(Received for publication, July 6, 1995; and in revised form, September 5, 1995 )
From the
The kinetics of ligand binding by Se155-4, an antibody
specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance.
Because trace amounts of oligomers in Fab and single-chain antibody
variable domain (scFv) preparations resulted in biphasic binding
profiles that were difficult to analyze, all kinetic measurements were
performed on purified monomeric fragments and, for certain mutant scFv,
dimeric forms. Results obtained with monomeric forms indicated that the
relatively low affinity of the antibody was due to rapid dissociation (k
0.25 s
). Dimeric
forms generally showed off-rates that were approximately 20-fold slower
and a 5-fold increase in association rate constants to approximately 2
10
M
s
. Although the association phases for scFv
dimers showed good curve fitting to a one component interaction model,
the dissociation phases were biphasic, presumably because the
availability and accessibility of sites on the antigen always leads to
some monovalent attachment. The fast off-rate for dimers was the same
as the monomer off-rate. Se155-4 IgG off-rates were very similar
to those observed for scFv dimer, whereas the on-rate was the same as
that obtained with Fab and scFv monomer.
The relatively weak affinities that characterize protein-carbohydrate interactions make understanding how biological processes are mediated by oligosaccharides difficult. However, it is becoming evident, particularly for certain plant and animal lectins, that specificity is achieved by a combination of multivalence and the geometry of the subunit arrangement. There is now a considerable amount of data available on structural and energetic aspects of protein-carbohydrate interactions(1, 2) . General structural features are the stacking of aromatic side-chains against the sugar rings, the presence of hydrogen bond networks in which the sugar OH groups act as both acceptors and donors, and the coordination of multiple hydrogen bonds by water molecules(1) . As for their energetics, titration microcalorimetry has indicated that these interactions are usually enthalpy-driven and that water reorganization, especially desolvation, is a key feature of complexation(2) . Kinetics of the interactions are less well known but are obviously important in understanding the subtleties of carbohydrate-mediated biological events. Without consideration of the time factor, analysis of these interactions from a biological standpoint is difficult. The development of surface plasmon resonance techniques has provided an opportunity to explore biomolecular interaction in real time.
We
have applied SPR ()technology to the analysis of antigen
binding by the antibody Se155-4. The specificity of Se155-4
is dominated by a 3,6-dideoxyhexose, abequose, presented by the
lipopolysaccharide O-antigen of Salmonella serogroup
B bacteria(3) . This polysaccharide repeating unit is built
from four hexopyranose units:
{
2)[
D-Abe(1
3)]
D-Man(1
4)
L-Rha(1
3)
D-Gal(1
}.
The structural (4) and energetic (5, 6) aspects of antigen binding to Se155-4
have been well characterized, and an efficient Escherichia coli expression system is available for the production of antibody
fragments(7, 8) .
Initial SPR investigations of antigen binding by wild-type Se155-4 scFv revealed a distinctly biphasic association profile(8) . A subsequent study on the isolation of scFv mutants with enhanced binding properties from complementarity-determining region-randomized phage display libraries showed that mutants were selected primarily on their propensity to form dimers(9) . It was observed that mutants that existed largely in the dimeric state displayed classical association kinetics, which in turn led to the realization that the wild-type biphasic association kinetics were the result of trace amounts of scFv dimer in the preparations. In this report we describe the detailed analysis by SPR of Se155-4 IgG, Fab, and scFv binding to immobilized antigen.
For the calculation of association
rate constants, samples were appropriately diluted in HEPES-buffered
saline and analyzed at several antibody concentrations. Samples which
gave a significant buffer-related RU change on injection were also
analyzed on a blank BSA surface. For the calculation of dissociation
rate constants, relatively high concentrations of antibody were used,
and the kinject command was used to inject 10 µl of 100 µM free trisaccharide,
-D-Gal(1
2)[
-D-Abe(1
3)]
-D-Man,
over the sensor chip surface immediately following the sample
injection.
where R is the response at time t, R
is the amplitude of the response, and k
is the dissociation rate constant.
Dissociation of two components can be treated as the sum two
independent events where each is described by an equation of the above
form. The association rate constant can then be derived using the
equation
where R is the response at time t, R
is the maximum response, C is the
concentration of ligate in solution, k
is the
association rate constant, and k
is the
dissociation rate constant. A two component association is treated as
the sum of two independent events, each described by an equation of the
above form.
Rate constants were also calculated by using linear transformations of the sensorgram data and the equation
where K is the slope of a
dRU/dt versus RU plot. A range of concentrations are analyzed,
and a plot of K
versus C has a slope of k
and a Y-intercept of k
.
Affinities were calculated from rate constants and from analysis of equilibrium binding. By measuring equilibrium resonance units as a function of ligand concentration, binding data can be analyzed by Scatchard plots using the equation
where R is the equilibrium resonance
units, R
is the resonance signal at saturation, C is the concentration of free protein, and K
is the association constant. A plot of R
/C versus C has a slope of
-K
.
