From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada
Received for publication, September 5, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Proaerolysin, the proform of the channel-forming
protein aerolysin, is secreted as a dimer by Aeromonas sp.
The protein also exists as a dimer in the crystal, as well as in
solution, at least at concentrations in the region of 500 µg/ml.
Recently it has been argued that proaerolysin becomes monomeric at
concentrations below 100 µg/ml and that only the monomeric form of
the protoxin can bind to cell surface receptors (Fivaz, M., Velluz,
M.-C., and van der Goot, F. G. (1999) J. Biol.
Chem. 274, 37705-37708). Here we show, using non-denaturing
polyacrylamide electrophoresis, chemical cross-linking, and analytical
ultracentrifugation, that proaerolysin remains dimeric at the lowest
concentrations of the protein that we measured (less than 5 µg/ml)
and that the dimeric protoxin is quite capable of receptor binding.
The channel-forming toxin aerolysin is secreted as an inactive
precursor called proaerolysin by bacteria in the Aeromonas family. Proaerolysin binds to
glycosylphosphatidylinositol-anchored receptors on sensitive
cells and is converted to aerolysin by proteases such as furin.
Aerolysin then oligomerizes, and the heptamers that are formed insert
into the bilayer, producing channels (1). Proaerolysin exists as
a dimer in the crystal, and the structure of the dimer has been solved
(2). In previous studies we have shown that proaerolysin is secreted as
a dimer by Aeromonas salmonicida (3), and
we have also found, using analytical ultracentrifugation and chemical
cross-linking, that it is a dimer in solution (4), at least at
concentrations in the range of 500 µg/ml (10 µM). The
dimeric form of the protein may have several advantages over the
monomer. First of all, it appears that only the dimer is recognized by
the Aeromonas secretion machinery, which is responsible for transit of proaerolysin across the outer membrane of the bacteria. Thus
dimerization in the periplasm is necessary for secretion by the
bacteria (3). Second, a large area of the surface of the protein
is protected in the dimer, and this could contribute to the unusual
protease stability of aerolysin (2, 5). Third, two regions of the
molecule appear to be involved in receptor binding, one at the top of
the large lobe and another in the small lobe (6). In the dimer the
large lobe of one monomer is much closer to the small lobe of the other
monomer than it is to its own small lobe (2). Finally, dimerization may
help control the oligomerization step in channel formation. The
monomers presumably must separate for oligomerization to occur because
the oligomer is heptameric (7, 8). The stability of the dimer may
reduce the risk that the protein will oligomerize in the absence of a target membrane.
Recently, it has been argued that proaerolysin is a monomer in solution
at concentrations below 100 µg/ml (~2 µM) and that it
is the monomer that binds to cell surface receptors (9). These
conclusions were based on the results of binding and cross-linking studies with radioiodinated proaerolysin, on
concentration-dependent changes in the elution profile of
proaerolysin using gel permeation chromatography with an agarose
matrix, and on the apparent inability of a variant of proaerolysin
(M41C) that is a covalent dimer to bind to surface proteins. In this
paper we present contradictory evidence and alternative explanations of
the data of the earlier study. We show that proaerolysin remains
dimeric in solution at the lowest concentrations that we measured (less
than 5 µg/ml) and that covalent proaerolysin dimers can indeed bind
to cell surfaces.
Materials--
Trypsin and trypsin inhibitor were obtained from
Sigma. Native proaerolysin and the proaerolysin variants were purified
according to our published procedure (10). All of the samples migrated with an apparent mass of 52 kDa on reducing
SDS-PAGE1 (data not shown).
Production of Proaerolysin Variants That Form Covalent
Dimers--
The production of M41C proaerolysin and the properties of
this variant have been described previously (3). Two other covalent variants were made in the same way. Briefly, the structure of the
native proaerolysin dimer was examined to locate single residues that
lie within a few Angstroms of each other in the dimer (as Met-41
does). Two residues, Ser-13 and Lys-185, were identified, and two
proaerolysin variants, S13C and K185C, were created by site-directed
mutagenesis and purified. Some of the purified S13C migrated in
nonreducing SDS-PAGE with the same apparent mass as M41C (~100 kDa),
indicating that an intermolecular bridge had formed between the
cysteines at position 13 in the two monomers (see Fig. 1A).
