From the Institute of Medical Biochemistry, Vienna Biocenter,
University of Vienna, Dr. Bohr Gasse 9/3, A-1030 Vienna, Austria and
the Institute of Analytical Chemistry, University of
Vienna, Währingerstrasse 38, A-1090 Vienna, Austria
Received for publication, September 1, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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The formation of complexes between the minor
receptor group human rhinovirus HRV2 and two recombinant soluble
receptor fragments derived from the human very low density lipoprotein
receptor (VLDLR) and containing ligand-binding repeats 1-3
(MBP·VLDLR1-3) or 1-8
(MBP·VLDLR1-8) fused to the carboxyl terminus of the
maltose-binding protein was analyzed by affinity capillary electrophoresis. At low molar ratios of receptor/virus, the peaks corresponding to substoichiometric complexes were broad indicating heterogeneity. When the receptors were present in molar excess with
respect to the virus, the peaks were sharp, suggesting saturation of
all binding sites. For the determination of the stoichiometry, constant
amounts of receptor were incubated with increasing amounts of virus,
and the peak areas corresponding to free receptor were measured and
plotted versus total virus concentration. Extrapolation of
the linear part of the resulting curve to zero concentration of free
receptor enabled quantitation of the molar ratios of the components
present in the complex. Using this method, we determined that about 60 molecules of MBP·VLDLR1-3 but only about 30 molecules of MBP·VLDLR1-8 were bound per virion.
Human rhinoviruses
(HRVs),1 members of the
picornavirus family, are small (~30 nm in diameter) icosahedral
particles composed of 60 copies each of the viral capsid proteins VP1
through VP4 and a positive strand RNA genome of about 7200 nucleotides
in length (1). The 102 serotypes recognized to date use three different
classes of receptors for cell entry. 91 serotypes (the major group)
bind to intercellular adhesion molecule 1 (ICAM-1, Refs. 2-4), 10 serotypes (the minor group) bind to several members of the low density
lipoprotein receptor (LDLR) family (5-7), and 1 serotype (HRV87) binds
to a glycoprotein of so far unknown function (8). ICAM-1 is a member of
the immunoglobulin superfamily with five immunoglobulin-like domains
making up its extracellular part. The LDLR family comprises a number of
membrane proteins all having various numbers of highly conserved
complement type A repeats of about 40 amino acids in length containing
six cysteines each, which exhibit extensive disulfide bridging (for
review, see Ref. 9). Whereas the structure of ICAM-1 is
known at atomic resolution (10, 11), only the structures of single
ligand-binding repeats have been determined by NMR (12) and x-ray
crystallography (13).
The binding site of ICAM-1 on HRV14 and on HRV16 has been characterized
by electron cryo-microscopy followed by image reconstruction techniques
(14, 15). This confirmed the earlier prediction of receptor attachment
occurring within the canyon, a cleft encircling the 5-fold axes of the
viral icosahedral symmetry (16, 17). In an attempt to determine the
binding site of LDL receptors on minor group viruses, we had previously
expressed soluble fragments of LDLR in Sf9 insect cells using
the baculovirus system (18, 19). Although these recombinant
minireceptors are extremely potent in protecting HeLa cells against
infection with various minor group virus serotypes, it was not possible
to demonstrate the formation of virus-receptor complexes in solution.
The earlier observation of comigration of the detergent-solubilized
chicken homolog of human VLDLR and HRV2 on sucrose density gradients
(6) prompted us to investigate whether minireceptors derived from human
VLDLR might be capable of forming stable virus complexes. We
demonstrate here that this is indeed the case. Using electron
cryo-microscopy, we have discovered that the receptor binding site does
not lie in the canyon; its exact location has been determined (20).
An icosahedron exhibits three kinds of symmetry-related sites. 60 equivalent sites result from 2-fold symmetry and 20 sites from 3-fold
symmetry, and 12 equivalent sites are present at the 5-fold axes.
Therefore, determining the number of receptor molecules attached to the
virion allows predictions of the geometric class of the binding
site. For example, we have previously shown that about 30 molecules of
the virus-neutralizing monoclonal antibody 8F5 can attach
to HRV2 (21); this was then confirmed with the finding that the
antibody binds bivalently to two epitopes related by 2-fold
icosahedral symmetry (22).
Scatchard analysis of data obtained from surface plasmon resonance
measurements revealed about 67 binding sites for ICAM-1 on the surface
of HRV14 (23), which is close to the theoretical 60 sites and
corresponds well with the data obtained by electron cryo-microscopy
showing that 60 receptors are attached to all 60 available sites (14).