Figure 1: Relative response versus time for the binding to immobilized BSA-O-polysaccharide of wild-type Se155-4 scFv containing trace amounts of dimer (A), monomeric wild-type scFv obtained by size-exclusion HPLC (B), and mutant B5-1, which exists almost entirely in the dimeric form (C). The concentrations are (from bottom to top) 1, 1.5, 2, 3, and 5 µM in A; 0.2, 0.4, 0.8, 1.2, and 1.6 µM in B; and 20, 40, 80, 120, and 200 nM in C.
Figure 2:
Effect on off-rate measurement of the
immediate injection of free trisaccharide following antibody injection.
Primary dissociation data for monomeric SK4 (A) and dimeric
B5-1 (B) in the absence () and presence (
) of
free trisaccharide are shown.
Figure 3: Curve fitting of monomeric wild-type scFv dissociation phase to the BIA evaluation single-component model. Dissociation data were collected in the presence of free trisaccharide and yielded the monomer off-rates given in Table 1.
The
dissociation data, collected in the presence of free trisaccharide, for
single-chain Fv containing trace amounts of dimer fit better to the
BIAevaluation biexponential decay function describing the dissociation
of two components from the immobilized antigen than to the single
exponential expression (Fig. 4, A and B). This
was also true for dimer (mutant B5-1) dissociation (Fig. 4, C and D) and is thought to result
from both monovalent and bivalent attachment of the dimers to the
surface. The availability and accessibility of sites on the repeating
epitope antigen would always lead to some monovalent attachment,
particularly at antibody concentrations that give binding approaching R. In each instance, the faster of the two
off-rates was quite similar to the rates obtained with pure monomers,
although the short linker scFv (SLA-1), IgG, and B4-3 had
somewhat slower rates (Table 1).
Figure 4: Curve fitting of wild-type scFv containing trace amounts of dimer (A and B) and B5-1 scFv (C and D) dissociation phases to the BIAevaluation single-component (A and C) and two-component (B and D) models. Analyses were performed on dissociation data collected in the presence of free trisaccharide ligand and generated the two off-rates, given in Table 1, for samples containing dimer.
Figure 5:
Nonlinear analysis of wild-type scFv and
B5-1 scFv association phases. Monomeric wild-type scFv (A) and wild-type scFv containing trace amounts of dimer (B) were fitted to the BIAevaluation one-component and
two-component models, respectively. B5-1 dimer (C) was
fitted to the one-component model. The monoexponential association
model was used to generate the k values presented
in Table 1.
The binding profiles obtained with IgG and scFv
preparations that were predominantly dimer were more amenable to
on-rate analysis. Using the nonlinear analysis method, the plotted
residuals indicated that the association data fit well to the single
exponential function (Fig. 5C). Association rate
constants were approximately 2 10
M
s
for the three
scFv dimers that were examined (Table 1). Interestingly,
Se155-4 IgG was observed to have an association rate constant
that was 3-5 times slower than the dimeric scFv (Table 1).
On-rates determined by linear transformation of the data and the
concentration dependence of binding were in excellent agreement with
those obtained by nonlinear fitting of the primary data (Table 1, Fig. 6).
Figure 6:
Linear regression analysis of kversus C plots for dimeric
B5-1 on-rate determination. The slopes of these plots yielded the
the k
values presented in Table 1. conc, concentration.
Figure 7: Scatchard plots for the determination of affinities from equilibrium binding. A, monomeric wild-type scFv. B, dimeric B5-1. C, Se155-4 IgG.
The kinetics of complex association and dissociation is an aspect of carbohydrate recognition by proteins that has been virtually inaccessible, but it is only when this component is also considered that a complete picture will emerge as to how carbohydrates exert their biological activity. It is possible to obtain rate constants for protein-carbohydrate interactions by fluorescence (16) and NMR (17) methods, but these approaches are not generally applicable. Shinohara et al.(18) demonstrated that SPR could be used to study the specificities of different lectins for various glycopeptides but did not attempt to analyze the contribution of lectin valence to observed rate constants. The present investigation represents the first comprehensive effort to derive kinetic constants for a protein-carbohydrate interaction by means of SPR and is another demonstration that SPR is a very useful tool in the study of low affinity interactions. Other examples are peptide/major histocompatibility complex binding to T-cell receptors (19, 20) and transient cellular interactions involving cell adhesion molecules (21) .