The rest of the protein migrated as a monomer (non-covalent dimers are
dissociated by the SDS in SDS-PAGE), indicating that intermolecular
bridge formation was not complete. Consistent with this result, the
S13C variant retained appreciable channel-forming activity, although it
was rendered more active by reduction with dithiothreitol (data not
shown). The K185C variant migrated entirely as a single band, more
slowly than the S13C and M41C dimers (see Fig. 1A),
presumably because the new intermolecular bridge prevented complete
unfolding in SDS. It migrated with the same apparent mass as native
proaerolysin under reducing conditions (data not shown). Like the M41C
variant, K185C was virtually inactive unless reduced.
Chemical Cross-linking--
Samples of proaerolysin in 150 mM NaCl, 20 mM HEPES, pH 7.4 were treated with
0.25 mM dithiobis[succinimidyl propionate] (DSP, Pierce)
at room temperature. After 30 min, 1 M Tris, pH 8, was added to a final concentration of 90 mM, and the incubation
was continued for 5 min before adding sample buffer for SDS-PAGE.
Binding to Human Erythrocytes--
Human erythrocytes from
outdated blood were washed with 150 mM NaCl, 20 mM phosphate, pH 7.4. The cells were resuspended to 4%
(v/v) in 150 mM NaCl, 10 mM polyethylene glycol
(average molecular weight, 8000), 17.5 mM HEPES, 1%
(w/v) bovine serum albumin, pH 7.4, containing either 5 × 10 Analytical Ultracentrifugation--
Proaerolysin was dialyzed
extensively against 150 mM NaCl, 20 mM HEPES,
pH 7.4 and loaded into aluminum Kel-F 12-mm double sector cells.
Sedimentation velocity runs were carried out in a Beckman Analytical
XL-A ultracentrifuge using an An-55 Al (aluminum) rotor. Sedimentation
equilibrium runs employed an An-60 Ti (titanium) rotor. Temperature and
speed conditions are indicated in the figure legends. A partial
specific volume of 0.745 cm3/g was used for the
calculations (4).
Sedimentation velocity scans were analyzed using XL-A Ultra Scan
version 4.1 sedimentation data analysis software (Borries Demeler,
Missoula, MT), which employs a published method of boundary analysis
(11). Sedimentation equilibrium scans were analyzed using a nonlinear,
least squares, curve-fitting algorithm (12), contained in the XL-A data
analysis software.
Electrophoresis and Blotting--
SDS-PAGE was carried out under
reducing or nonreducing conditions (13). Non-denaturing gel
electrophoresis was performed in 10% gels in the absence of reducing
agents (14). When necessary, proteins were blotted onto nitrocellulose
and detected using a mouse anti-proaerolysin monoclonal antibody and
anti-mouse horseradish peroxidase (15). Blots were developed by
enhanced chemiluminescence (PerkinElmer Life Sciences).
Covalent Proaerolysin Can Bind to Cell Surfaces--
Fivaz
et al. (9) reported that the M41C variant of proaerolysin
was apparently unable to bind to proteins from baby hamster kidney cells and drew the conclusion that the proaerolysin dimer must
dissociate for binding to occur. We compared the binding of native
proaerolysin to human erythrocytes with the binding of the three
covalent variants, including M41C. A blot of the membrane-associated
proteins separated by nonreducing SDS-PAGE is shown in Fig.
1B. For comparison, a
Coomassie-stained nonreducing SDS-PAGE gel of the proteins used is
shown in Fig. 1A. Dimers of both S13C and K185C were readily
detected in amounts comparable with the amount of bound native monomer,
indicating that both covalent dimers could bind to the erythrocyte
membranes. Much less M41C was detected, suggesting that this variant
binds more poorly than wild type and the S13C and K185C dimers, a
result that is consistent with the previous finding (9).