Nevertheless, steric hindrance, low affinity, or other constraints
might lead to lack of complete occupancy of all theoretically available
sites. Therefore, knowledge of the stoichiometry can indicate whether
the number of receptors attached to the virus is compatible with the
theoretical value or whether the receptor attaches in an unexpected way.
Electrophoretic separation of analytes by capillary electrophoresis
(CE) is based on the differences of mobilities in a given buffer
solution. With this method, affinity interactions can be studied in
solution, and no attachment of any of the components to a solid support
is required (which is the case for enzyme-linked immunosorbent assay
(ELISA)-type formats or for surface plasmon resonance methodology). As
a consequence, all available sites should be equally accessible.
Attachment of antibody or receptor molecules to the viral surface
results in a change in the molecular mass of the particle and/or in its
charge giving rise to an alteration of the electrophoretic mobility
(24). The method appears thus ideally suited for the analysis of
complex formation not only because of its resolution power, but also
because the amounts of material required for analysis are extremely low
(the nanogram range).
In a series of articles we have previously demonstrated the separation
of rhinoviral particles from contaminants (25), the resolution of
native virus and subviral particles (26), and the detection of
antibody-virus complexes (24) by CE. Using this technique, we show here
that a recombinant soluble very low density lipoprotein receptor
fragment, which is fused to the carboxyl terminus of maltose-binding
protein and comprises ligand-binding repeats 1 to 3 (MBP·VLDLR1-3) binds HRV2 with a stoichiometry of about
1:60. A recombinant receptor protein containing repeats 1-8
(MBP·VLDLR1-8) attaches to the virus with a
stoichiometry of about 1:30. This suggests that the presence of the
additional five repeats is either sterically hindering the attachment
of other receptor molecules or that this larger molecule possesses additional binding sites for the virus, which are presumably
contributed by the extra repeats.
Chemicals--
All chemicals were obtained from Sigma unless
specified otherwise. The background electrolyte (BGE) was 100 mM boric acid containing 10 mM SDS; it was
adjusted to pH 8.3 with 1 M NaOH. Samples were dissolved in
a buffer solution corresponding to half-diluted BGE without SDS added
but containing o-phthalic acid (20 µg/ml) as an internal
standard. Buffers were filtered through a 0.45-µm cellulose nitrate
membrane before use. All solutions were centrifuged for 2 min in a
tabletop centrifuge at 5000 × g prior to CE analysis.
Virus Preparation--
Human rhinovirus serotypes 2 and 14, as
originally obtained from the American Type Culture Collection, were
produced and purified from infected cell pellets as described
previously (25, 27). The concentration was determined
spectrophotometrically using an extinction coefficient of 77 at 260 nm
(A260) for a 1% w/v solution (28) and taking
into account any contaminants with absorption at 260 nm as identified
by CE (24). Purified virus was suspended in 50 mM Tris-HCl,
pH 7.4, at an approximate concentration of 3 mg/ml and kept at
Receptor Preparation--
The cDNAs encoding repeats 1-3 or
1-8, respectively, were amplified from pVLDLR (29) by polymerase chain
reaction, cloned into pMalc2b (New England BioLabs) and expressed as
detailed elsewhere (30). Making use of the carboxyl-terminal appended
hexa-His tag the proteins were enriched from the low speed supernatants of the bacterial lysates and folded on GST·RAP (glutathione
S-transferase receptor-associated protein)-Sepharose as
described previously (30). Pure monomeric forms of the fusion proteins
were obtained by size-exclusion chromatography on Superdex 200 columns
in TBSC (150 mM NaCl, 2 mM CaCl2,
25 mM Tris-HCl, pH 7.5). Protein concentrations were
determined by the Bradford method (31) and from
A280 based on the calculated molar extinction
coefficients. The difference between the methods was within 5%.
Instrumentation--
An automated HP3D Capillary Electrophoresis
System (Hewlett Packard, Waldbronn, Germany) was used throughout. It
was equipped with an uncoated fused-silica capillary (Composite Metal
Service Ltd., 51.5-cm effective, 60.0-cm total length, 50-µm inner
diameter) packed in a standard Hewlett Packard cassette and
thermostated at 20 °C during all experiments. Injection was
performed at 50 millibar pressure for 9 s. Between all runs the
capillary was conditioned by aspirating 100 mM NaOH, water,
and BGE for 2 min each applying ~950 millibar pressure. The detector
signals were recorded at 205 nm. In specified cases, fast spectral
scanning mode was used. Positive polarity mode (negative pole is placed at the capillary outlet) with 25 kV was used for all experiments.
