(Received for publication, January 16, 1997, and in revised form, February 21, 1997)
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada
Aerolysin is a channel-forming protein secreted
by virulent Aeromonas spp. Some eucaryotic cells, including
T-lymphocytes, are sensitive to very low concentrations of the toxin
(<109 M). Here we show that aerolysin binds
selectively and with high affinity to the glycosylphosphatidylinositol
(GPI)-anchored surface protein Thy-1, which is found on T-lymphocyte
populations as well as in brain. Less than 1 ng of purified Thy-1 could
be detected by probing Western blots with the toxin. Mutant T-cell
lines that lack the ability to add GPI anchors to Thy-1 and other
surface proteins were much less sensitive to aerolysin, as were
wild-type cells that were pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins.
Phosphatidylcholine/cholesterol liposomes containing purified Thy-1 in
their membranes were much more sensitive to aerolysin than protein-free
liposomes.
Aerolysin is one of the best studied of all of the bacterial cytolytic toxins. The protein is secreted as a 52-kDa inactive precursor called proaerolysin by a number of Aeromonas spp., and it is known to be required for the virulence of Aeromonas hydrophila in mice (see Ref. 1 for a recent review). Proaerolysin exists as a freely soluble dimer in solution. It is converted to the active form of the protein by proteolytic processing near the C terminus. Aerolysin is also a soluble dimer; however, in contrast to the protoxin form, it is capable of oligomerization, producing heptameric structures that transform the protein into an insertion-competent state.
Cells that contain a receptor for aerolysin are much more sensitive to the toxin than other cells. Among erythrocytes that have been compared, those of the rat are most sensitive (2, 3), and we have shown that this is because they contain a 47-kDa glycoprotein that binds both proaerolysin and aerolysin with considerable affinity (4). Presumably the primary function of the receptor is to concentrate the toxin on the cell surface, thereby indirectly promoting oligomerization. However, it is also possible that receptor binding has a more direct role in oligomerization. For example, it could facilitate dissociation of the aerolysin dimer, which is presumably an essential step in formation of the heptamer.
Most studies with cytolytic toxins have been carried out with erythrocytes because of their uncomplicated structure and metabolism, the ease with which they can be obtained free of other cells, and the simplicity of hemolytic assays. However, there is no reason to believe that these cells would be the primary targets of the bacterial pathogens that secrete cytolysins. Components of the immune system are more obvious candidates for toxin targeting. In this report, we show that Thy-1, a major surface glycoprotein of rodent T-lymphocytes, is a high affinity receptor for aerolysin.
The murine lymphoma cell lines BW5147.3 and
BW5147.3(Thy-1 e).10 were purchased from the American
Type Culture Collection (Rockville, MD). The cell lines EL4 and
EL4(Thy-1
f) were generously supplied by R. Hyman (Salk
Institute). All cell lines were maintained in Dulbecco's modified
Eagle's high glucose medium supplemented with 10% bovine fetal clone
I serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in 5%
CO2 at 37 °C.
Cells (5 × 105/ml)
in complete Dulbecco's modified Eagle's medium were incubated with
7 × 1010 M aerolysin for 1 h at
37 °C. Live/dead counts were made using a hemocytometer after adding
an equal volume of 0.1% trypan blue in phosphate-buffered saline.
Killing of EL4 cells by aerolysin was also measured using the membrane-impermeant probe Po-Pro 1 (Molecular Probes, Inc.), which fluoresces when bound to nucleic acids made available by the action of aerolysin on the cells. The time dependence of cell death was measured at various aerolysin concentrations with a Photon Technology QM-1 spectrofluorometer. Excitation and emission wavelengths were 435 and 455 nm, respectively, and slit widths were 4 nm.
SDS-PAGE1 and Detection of Aerolysin-binding Proteins by Western BlottingSDS-PAGE was
carried out by the method of Neville (5). Gels were blotted onto
nitrocellulose and developed by sandwich Western blotting as described
by Gruber et al. (4), which involves probing blots with
2 × 108 M proaerolysin, followed by
polyclonal anti-aerolysin antibody and anti-rabbit horseradish
peroxidase. Blots were then developed by enhanced chemiluminesence
(Amersham Corp.).