The data presented here show
that extreme care must be exercised in the interpretation of SPR data
for protein-sugar interactions, largely because of the repetitive
nature of most carbohydrate ligands. Failure to recognize bivalent and
multivalent interactions can lead to misinterpretation of results and
overestimations of the intrinsic affinities of these systems. Others
have also cautioned that multimeric interactions may be a common source
of incorrect results when measuring low affinity interactions by
SPR(21) . Another important precaution is that samples be run
on control surfaces, because at the protein concentrations required for
analysis of low affinity binding, the amounts of free protein entering
the dextran matrix of the sensor chip are sufficient to give a
substantial increase in RUs. When properly collected, the SPR data
yielded affinity measurements that were in good agreement with those
obtained by titration microcalorimetry. Using equilibrium binding data
obtained by SPR, the Kvalues of
Se155-4 Fab and wild-type scFv were estimated to be 6.5
µM and 6.8 µM, respectively, for interaction
with O-polysaccharide, compared with microcalorimetry values
of 4.8 µM for Fab and 7.7 µM for scFv, with
trisaccharide ligand.
The observation that the biphasic association profiles obtained with some scFv preparations are characteristic of monovalent fragments containing trace amounts amounts of dimer or higher oligomer necessitates some reinterpretation of earlier results for Se155-4 binding(8, 9) . It is now clear that previous analyses of biphasic association profiles were conducted on the part of the sensorgram resulting from binding of the minor dimeric component. Although the faster on-rates exhibited by dimers partially compensated for the overestimation of binding analyte concentrations, the reported on-rates for the wild-type Se155-4 scFv were, nonetheless, somewhat lower than the actual monomer values.
The
results presented here address the question of how much of an affinity
gain is conferred by the presence of a second combining site. Crothers
and Metzger (22) proposed that the observed affinity of a
molecule such as IgG is the product of two binding constants, the first
being that of a monovalent fragment of the antibody and the second
being the dimensionless constant for the second site, once the first
site is occupied. A gain is only obtained if the antigen is multivalent
or attached to a surface. However, Jencks (23) has pointed out
that the binding energy of a dimeric molecule contains an interaction
energy term and is not simply the sum of the two component binding
energies. A comparison of monomeric and dimeric Se155-4 scFv
showed that divalency led to a 5-fold increase in association rate. A
similar finding was reported for IgG3 binding to a multivalent
carbohydrate antigen, and the effect was attributed to the tendency of
IgG3 to aggregate(24) . However, Se155-4 IgG did not
exhibit a significantly faster on-rate than the monovalent forms. This
may reflect a more unfavorable interaction energy because of less
flexibility or more unfavorable steric interactions associated with the
IgG form. The dissociation data, collected in the presence of free
trisaccharide, indicate that divalency decreases off-rates
approximately 20-fold, which in combination with the faster on-rates
translates into a 100-fold increase in functional affinity due to scFv
bivalency. However, the dimer dissociation rates in the absence of free
ligand, which are approximately 2 10
s
(9) , may give a more accurate
indication of the effect of divalency in vivo. If these
off-rate values are used in affinity calculations, a 1000-fold gain is
conferred by scFv bivalency.
In some instances at least, the
considerable amount of monovalent attachment of bivalent molecules as
binding approaches R enables the calculation of
monovalent or intrinsic affinities. Although it is difficult to derive
on-rates from the association phases of sensorgrams for analyte
concentrations giving near R
binding, analysis
of the dissociation phases yields both monomeric and dimeric off-rates.
Analysis of binding at low analyte concentration gives accurate
measurement of dimer on-rates. For Se155-4 IgG, the fact that the
IgG on-rate was the same as that of the monovalent Fab fragment made it
possible to calculate an an intrinsic affinity from rate constants that
was in good agreement with that obtained by microcalorimetry. This
should be possible for other multivalent interactions with low
intrinsic affinities, such as lectin-carbohydrate binding, provided the
off-rates are not too rapid to be measured by SPR and that the monomer
and dimer on-rates are similar.
The extremely fast dissociation rates were the most striking feature of the binding kinetics described here. Presumably, this is typical of protein-carbohydrate interactions in general and is the reason for their low intrinsic affinities. Fluorescence (16) and NMR (17) methods have given on-rates for other protein-carbohydrate interactions that are similar to those reported here. These methods have also indicated that fast off-rates are responsible for the low affinities of protein-carbohydrate interactions and that differences in binding constants are primarily the result of differences in off-rates. It is not known if there is a biological reason for these fast rates or if they result from the types of atomic interactions that drive protein-carbohydrate interactions. It may reflect the fact that higher intrinsic affinities are not necessary because repeating epitopes and clustering of glycoproteins and glycolipids on membrane surfaces give large effective ligand concentrations and permit adequate binding through multivalent attachment. Monomeric affinities that are not higher than those needed for exertion of biological function may safeguard against self-recognition.
In summary, SPR has been shown to provide new insights into the subtleties of protein-carbohydrate interactions. The affinity gain conferred by bivalency has been quantitated, and it was shown that scFv dimers offer an advantage over whole antibodies in this regard. The observation that rapid off-rates are responsible for the low intrinsic affinity of Se155-4 and probably carbohydrate-binding proteins in general raises the possibility of engineering higher affinity by manipulating the dissociation rate constants.
This is National Research Council of Canada Publication 39507.