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INTRODUCTION |
Histidine-rich glycoprotein
(HRG)1 is a 75-kDa protein
that exists at relatively high levels in the plasma of many vertebrate species including humans (1) and has been the subject of investigation by a number of laboratories. Although the exact physiological function
of HRG is unknown, a number of ligands for the molecule have been
identified; these include heme (2), divalent metal ions (2), heparin
(3, 4), heparan sulfate (5), plasminogen (6, 7), fibrinogen (8), and
thrombospondin (9). HRG has been proposed to have a modular structure
allowing it to bind several ligands independently (10), and evidence
suggests that the interaction of HRG with glycosaminoglycans is
regulated by metal ions and pH (11). HRG also has been found to be a
regulator of heparin binding growth factor action (5), lymphocyte
proliferation (12), and lymphocyte cell adhesion (13-15). Recently, we
showed that HRG binds strongly to immunoglobulin (IgG) and inhibits the formation of insoluble immune complexes (IICs) between ovalbumin and
polyclonal rabbit anti-ovalbumin IgG in vitro (16). These latter findings have implicated HRG in regulating IIC formation in vivo, particularly in relation to patient suffering from
autoimmune diseases like rheumatoid arthritis and systemic lupus
erythematosus, where there is an excessive production of autoantibodies
and the formation of IICs (17-19). The failure of IICs to be cleared
from the blood circulation in these patients may result in the
deposition of IICs in specific target tissues, and this may be an
important factor in the pathogenesis of these diseases (20). The
associated pathology may be caused, at least in part, by the ability of
the deposited IICs to activate the complement pathway and to induce inflammation in the target tissue and/or by tissue injury resulting from decreased nutrient transport (21).
Immunoglobulin molecules consist of two identical heavy (H)- and light
(L)-chains, with the variable regions of the H- and L-chains
associating to form the antigen binding site of the antibody. There are
nine types of H-chains in humans (µ, 
, 
1, 2, 3, 4,
1, 2, and 
) that define a range of classes and subclasses. The
functional role of each of the H-chains, with the exception of the chain, is well characterized (22). In addition, in many species there
are two types of L-chains, termed 
and 
, but unlike the different
H-chain types, their function is unknown (22). Our recent finding that
HRG binds to human IgG with high affinity and inhibits the formation of
IICs (16) has raised the question of whether HRG binds with the same
affinity to the different IgG subclasses (which can possess either or L-chains). In the present study we use optical biosensor
techniques to study the binding of HRG to several different subclasses
of IgG and also to IgM. Our results show that HRG has a differing
affinity for the various IgG subclasses but that the L-chain type of
IgG also has a marked effect on HRG binding. Thus, the affinity of HRG
for the forms of IgG1 and IgG2 is at least 10-fold greater than for
the forms of IgG1 and IgG2 (and other IgG subclasses), with these
interactions being significantly affected by the presence of
Zn2+. The results provide strong evidence that the binding
of HRG to IgG molecules is profoundly influenced by both the H- and
L-chain isotype of the IgG molecule, an observation that may have
relevance to the ability of HRG to inhibit the insolubilization of
IgG-containing immune complexes (IC).
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MATERIALS AND METHODS |
Reagents--
Human myeloma IgG1, IgG1, IgG2, IgG2,
IgG3, IgG3, and IgG4, were purchased from Sigma; IgG4 and
IgM were from Calbiochem-Novabiochem and Accurate Chemicals,
respectively. Bence Jones (BJ) proteins were a generous gift from Dr.
Bob Raison, University of Technology, Sydney. Human IgG (isolated from
pooled human serum), bovine serum albumin (BSA, fraction V), aprotinin,
polyoxyethylenesorbitan monolaurate (Tween 20), phosphate-citrate
buffer with sodium perborate capsules and 2,2'-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) diammonium (ABTS) were
purchased from Sigma. Carboxymethyl dextran cuvettes for the IAsys
biosensor, 1-ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDC),
N-hydroxysuccinimide (NHS), and ethanolamine were purchased
from Fisons Affinity Sensors, Cambridge, UK. Streptavidin (STP) was
purchased from Progen Industries, Australia. Sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC-biotin), gentle Ag/Ab elution buffer
(a cuvette regeneration buffer gentle to the dextran matrix), and
horseradish peroxidase (HRP)-conjugated STP (HRP-STP), were purchased
from Pierce.