CE Separations--
Appropriately diluted virus (10 µl) was
mixed with 10 µl of purified receptor resulting in the molar ratios
indicated in the figures. Incubation was for 60 min at room temperature.
Analysis of Complexes between HRV2 and MBP·VLDLR1-3
by Capillary Electrophoresis--
To assess whether stable complexes
between HRV2 and MBP·VLDLR1-3 were formed in solution, a
constant amount of HRV2 and increasing amounts of
MBP·VLDLR1-3 were mixed, incubated at room temperature,
and analyzed by CE. As depicted in Fig.
1, addition of the receptor to HRV2 led
to a decrease in the virus peak. The peak first became broader,
suggesting the presence of a heterogeneous population of virus-receptor
complexes, but upon further addition of receptor, the complex peak
gradually shifted toward longer migration times and became sharper and
more symmetric again with a concomitant appearance of a peak
corresponding to free receptor. Obviously, under conditions of excess
MBP·VLDLR1-3 the fully saturated complex is formed (Fig.
1, bottom trace). Spectral analysis of the peak assigned as
virus-receptor complex revealed a significant increase of the ratio of
the signals measured at 205 and 260 nm as compared with the ratio of
these values for virus alone. Absorbance at 205 nm is nonspecific,
whereas absorbance at 260 nm indicates the presence of nucleic acid.
For pure HRV2, the ratio of
A205/A260 was found to be
about 6 (as determined for the virus peak in Fig. 1, top
trace) whereas it had increased to a value of about 10 in the peak
corresponding to the complex (as determined from Fig. 1, bottom
trace). This clearly supports the assumption of an increase in
protein content in the complex with respect to the virus. As expected,
there is still an absorption maximum at 260 nm, indicating the presence
of the genomic RNA (spectra not shown).
VLDLR does not bind major receptor group HRVs (8). To assess whether
complex formation was specific, a member of the major group, HRV14, was
incubated with MBP·VLDLR1-3 and analyzed by CE under
conditions identical to those used for analysis of HRV2. As depicted in
Fig. 2, the peak of free receptor was
seen to be clearly separated from the peak corresponding to free HRV14. When compared with the control, no change of migration time or shape of
the virus peak was noted upon incubation with receptor. This
unambiguously indicates the absence of binding of this recombinant receptor to HRV14 and proves the specificity of the interaction with
HRV2.
Determination of Binding Stoichiometry--
To determine the
stoichiometry of the components present in the complex between
MBP·VLDLR1-3 and HRV2, a constant amount of receptor (10 µl at 5.3 µM) was incubated with increasing amounts of
virus (10 µl from 0 to 0.076 µM), and analysis of
complexes formed was carried out by CE (electropherograms not shown).
The peak area of free MBP·VLDLR1-3, normalized to that
of the internal standard, was then plotted versus the total
concentration of HRV2 present in the mixture. The resulting curve was
linear up to 0.01 µM HRV2 (which corresponds to a molar
ratio of virus and receptor of 1:145). At lower ratios the slope
decreases; this is indicative of saturation of binding (Fig.
3). Extrapolation of the linear part of
the curve to zero concentration of receptor (intercept with the
x axis) enables quantitation of the amount of virus required
to bind all receptor molecules present in the mixture. The binding
stoichiometry, as derived from the ratio of these two values was found
to be 61±2:1 for the complex MBP·VLDLR1-3·HRV2. The
error of the ratio is expressed as the S.D. obtained from the estimate
of the intercept by linear fit.
To ensure that the data used for extrapolation are derived under
conditions where the majority of receptor binding sites are occupied,
we plotted the effective mobility of the complex versus the
ratio of receptor and virus in the incubation mix (Fig.
4). The curve was then fitted using the
rectangular hyperbolic equation, which allowed determination of
mobility at 100% saturation. From this, it becomes clear that the
linear part of the curve (Fig. 3), which was used for the
extrapolation, represents data points within at least 95%
saturation (at a virus concentration of 0.1 µM under the
conditions given in Fig. 3).