Tissues were quickly excised and homogenized in 5 volumes of 50 mM Tris-HCl, 0.16 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 µM pepstatin A, pH 8, using a Polytron homogenizer (Brinkmann Instruments) at 4 °C. Protein was determined as described by Markwell et al. (6).
N-Deglycosylation of GlycoproteinsAn equal volume of 40 mM NaH2PO4, 100 mM
EDTA, 1% SDS, and 10% -mercaptoethanol, pH 7.5, was added to 5 µl of 4.5 × 106 cells/ml in phosphate-buffered
saline, and the mixture was boiled for 2 min. After cooling to room
temperature, 3.3 µl of a protease inhibitor mixture consisting of 0.6 mM phenylmethylsulfonyl fluoride, 60 µg/ml aprotinin, 120 µM leupeptin, and 12 µM pepstatin A were added, followed by 2.5 µl of 10% octyl glucopyranoside and 7.5 µl
of peptide N-glycosidase F (Oxford Glycosystems) containing 1.5 units of the enzyme. A control incubation was also carried out in
which 7.5 µl of buffer were added in place of the enzyme. After
18 h at 37 °C, sample buffer was added, and aliquots were separated by SDS-PAGE and sandwich Western-blotted.
Affinity-purified Thy-1 from rat thymus (7) was a generous gift from Dr. R. McMaster (University of British Columbia). The only band visible on a silver-stained SDS-polyacrylamide gel corresponded to the monomeric protein. In addition to the monomer, a small band corresponding to the dimer was detected by sandwich Western blotting.
Liposome Preparation and Incorporation of Thy-1Dried lipid
films were prepared from a mixture of egg yolk
L--phosphatidylcholine (Avanti Polar Lipids, Inc.) and
cholesterol (mole ratio of 7:1) and rehydrated in 2 ml of 100 mM carboxyfluorescein in 20 mM HEPES and 150 mM NaCl, pH 7.4. Large unilamellar vesicles (liposomes)
were prepared by extrusion through 25-mm polycarbonate filters using an
Extruder (Lipex Biomembranes Inc., Vancouver, British Columbia, Canada)
as described by Hope et al. (8). The liposome concentration
was determined by measuring the phosphate content using a standard
assay (9).
Thy-1 was incorporated into the liposomes following the procedure of Rigaud et al. (10). Briefly, 250 µl of Thy-1 (42.5 µg) in 10 mM Tris, pH 8.0, containing 1% octyl glucoside were added to 500 µl of carboxyfluorescein-entrapped liposomes (1.3 µmol of lipid). As a negative control, 250 µl of 10 mM Tris, pH 8.0, containing 1% octyl glucoside were added to 500 µl of carboxyfluorescein-entrapped liposomes. To both samples were added 250 µl of a 2.2% stock of octyl glucoside in 20 mM HEPES and 150 mM NaCl, pH 7.4, so that the final octyl glucoside concentration was 0.8%. After overnight dialysis against 20 mM HEPES and 150 mM NaCl, pH 7.4, the liposomes were passed over a Sephacryl S-300 column (18 ml) to remove unincorporated Thy-1.
Liposome Release AssayCarboxyfluorescein release was
monitored spectrofluorometrically. The excitation wavelength was set at
490 nm, and the emission wavelength at 520 nm. A 4-nm slit width was
used for both monochromators. Small aliquots of the liposome
preparations (21.5 nmol of lipid) were added to 3 ml of 20 mM HEPES and 150 mM NaCl, pH 7.4, and carboxyfluorescein release was followed with time. Activated aerolysin was added to a concentration of 4 × 108
M at 2 min. Total entrapped carboxyfluorescein in the
liposomes was measured by adding Triton X-100 to a 0.1% (w/v) final
concentration at the end of each run. All of the experiments were
carried out at room temperature.
Five-hundred µl of 9 × 107 EL4 cells/ml were incubated with 200 milliunits of phosphatidylinositol-specific phospholipase C (PI-PLC; Boehringer Mannheim) for 2 h at 37 °C in phosphate-buffered saline. A control sample was incubated without the enzyme. The cells were subsequently pelleted at 80,000 rpm for 20 min at 4 °C in a Beckman TLA 100.2 rotor. Aliquots of the supernatants and pellets were used for the sandwich Western blotting procedure.