Purification of Native Human HRG--
Native human HRG of 75-kDa
molecular mass was purified from fresh human plasma as described
previously (23) by equilibrating a phosphocellulose column with loading
buffer containing 10 mM sodium phosphate (pH 6.8) that
contained 1 mM EDTA and 0.5 M NaCl. The plasma
was mixed with EDTA and NaCl to final concentrations similar to the
loading buffer and with 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride (AEBSF) (ICN Pharmaceutical Inc., Costa Mesa, CA) at 100 µg/ml and aprotinin (a trypsin inhibitor) at 2 µg/ml. The plasma
was passed through the equilibrated column, and unbound protein was
removed by extensive washing of the column with the loading buffer.
Bound HRG was then eluted from the column using the same buffer
containing 2 M NaCl.
Biotinylation of Proteins--
The proteins to be biotinylated
(e.g. IgG subclasses, HRG, IgM, BSA, BJ proteins) were
dissolved in 10 mM sodium phosphate buffer (pH 7.2)
containing 150 mM NaCl (PBS) at a concentration of 1 mg/ml
and then reacted with 1 mg/ml NHS-LC-biotin for 30 min at room
temperature. The reaction was stopped by the addition of Tris-HCl
buffer (pH 8.0) to a final concentration of 100 mM. Unreacted biotin was removed by washing the sample extensively (five
cycles of concentration and dilution) in a Centricon 10 microconcentrator (Amicon Inc) before storing the biotinylated proteins
at 
20 °C in small aliquots until use.
Preparation of BJ Proteins--
The lyophilized and BJ
proteins (BJ and BJ) were dissolved in PBS. SDS-polyacrylamide
gel electrophoresis analysis showed that BJ consisted of two bands
of molecular masses ~ 22 kDa and ~ 44 kDa, whereas the
BJ consisted of only a single band of molecular mass ~ 44 kDa. These proteins were subjected to gel filtration using a Superose
12 fast protein liquid chromatography column to separate the monomeric
from the dimeric form of each protein, particularly for BJ; BJ
showed only a single peak, demonstrating that it existed exclusively in
a dimer form. The dimeric form of each protein was biotinylated for use
in all ELISA and biosensor studies.
Binding of IgG Subclasses, IgM, and BJ Proteins to Immobilized
HRG--
The binding of biotinylated forms of human IgG subclasses
(myeloma-derived), IgM, and BJ proteins to immobilized human HRG was
examined by ELISA. HRG (20 µg/ml) was immobilized on Maxi-Sorb ELISA
trays by incubating in 0.1 M NaHCO3 buffer (pH
9.6) for 1 h at 37 °C. The trays were blocked with PBS
containing 10 mg/ml BSA and 3 mM NaN3
(PBS-BSA-Az) for 1 h at 37 °C and then incubated with
biotinylated proteins in the concentration range between 1.6 and 100 µg/ml in PBS-BSA-Az for 3 h at 37 °C. Unbound proteins were
washed away three times with PBS containing 10 mg/ml BSA and 0.05%
Tween 20 (PBS-BSA-T). Bound biotinylated proteins were detected by
incubating the trays with HRP-STP (in 1/500 dilutions) in PBS
containing 10 mg/ml BSA (PBS-BSA) for 1 h at 37 °C. This was
followed by incubating the trays with the colorimetric HRP substrate
ABTS (0.2 mg/ml) for 30 min at 37 °C in a 0.05 M sodium citrate, 0.1 M sodium phosphate buffer (pH 5.0) containing
0.03% (v/v) sodium perborate (a substitute for hydrogen peroxide). The absorbance of the solution of each well was measured at 405 and 490 nm
using an ELISA plate reader (dual wavelength measurement). The results
showed that the binding of these proteins to immobilized HRG was
concentration-dependent. There was no color development in
control experiments using trays coated with BSA instead of HRG or when
the trays were incubated with biotinylated BSA (b-BSA) instead of the
biotinylated proteins. These control experiments, therefore, indicated
that the binding of these proteins was specific for HRG.
Determination of Binding Constants Using the Biosensor--
An
IAsys resonant mirror biosensor (Affinity Sensors, Cambridge, UK) (24,
25) with a carboxymethyl dextran-sensing cuvette was used to determine
the kinetic constants and affinities of the binding of HRG to
immobilized ligands (e.g. IgG, IgM, and BJ proteins). Except
where indicated, all experiments were performed in PBS-BSA-T and at a
temperature of 25 °C. The BSA (1%, w/v) was included in the buffer
to reduce any nonspecific binding of the proteins and also to buffer
the effects of any added Zn2+ (in the experiments where
Zn2+ was included), since a large proportion of plasma
Zn2+ is bound to albumin (26). The reaction vessel was
stirred continuously by the aid of a propeller. Binding was measured at
2-s intervals, and the readout from the biosensor was in units of
arc-s. Each binding reaction was routinely followed for 5 min. All
binding experiments were performed at least in duplicate. The Fast Fit program supplied by Fisons was used to evaluate the kinetic constants (27).