Assessment of Complex Formation between HRV2 and
MBP·VLDLR1-8 by CE--
In earlier experiments, we have
shown that repeats 3-5 of LDLR were sufficient to protect HeLa cells
against infection with HRV2, whereas minireceptors with only two
repeats failed to protect the cells but were still able to recognize
virus in ligand blots (19). Using the same assay, we have further shown
that bacterially expressed MBP·VLDLR fusion proteins with repeats
1-3 and with repeats 1-8 are active in virus binding, whereas a
minireceptor with repeats 4-6 is inactive (30). This raised the
question of whether the entire ligand binding domain encompassing eight repeats might have only one single binding site for the virus or
whether a second binding site, presumably residing within the carboxyl-terminal repeats, was present in this receptor. In this latter
case, the receptor might allow for cross-linking of multiple virions or
might attach bivalently to any of the symmetry-related receptor binding
sites present on the viral surface. We thus asked whether the
stoichiometry of the components in the complex between HRV2 and
MBP·VLDLR1-8 was different from the complex of HRV2 with
the smaller receptor.
MBP·VLDLR1-8 was incubated with HRV2 under the same
conditions as used for MBP·VLDLR1-3 (see above and Fig.
1) with the exception that the molar ratios between virus and receptor were somewhat different (Fig. 5). Again,
the complex forming at low ratios appeared as a broad peak, which
became sharper upon increasing the receptor concentration. As mentioned
above, this might indicate the transition from a heterogeneous
population of complexes with various degrees of receptor saturation to
homogenous complexes with all virions carrying the same number of
receptor molecules. Nevertheless, comparison of the
HRV2·MBP·VLDLR1-3 complexes (Fig. 1) reveals some
differences. The peaks of the complexes of HRV2 with the larger
receptor are broader at all ratios and appear more heterogeneous.
Furthermore, already at a molar ratio of 1:8 between virus and
MBP·VLDLR1-8, a substantial change in the migration is
apparent. Such an obvious difference cannot be explained exclusively by
the different molecular masses of the receptor fragments (~59
kDa for MBP·VLDLR1-3 versus 81 kDa for
MBP·VLDLR1-8) as the contribution of the difference in
the receptor molecular masses to the total mass of the complex is
negligible (e.g. only 2% in this particular case). It might therefore rather reflect higher affinity of the virus for
MBP·VLDLR1-8 as compared with
MBP·VLDLR1-3 resulting in a larger number of receptor
molecules bound per virion under equilibrium conditions.
These differences can be better appreciated from the plot of effective
mobilities of both complexes versus the ratios of receptors and virus in the incubation mix (Fig. 4). From this representation, the
steep increase in the mobility is clearly visible for the larger
receptor. This might be taken to indicate higher affinity (see
"Discussion"). Disregarding the region of very low molar ratios of
receptor/HRV2, both curves have the form of a classical binding
isotherm attaining a plateau at seemingly full saturation and can be
fitted using a rectangular hyperbolic function. The deviation from the
hyperbolic appearance might indicate that binding sites on the viral
surface exhibit somewhat different affinities for the receptors.
Different Stability of the Complexes between HRV2 and
MBP·VLDLR1-3 versus MBP·VLDLR1-8--
In
previous work (25), we discovered that the presence of low
concentrations of SDS is required for a reliable separation of virus
from contaminants or from virus-antibody complexes. Fortunately, the
virus-receptor complexes turned out to be stable in SDS at the
concentrations used. This is reminiscent of the finding that the
receptors bind virus in ligand blots made after polyacrylamide gel
electrophoresis in the presence of SDS, provided that no reducing agent
is added. Assuming a correlation between attachment mode and stability
of the complexes in the presence of SDS, the two different receptor
fragments were incubated with HRV2 under identical conditions.
Dissociation of preformed complexes by the addition of SDS was then
investigated by CE. When preformed MBP·VLDLR1-3·HRV2 complexes were incubated at room temperature in 10 mM SDS,
dissociation was clearly evident at about 20 min, whereas the
MBP·VLDLR1-8·HRV2 complexes failed to dissociate even
when incubated for several hours in the presence of 20 mM
SDS (data not shown). This is taken to indicate that the latter
receptor binds more strongly to the virus under these particular conditions.
Stoichiometry of the Reaction between HRV2 and
MBP·VLDLR1-8--
The stoichiometry of the components
in the complex between HRV2 and MBP·VLDLR1-8 was
determined as described for the binding of MBP·VLDLR1-3
to HRV2. A constant amount of receptor was incubated with an increasing
amount of HRV2, and each sample was analyzed by CE (electropherograms
not shown). The peak area of free MBP·VLDLR1-8 was then
plotted versus the total virus concentration in the mixture
(Fig. 6). This resulted in a hyperbolic relationship with an initial linear range up to ~0.013
µM HRV2 (which corresponds to a ratio of receptor to
virus of 140:1). Again, the data points in the linear part of the curve
correspond to at least 95% saturation of virus with receptor as
determined in the same way as for MBP·VLDLR1-3 (see Fig.