Proaerolysin Dimers Can Be Cross-linked at Low Protoxin
Concentrations--
Previously we have found that treating native
proaerolysin with dimethylsuberimidate produces cross-linked dimers;
however, we did not try protein concentrations lower than ~500
µg/ml or 10 µM (4). Fivaz et al. (9)
used glutaraldehyde and proaerolysin concentrations as low as 50 µg/ml to study dimer cross-linking. They concluded that cross-linking
was concentration-dependent and that this was an indication
that the dimer was dissociating as the protein concentration was
lowered (9). They drew this conclusion despite the fact that their
results appear to show that proportionally as much cross-linked dimer
was formed at 50 µg/ml proaerolysin as at 100 µg/ml. In preliminary
experiments we found that DSP cross-links proaerolysin much more
efficiently than either dimethylsuberimidate or glutaraldehyde, and we
used this reagent to look for evidence of the presence of dimers at proaerolysin concentrations as low as 4 µg/ml. The results in Fig.
2 show that even at the lowest
concentration, nearly all of the protoxin migrated with an apparent
mass of ~100 kDa after treatment with DSP, indicating that little or
none of it was dissociated into monomers and contradicting the
conclusion of the glutaraldehyde study (9).
Proaerolysin Migrates as a Dimer in Non-denaturing Gels at All
Concentrations--
Our ability to cross-link proaerolysin dimers at
low protoxin concentrations led us to believe that the protein might be
stable enough to remain dimeric during non-denaturing gel
electrophoresis. The results in Fig.
3A show that this is the case.
Native proaerolysin migrated with the same mobility as M41C and K185C,
both of which are covalent dimers under these conditions.
Diluting the protein to concentrations as low as 0.1 µg/ml had no
effect on its mobility (Fig. 3B)
Sedimentation Velocity Centrifugation Indicates That Proaerolysin
Is a Dimer--
The sedimentation velocity analysis of proaerolysin at
16 and 80 µg/ml is illustrated in Fig.
4. It is clear that the protein was very
homogeneous at both concentrations, as indicated by the convergence of
the lines in the "fan plots" (11). The sedimentation coefficient
was 5.2 ± 0.1 S at both protein concentrations, in agreement with
the previous result obtained with 500 µg/ml proaerolysin (4). Thus
there is no significant change in the sedimentation coefficient or the
homogeneity of proaerolysin, and hence no evidence for dimer
dissociation, in the concentration range between 16 and 500 µg/ml.
Proaerolysin Has the Mass of a Dimer by Sedimentation Equilibrium
Centrifugation--
Sedimentation equilibrium analysis confirmed that
proaerolysin is entirely dimeric at 16 µg/ml. At this concentration
the data could be fitted to a single ideal component model of molecular mass 94.9 kDa (Fig. 5A), very
similar to the mass of 95.6 kDa we have obtained previously at higher
protein concentrations (4). At the higher concentration of 91 µg/ml,
the best fitting line for a monodisperse sample gave a molecular mass
of 95.2 kDa (Fig. 5B); however the fit to a single ideal
component model was not as good as it was at the lower concentration.
This is indicated by the nonrandom trend exhibited by the residuals.
The systematic pattern observed at the higher concentration is
indicative of the presence of some aggregation (16).
The results of the present study of the state of proaerolysin in
solution lead to different conclusions than those reached by Fivaz
et al. (9). Our data indicate that the protoxin is a dimer,
even at very low concentrations, and that dimer dissociation is not
required for binding to surface receptors.
We believe that several factors may account for differences between our
results and conclusions and those of Fivaz et al. (9). These
authors observed that the M41C variant did not bind to surface proteins
and they interpreted this to mean that dimer separation is necessary
for binding. A more likely explanation is that the M41C substitution
reduces the ability of the protein to interact with the receptor. The
mutation is in the carbohydrate binding fold or aerolysin
pertussis toxin domain of proaerolysin, which we have identified
previously (17). We have shown that changing other amino acids in this
region can have pronounced effects on receptor binding (6).