Liposomes (86 nmol of phosphatidylcholine) containing reconstituted Thy-1 (0.85 µg) and control liposomes containing no protein were treated with 600 milliunits of PI-PLC for 60 min at room temperature. Liposomes containing Thy-1 incubated under the same conditions without PI-PLC also served as controls.
We have shown that there is a 47-kDa glycoprotein in
rat erythrocyte membranes that binds aerolysin with high affinity (4). The protein can be detected by exposing Western blots of membrane proteins to low concentrations of proaerolysin (109
M) and probing with an anti-aerolysin antibody after
washing to remove unbound protein (4). We decided to screen a number of
mouse tissues in a similar way for proteins that might bind proaerolysin. The tissues were homogenized and dissolved directly in
sample buffer for SDS-PAGE. Bands indicating aerolysin binding were
observed in all of the tissues, but by far the most intense were those
observed in the region of 30 kDa in the lanes containing thymus
and brain (Fig. 1).
T-cell Lines Also Contain a 30-kDa Aerolysin-binding Component
The appearance of an aerolysin-binding protein on blots
of homogenized thymus prompted us to examine several T-cell lymphomas in the same way as we screened the tissues. The results for two cell
lines, EL4 and BW5147.3, are shown in Fig. 2. It is
clear that a protein corresponding to those observed in the brain and thymus is also found in these T-cells.
The T-cell Aerolysin-binding Protein Is GPI-anchored
We have obtained evidence that the rat erythrocyte receptor is anchored to the cell surface by glycosylphosphatidylinositol attached to its C terminus (1). To determine if the 30-kDa protein in the T-cells might be anchored in a similar way, we took advantage of the fact that there are EL4 and BW5147.3 cell lines that lack the ability to attach these anchors to any of their surface proteins (11). When proteins from these cells were studied by the Western blotting technique, it was clear that they contained far lower quantities of the 30-kDa species (Fig. 2).
GPI anchoring of the 30-kDa protein found on normal EL4 cells,
suggested by the experiment with the mutant cells, was confirmed by
treating the wild-type cells with PI-PLC. This enzyme is known to
release many proteins anchored to cell membranes in this way (12). The
results in Fig. 3 show that reaction with the enzyme led
to a pronounced decrease in the amount of cell-associated aerolysin-binding protein, which appeared in the soluble fraction after
cell pelleting. This allows us to conclude that this
proaerolysin-binding protein is GPI-anchored and also that the lipid
portion of the anchor for this protein is a single molecule of
diacylglycerol. Several mammalian GPI-anchored proteins, such as human
acetylcholinesterase (13) and Trypanosoma brucei procyclic
acid repeat protein (14), are known to contain an additional acyl chain
esterified to the inositol moiety. These proteins are not released by
phospholipase C treatment (13).
N-Glycosidase Treatment Suggests That the T-cell Protein Is Thy-1
More than 100 different proteins are known to be attached
to the plasma membrane by GPI anchors, and several have been reported to be present on T-lymphocytes (15). Of those for which information is
available, we could find only two that have molecular masses in the
region of 30 kDa. One of these is RT6, which is not expressed by EL4
cells. The other is Thy-1, a glycoprotein that migrates as two or more
bands in the region of 30 kDa on SDS-polyacrylamide gels. It is the
smallest known member of the immunoglobulin superfamily (16), and in
mice and rats, it is a major cell-surface component of thymocytes and
brain (17). Thy-1 is known to be N-glycosylated at three
positions, and it is not O-glycosylated (18). The molecular mass of the protein after peptide N-glycosidase F treatment
should be ~14.5 kDa. The results in Fig. 4 show that
when EL4 cell samples were treated with peptide
N-glycosidase F, the aerolysin-binding component was reduced
to a single band migrating with an apparent mass in this range. This
result supports the view that the aerolysin receptor is Thy-1. In
addition, it eliminates the possibility that it is RT6 since the
molecular mass of the amino acid chain of this protein is much higher
(19). The possibility that the 47-kDa rat erythrocyte receptor we have
described (4) is simply a more heavily glycosylated derivative of the
30-kDa glycoprotein in the T-cell and brain is also excluded by this
result. The molecular mass of the erythrocyte proaerolysin-binding
protein is reduced only to 32 kDa by peptide N-glycosidase F
treatment (4). We have recently obtained the N-terminal sequence of the
erythrocyte receptor, which indicates that the protein is related to a
small family of GPI-anchored proteins that are involved in
ADP-ribosylation reactions.2 Interestingly,
there are no apparent sequence similarities between these proteins and
Thy-1.
