduced the possibility of further roles
for GPI-anchored proteins (Haynes et al.,
1998). This subunit appears to
be GPI-anchored, on the basis of its deduced amino acid sequence. A trivial
explanation for the absence of chemoresponse to chemical stimuli could be the
inhibition of this channel rather than blockade of chemoreceptors by the
anti-A and anti-B antisera, since an ability to reverse swimming direction in
response to Ca2+-based action potentials is required for
chemoresponsiveness (Van Houten,
1978
). Inhibition of the
Ca2+ channel would cause a global effect on attraction
chemoresponses. However, since the inhibition of chemoresponses by the
antisera is selective, and not all responses to stimuli are affected, as would
be expected if the Ca2+ channel were inhibited, our results suggest
a more specific effect of the anti-A and anti-B antisera on receptors.
An alternative approach to demonstrating that the chemoreceptors are GPI-anchored would be to treat whole cells with PI-PLC. The volumes that would be needed for pre-treatment of the cells before T-maze assays make this prohibitively expensive, and this approach might fail if the PLC treatment removed many, but not all, of the GPI-anchored proteins. Considering the preponderance of the large surface antigens, especially on the cilia, we would predict that most of the proteins removed in partial digestions would be the surface antigens. Indeed, incubation of whole cells in PLC is not sufficient to remove enough surface antigen to protect the cells from immobilization by anti-A and anti-B antisera (J. L. Van Houten, unpublished observations).
The strongest inhibition of the chemoresponse by the antisera is of the
folate chemoresponse. In the past, we have found that the folate-binding
proteins on the surface of P. tetraurelia are difficult to solubilize
in non-ionic detergents, which is a characteristic of GPI-anchored proteins
(Sasner and Van Houten, 1989).
Interestingly, the folate-uptake-binding proteins (folate receptors) of
mammalian cells are GPI-anchored (Antony,
1996
; Rothberg et al.,
1990
). If the P.
tetraurelia folate chemoreceptor is GPI-anchored, its signal transduction
pathway must include other membrane proteins that interact with the
GPI-anchored protein and rapidly signal to the interior of the cell (uptake
through potocytosis or other endocytotic pathways would be too slow). It will
be interesting to look for these interacting proteins among the other proteins
of the surface membranes.
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Acknowledgments |
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