Fivaz et al. (9) also found that they were unable to
cross-link proaerolysin dimers with 3,3'-dithiobis[sulfosuccinimidyl propionate] after exposing baby hamster kidney cells to native proaerolysin that had been radioiodinated using IODO-GEN. They concluded that this was because dimers had dissociated and that only
monomers were bound to surface receptors (9). However, even in control
experiments using protoxin concentrations at which they readily
observed dimer with glutaraldehyde, very little cross-linked dimer was
produced with 3,3'-dithiobis[sulfosuccinimidylpropionate]. This may
indicate that this reagent is a poor cross-linking agent for
proaerolysin. The use of radioiodinated proaerolysin may also have
affected the outcome of their attempt to cross-link dimers to surface
proteins. We have found that the use of IODO-GEN results in a
product that is less active than normal
proaerolysin,2 perhaps
because one or more tyrosines important for binding are affected. Two candidates are Tyr-61 and Tyr-162, because variants in
which these residues are replaced bind more poorly to
glycosylphosphatidylinositol-anchored proteins than does native
proaerolysin (6).
Fivaz et al. (9) noted an increase in the elution volume
from a Sephadex gel filtration column with decreasing concentration of
the proaerolysin applied and concluded that this was evidence that the
dimer dissociated at low concentrations. We have found that
proaerolysin will bind to agarose-based matrices such as Sephadex under
a variety of conditions and that this can lead to increased retention
with decreased concentration (data not shown here). It is possible that
this kind of interaction may account for their observations.
The dimeric state of proaerolysin in solution, which we confirm here,
is consistent with several things we know about the protein, including
its secretion, its stability to proteases, and the extensive
interaction between monomers in the crystal (2). Nevertheless, some
time after activation by proteolytic nicking, the monomers must
separate, not only because there are an odd number of monomers in the
oligomer, but because all of the monomers are thought to be in the same
orientation in the oligomer, whereas they are in opposite orientations
in the dimer (2). How and when dimer separation occurs remains to be established.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 M native proaerolysin or one
of the proaerolysin variants. After 30 min of incubation on ice, the
cells were pelleted and lysed by suspending them in ice-cold 5 mM sodium phosphate, pH 8.0. The membranes were pelleted
and washed in the phosphate buffer. They were then dissolved in sample
buffer, separated by SDS-PAGE, and blotted to detect proaerolysin that
had bound.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mobilities and binding of covalent
proaerolysin dimers. A, Coomassie-stained gel
illustrating the mobilities of the proaerolysin variants compared with
native proaerolysin under nonreducing conditions. Under reducing
conditions all of the proteins had the same mobility. B,
binding of proaerolysin variants to human erythrocytes. After exposing
erythrocytes to the different proaerolysins, membranes were recovered
and separated by nonreducing SDS-PAGE. Native proaerolysin and the
variants were detected by Western blotting. The positions of 116- and
80-kDa molecular mass markers are identified.wt,
wild type.
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Fig. 2.
Cross-linking of proaerolysin with DSP.
Proaerolysin at 20 µg/ml (lane 1) or 4 µg/ml (lane
2) was treated with DSP, electrophoresed, and blotted as described
under "Experimental Procedures." Untreated proaerolysin is in the
third lane. The positions of three molecular mass
markers are indicated.
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Fig. 3.
Proaerolysin migrates as a dimer in
non-denaturing gels regardless of concentration. A,
Coomassie-stained gel comparing the mobilities of native proaerolysin
and the covalent proaerolysin dimers M41C and K185C. B,
Western blot comparing the mobilities of several concentrations of
native proaerolysin (in µg/ml) with M41C
proaerolysin.wt, wild type.
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Fig. 4.
Sedimentation velocity analysis of
proaerolysin. In A the A230 was
0.2, and in B the A280 was 0.2. The
run was performed at 20 °C and 42,000 rpm in 150 mM
NaCl, 20 mM HEPES, pH 7.4. The analysis was carried out
according to the method of van Holde and Weischet (11) in which the
lines converging toward a common s20,w
(Svedbergs) value and the number of lines are proportional to the
fraction of sample represented. Time is shown in seconds.