Picogram Amounts of Purified Thy-1 Are Detected Using Aerolysin on Western Blots
To confirm that aerolysin binds Thy-1, a blot
containing varying amounts of the purified rat thymus protein in the
nanogram and picogram range was probed with proaerolysin (Fig.
5). It may be seen that proaerolysin binding provides a
very sensitive method to detect Thy-1 after Western blotting. Less than
1 ng of the GPI-anchored protein could be detected in this way.
Thy-1 from Several Species Binds Aerolysin
The results in
Fig. 6 show that Thy-1 in the brains of a number of
species may bind proaerolysin. Thus, rabbit, human, and pig brains
contain proteins of corresponding size and in similar amounts to the
mouse. Comparable binding intensity was not observed in cow and sheep
brains. However, we did not determine whether this is because Thy-1
levels are low in the brains of these species (none of the commercially
available anti-Thy-1 antibodies we could find will detect Thy-1 on
Western blots) or whether it is because the cow and sheep Thy-1
proteins differ from the protein from the other species in some way
that affects proaerolysin binding.
Cells That Lack GPI-anchored Proteins Are Less Sensitive to Aerolysin
We used a simple assay to determine if EL4 cells
containing surface GPI-anchored proteins are more sensitive to
aerolysin than cells without these determinants. EL4 cells were
incubated with 1 µM Po-Pro 1, a membrane-impermeant probe
that fluoresces when it intercalates with double-stranded nucleic acid.
When aerolysin is added to wild-type cells, there is an increase in
fluorescence after a delay that we have found depends on the
concentration of the toxin (data not shown). Live/dead cell counts
carried out in parallel showed that all of the cells are dead by the
time the fluorescence curve plateaus. As little as 1010
M aerolysin leads to 100% killing of wild-type cells in
<1 h at 37 °C (data not shown). The results of an experiment
comparing the effect of a much higher concentration of aerolysin
(1.8 × 10
8 M) on wild-type and
EL4(Thy-1
f) cells are shown in Fig. 7. It
is clear that this toxin concentration, nearly 100-fold higher than
that needed to kill wild-type cells, had no measured effect on the
mutant cells. The survival of the mutant cells was confirmed
independently by live/dead cell counts. Comparable experiments showed
that BW5147.3(Thy-1
e).10 cells, which also lack
GPI-anchored proteins, were much less sensitive than the corresponding
wild-type cells.
Results similar to those in Fig. 7 were obtained when we compared EL4 cells with cells that had been pretreated with PI-PLC. The enzyme-treated cells had greatly reduced sensitivity to the toxin.3
Liposomes Containing Thy-1 Are More Sensitive to AerolysinThe results of the experiments comparing wild-type and
mutant cell lines were convincing evidence that GPI-anchored proteins confer aerolysin sensitivity to T-lymphocytes. However, since these
cells may contain several different proteins anchored in this way, we
could not conclude that Thy-1 itself was acting as an aerolysin
receptor. We have shown previously that incorporation of the rat
erythrocyte receptor into planar bilayers increases their sensitivity
to aerolysin (4). Since GPI-anchored proteins are easily incorporated
into liposome membranes, we used entrapped dye release from large
unilamellar vesicles as an assay instead of the more cumbersome planar
bilayer assay to determine the effect of purified Thy-1 on the ability
of aerolysin to form channels. The results in Fig.