5). When this linear part of the curve was extrapolated to zero
concentration of free receptor (intercept with x axis), the
amount of virus needed to completely bind the given amount of receptor
is obtained. The stoichiometry for the complex
MBP· VLDLR1-8·HRV2 as derived from these data was 29 ± 2:1. This is about half of the value seen for
MBP·VLDLR1-3 (see above).
Analysis of the binding stoichiometry between a prototype virus of
the minor receptor group of human rhinoviruses (HRV2) and a member of
the low density lipoprotein receptor family (VLDLR) was analyzed by CE.
Previous attempts at isolating defined complexes between various
recombinant minireceptors derived from LDLR and expressed in the
baculovirus system failed possibly because of extensive aggregation
(18) or too low affinity (19). Based on the observation of comigration
of the chicken ovarian homolog of this receptor with HRV2 on sucrose
density gradients (6) and that of a strong reaction of a VLDL receptor
fragment shed from HeLa cells with HRV2 in ligand blots (7, 32), we
reasoned that this receptor might exhibit higher affinity toward minor group HRVs than the LDL receptor. Making use of two different VLDL
receptor fragments, one encompassing repeats 1-3 and the other the
whole ligand binding domain with repeats 1-8 expressed as
maltose-binding protein fusions in bacteria (30), we analyzed the
binding stoichiometry by capillary electrophoresis. We thus obtained
circumstantial evidence for the longer receptor binding more strongly
to HRV2 and found that about 60 MBP·VLDLR1-3 molecules
were accommodated on the viral surface, but only about 30 molecules of
MBP·VLDLR1-8 bound to HRV2. This leaves us with the
question of whether the additional repeats inhibit binding of 60 copies
of the receptor (as seen for MBP·VLDLR1-3) because of
steric hindrance or whether they are able to simultaneously attach to
another site related by icosahedral symmetry. There are some arguments
in favor of the latter interpretation. (i) The interaction between
MBP·VLDLR1-8 and HRV2 is clearly stronger than that of
MBP·VLDLR1-3 as shown by the lower concentration
required to appreciably shift the viral peak. (ii) The complete absence
of aggregation indicates that MBP·VLDLR1-8 is not able
to bind to two virions simultaneously, and (iii) a receptor fragment
containing only repeats 4-6 does not bind HRV2 (30) and might act as
an inert spacer between repeats 1-3 and repeats 6-8. Furthermore,
there are nine additional amino acids between repeats 5 and 6 that have
been proposed to impart some flexibility to the molecule allowing the
amino terminus and the carboxyl terminus of the molecule to approach
each other (33). Indeed, a recent model of the LDL receptor, as
obtained by electron cryo-microscopy image reconstruction, suggests
that the ligand-binding repeats are not arranged linearly, but there
might be a kink between repeats 3 and 4 (34). This could result in a
conformation of the molecule, which allows for the simultaneous
attachment of amino-terminal and carboxyl-terminal repeats to two
symmetry-related viral binding sites. The recent structure
determination of a concatamer of ligand-binding repeats 1 and 2 of
human LDLR by NMR technology (35) revealed that these two repeats could
move freely with respect to each other, and it is thus likely that the
whole molecule can adopt various conformations. This might be one of
the reasons for its potential to interact with a number of structurally
unrelated ligands. Based on an approximate length of 3 nm for one
repeat, the total length of the ligand binding domain can be estimated to be about 24 nm. If only two repeats might be available to function as spacers (with the remaining six being engaged in interactions with
the virus) the distance between the viral 5-fold axes (about 16 nm)
might not permit binding over the 2-fold axes of viral icosahedral
symmetry. However, other modes of attachment have also been observed.
In the case of rabbit hemorrhagic disease, on virus-like
particles, which exhibit 180 equivalent monoclonal antibody binding
sites, steric hindrance prevents the simultaneous occupation of 2-fold
symmetry-related sites, and the antibodies bind bivalently over local
3-fold axes resulting in 50% occupation (36). If
MBP·VLDLR1-8 indeed adopts a conformation with a pseudo
2-fold symmetry and binds bivalently, it will be of great interest to
determine the nature of the interactions between the viral
symmetry-related sites and the two clusters of ligand-binding repeats
located at the termini of the receptor molecule.