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Fig. 5.
Sedimentation equilibrium analysis of
proaerolysin. The A230 was 0.24 in
A, and the A280 was 0.22 in
B. The protein concentration in A was 16 µg/ml,
and in B the protein concentration was 96 µg/ml. The
continuous lines indicate the fitting to a thermodynamically
single ideal species of mass 94.9 kDa (A) and 95.2 (B). The top plots show the 2
residuals (18) as a function of the radial distance. The run was
performed at 6 °C and 15,000 rpm using 150 mM NaCl, 20 mM HEPES, pH 7.4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
On sabbatical leave from the University of the Basque Country
(Bilbao, Spain), supported by the Spanish Ministry of Education and Culture.
§ To whom correspondence should be addressed. Tel.: 250-721-7081; Fax: 250-598-6877; E-mail: tbuckley@uvic.ca.
Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M008097200
2 R. Barry, S. Moore, A. Alonso, J. Ausió, and J. T. Buckley, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: SDS-PAGE, SDS-polyacrylamide gel electrophoresis; DSP, dithiobis[succinimidyl propionate].
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REFERENCES |
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1. | Buckley, J. T. (1999) in Comprehensive Sourcebook of Bacterial Protein Toxins (Alouf, J. E. , and Freer, J. H., eds) , pp. 362-372, Academic Press, New York |
2. | Parker, M. W., Buckley, J. T, Postma, J. P. M., Tucker, A. D., Leonard, K., Pattus, F., and Tsernoglou, D. (1994) Nature 367, 292-295[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hardie, K. R., Schulze, A., Parker, M. W., and Buckley, J. T. (1995) Mol. Microbiol. 14, 1093-1011 |
4. |
van der Goot, F. G.,
Ausió, J.,
Wong, K. R.,
Pattus, F.,
and Buckley, J. T.
(1993)
J. Biol. Chem.
268,
18272-18279 |
5. | Garland, W. J., and Buckley, J. T. (1988) Infect. Immun. 56, 1249-1253[Medline] [Order article via Infotrieve] |
6. |
Mackenzie, C. R.,
Hirama, T.,
and Buckley, J. T.
(1999)
J. Biol. Chem.
274,
22604-22609 |
7. | Wilmsen, H. U., Pattus, F., Tichelar, W., Buckley, J. T., and Leonard, K. (1992) EMBO J. 11, 2457-2463[Abstract] |
8. | Moniatte, M., van der Goot, F. G., Buckley, J. T., Pattus, F., and van Dorsselaer, A. (1996) FEBS Lett. 384, 269-272[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Fivaz, M.,
Velluz, M. C.,
and van der Goot, F. G.
(1999)
J. Biol. Chem.
274,
37705-37708 |
10. | Buckley, J. T. (1990) Biochem. Cell Biol. 68, 221-224[Medline] [Order article via Infotrieve] |
11. | van Holde, K. E., and Weischet, W. O. (1978) Biopolymers 17, 1387-1401 |
12. | Johnson, M. L., Correia, J. J., Yphantis, D. A., and Halvorson, H. R. (1981) Biophys. J. 36, 575-588[Abstract] |
13. |
Neville, D. M.
(1971)
J. Biol. Chem.
246,
6328-6334 |
14. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
15. |
Diep, D. B.,
Nelson, K. L.,
Raja, S. M.,
Pleshak, E. N.,
and Buckley, J. T.
(1998)
J. Biol. Chem.
273,
2355-2360 |
16. | McRorie, D. K., and Voelker, P. J. (1993) Self-associating Systems in the Analytical Ultracentrifuge , Beckman Instruments Inc., Fullerton, CA |
17. |
Rossjohn, J.,
Buckley, J. T.,
Hazes, B.,
Murzin, A. G.,
Read, R. J.,
and Parker, M. W.
(1997)
EMBO J.
16,
3426-3434 |
18. | Straume, M., and Johnson, M. L. (1992) Methods Enzymol. 210, 87-105[Medline] [Order article via Infotrieve] |