8A show that nanomolar concentrations of
aerolysin cause the release of carboxyfluorescein from liposomes
containing incorporated Thy-1, but not from control liposomes
containing only lipid. As expected, when Thy-1 was removed from the
membranes by treating the liposomes with PI-PLC, their sensitivity to
aerolysin was abolished (Fig. 8B).
Thy-1 is the major protein on the surface of rodent T-cells, and for this reason alone, it is an ideal target for a bacterial toxin like aerolysin. The T-cells of other species contain variable amounts of the protein. For example, in the human, progenitor cells are the richest lymphocyte source (20). The brains of many species are also rich in Thy-1, but it is unlikely that this could be significant in Aeromonas infections. Interestingly, despite the fact that bovine thymocytes (21) are known to contain a homologue of the rat and human Thy-1 proteins, we found little or no aerolysin binding after blotting cow (or sheep) brain samples when compared with the other species we tested. Presumably, this means that the glycoprotein in these two species differs from Thy-1 of the others in some region of the structure that is crucial for proaerolysin binding.
Our results tell us several things about the interaction of aerolysin with Thy-1. The fact that the receptor could easily be detected in cell-free supernatants following treatment with PI-PLC by probing blots with proaerolysin (Fig. 3) is evidence that the diglyceride portion of the anchor is not required for binding. Similarly, since binding was not reduced when the N-linked sugars were removed (Fig. 4), it is clear that this portion of Thy-1 is also not involved in the interaction with the protoxin.
GPI-anchored glycoproteins are thought to be capable of much higher lateral mobility in the plasma membrane than proteins with conventional peptide transmembrane regions (22), and this may be an advantage in promoting channel formation by aerolysin. Before binding, the toxin is a water-soluble dimer, and to form membrane channels, it must oligomerize and form insertion-competent heptamers (1). Presumably, the toxin, once bound to the receptor, moves laterally in the membrane to form the oligomer. Clearly, the abundance of Thy-1 on murine T-cells should facilitate binding, and the lateral mobility of the receptor should facilitate oligomerization.
It is also easy to speculate that binding to a small protein molecule like Thy-1, close to the cell surface and lipid-anchored, may make it easier for the aerolysin oligomer to insert into the bilayer. Interestingly, such a function has already been proposed for CAMPATH-1, a small GPI-anchored polypeptide on human lymphocytes (23). Monoclonal antibodies against this molecule are very effective for complement-mediated cell lysis (24).
Thy-1 is not the only membrane protein that can act as a receptor for
aerolysin. We have previously characterized such a protein in the rat
erythrocyte (4), and the comparison of mouse tissues by sandwich
Western blotting in Fig. 1 identifies a few other bands that may
represent proteins with affinity for the toxin. Our recent evidence
indicates that the rat erythrocyte protein is a member of another
family of proteins that are unrelated to Thy-1, except that they all
also contain GPI anchors.2 It is worth noting that
Bacillus thuringiensis -toxin has recently been shown to
bind to a GPI-anchored aminopeptidase in the midgut of the insect
Manduca sexta (25).
Like many other toxins, aerolysin has long been classified as a channel-forming hemolysin, and it is clear that it destroys erythrocytes by breaching the permeability barrier. The mechanism by which it causes death in other cells, such as T-lymphocytes, is worth further study. It seems likely that at high concentrations, where channels would be formed at a rate that would overwhelm the cell, the cause of death may be comparable to that in the erythrocyte. However, at low toxin concentrations, other causes of death may be more important, and they may result from the binding of the toxin to receptors like Thy-1. Although the functions of many GPI-anchored proteins are unknown, there is evidence for some that clustering results in the generation of an intracellular signal. For example, clustering of Thy-1 molecules as a result of binding by an anti-Thy-1 antibody has been shown to cause lymphocyte apoptosis (26). Since oligomerization of aerolysin should promote clustering of Thy-1 in the same way as the antibody, we should expect to see aerolysin apoptosis under some conditions. Aerolysin should prove to be a useful tool in the study of apoptosis and in related studies of the function of Thy-1 and other GPI-anchored proteins.
We are grateful to R. Hyman for generously providing some of the strains. We thank Dr. R. McMaster for providing purified rat thymus Thy-1.