Recent data of cryo-electron microscopy image reconstruction of
complexes between HRV2 and MBP·VLDLR1-3 point to a
binding site different from the canyon floor with five receptor
molecules attaching very close to the 5-fold axes of symmetry (20).
Structure determination of complexes between HRV2 and the receptor
containing all eight ligand binding repeats will finally clarify
whether the lower stoichiometry of the larger receptor results from
bivalent binding or only from steric effects.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Formation of complexes between
MBP·VLDLR1-3 and HRV2. A constant amount of HRV2
(10 µl at 0.03 µM) was incubated with increasing
amounts of MBP·VLDLR1-3 (10 µl ranging from 0 to 8.75 µM) and analyzed by CE. The molar ratios between HRV2 and
MBP·VLDLR1-3 are indicated on the right.
IS, internal standard.
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Fig. 2.
No complex is formed between HRV14 and
MBP·VLDLR1-3. Upper trace, CE analysis
of HRV14 (0.03 µM); lower trace, HRV14 (10 µl at 0.03 µM) and MBP·VLDLR1-3 (10 µl
at 1.8 µM) were mixed, incubated, and analyzed by CE. The
ratios of the components in the mixtures are indicated on the
right. IS, internal standard.
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[in a new window]
Fig. 3.
Determination of the stoichiometry of HRV2
and MBP·VLDLR1-3 in the saturated complex.
MBP·VLDLR1-3 (10 µl at 5.3 µM) was
incubated with 10 µl of HRV2 at concentrations ranging from 0 µM to 0.076 µM. The mixtures were then
analyzed by CE. The peak area corresponding to free receptor
versus the concentration of total HRV2 present in the
mixtures is shown. Each point represents the mean of two independent
experiments (typical relative span of 6.5%). The molar ratios of
receptor/virus are indicated for each point.
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Fig. 4.
Dependence of the effective mobility of the
complexes between HRV2 and two different receptor fragments from the
receptor/virus molar ratios. The effective mobility (total
mobility minus electroosmotic mobility) of the complexes formed at
various molar ratios between receptor and virus are shown for
MBP·VLDLR1-3 and for MBP·VLDLR1-8,
respectively. Data were taken from experiments carried out essentially
as described in Figs. 1 and 4. The mean of two independent experiments
carried out in duplicate (typical relative span of 4%) is shown for
each receptor.
View larger version (23K):
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Fig. 5.
Formation of complexes between
MBP·VLDLR1-8 and HRV2. A constant amount of HRV2
(10 µl at 0.026 µM) was incubated with increasing
amounts of MBP·VLDLR1-8 (10 µl ranging from 0 to 3.6 µM) and were analyzed by CE. The molar ratios between
HRV2 and MBP·VLDLR1-8 are indicated on the
right. IS, internal standard.
View larger version (12K):
[in a new window]
Fig. 6.
Determination of the stoichiometry of HRV2
and MBP·VLDLR1-8 in the saturated complex.
MBP·VLDLR1-8 (10 µl at 3.6 µM) was
incubated with 10 µl of HRV2 at concentrations ranging from 0 µM to 0.1 µM. The mixtures were then
analyzed by CE. The total concentration of HRV2 present in the mixtures
versus the normalized peak area corresponding to free
receptor is shown. Each point represents the mean of two independent
experiments (typical relative span 4.5%). The molar ratios of
receptor/virus are indicated for each point.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Irene Goesler for viral preparations and excellent tissue culture work.
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FOOTNOTES |
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* This work was supported by Austrian Science Foundation Grants P-12269-MOB (to D. B.) and P-13504-CHE (to E. K.).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.
§ Present Address: Lambda, Labor für Molekularbiologische DNA-Analysen Ges. m. b. H., Industriestrasse 6, A-4240 Freistadt, Austria.
¶ To whom correspondence should be addressed. Tel.: 43 1 4277 61630; Fax: 43 1 4277 9616; E-mail: dieter.blaas@univie.ac.at.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M008039200
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ABBREVIATIONS |
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The abbreviations used are: HRV, human rhinovirus; CE, capillary electrophoresis; LDLR, low density lipoprotein receptor; VLDLR, very low density lipoprotein receptor; BGE, background electrolyte; ICAM-1, intercellular adhesion molecule 1; MBP, maltose-binding protein; GST, glutathione S-transferase.
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REFERENCES |
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