Coupling of STP to the Dextran Matrix--
STP was coupled via
-amino groups to the carboxymethyl dextran-sensing surface of the
IAsys biosensor cuvette using EDC and NHS (24, 25). This was done by
equilibrating the cuvette in PBS buffer containing 0.05% Tween 20 (PBS-T) and then reacting the cuvette with a mixture of EDC/NHS for 7 min. Unreacted EDC/NHS was washed away with PBS-T followed by three
washes with 0.01 M sodium acetate buffer (pH 4.5). STP (50 µg/ml) in indicated acetate buffer was added to the cuvette and
allowed to react with the activated carboxyl groups for 5 min.
Uncoupled STP was removed by washing with the acetate buffer, and
unreacted succinimidyl groups were blocked by incubating with
ethanolamine (1 M, pH 8.5) for 2 min. The cuvette was
washed three times with the acetate buffer and then washed with PBS-T
followed by a wash with 10 mM HCl to remove any
noncovalently bound protein. A biosensor response of ~500 arc s for
the immobilized STP was observed. According to the data provided by the
manufacturer this response represents the coupling of 3 ng of
STP/mm2 of sensing surface.
Binding of Biotinylated Proteins to the Biosensor
Surface--
Biotinylated proteins (e.g. b-IgG subclasses
or biotinylated IgM (b-IgM)) were coupled to the immobilized STP as
follows. Biotinylated protein (50 µg/ml) in PBS-T (e.g.
IgG or HRG) was added to the STP-coupled dextran cuvette equilibrated
in PBS-T and allowed to bind to the immobilized STP for 5 min.
Nonspecifically bound biotinylated protein was removed by washing three
times with PBS-T, followed by three washes with 10 mM HCl
for 2 min (for 3 cycles).
As noted previously (16), our studies indicate that the ability of HRG
to bind to immunoglobulins is lost when the HRG is biotinylated and
immobilized by binding to streptavidin that has been covalently
attached to the biosensor surface. Similarly, the ability of HRG to
bind IgG is lost when the HRG is immobilized directly by the use of the
homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate
(BS3). Interestingly, HRG immobilized by these procedures is still able
to bind the complement component C1q (16), suggesting that some
functions of HRG are unaffected by the immobilization. The most likely
explanation for these observations is that the free -amino groups on
the HRG involved in biotinylation and cross-linking reactions are
located close to the immunoglobulin binding region(s), and that this
region(s) is sterically hindered upon immobilization of the HRG.
Biosensor studies of the binding of HRG to immunoglobulins, therefore,
could only be carried out by examining the binding of soluble HRG to
immobilized immunoglobulins.
Binding of Protein Ligands to Proteins on the Biosensor
Surface--
Before immobilization of the biotinylated proteins on the
STP-dextran, nonspecific binding between the protein ligands and the
STP-dextran was assessed by the addition of different amounts of the
nonbiotinylated protein ligands in PBS-BSA-T to the cuvette. Under
these conditions no significant level of nonspecific binding of any of
the protein ligands used in this study could be detected.
Preliminary experiments were performed to establish the concentration
range of protein-ligand suitable for kinetic analysis. PBS-T was added
to the protein-coupled cuvette to establish a base line (5 min), and
protein-ligand was then added in PBS-T at different concentrations.
Binding of the protein-ligand was studied by monitoring the association
phase for 5 min. Subsequently, the cuvette was washed with PBS-T, and
the dissociation phase was monitored for 5 min. Bound protein-ligand
was removed (cuvette regeneration) by washing with either 10 mM HCl or the gentle Ag/Ab elution buffer (Pierce). The
base line was then re-established after washing the cuvette with PBS-T.
As previously (16), we found no evidence that the 10 mM HCl
cuvette regeneration wash significantly affected the ability of the
proteins to subsequently interact with immobilized proteins on the
cuvette surface.
Evaluation of the Kinetic Constants--
The IAsys biosensor was
provided with a digital DECpc 450D2LP computer. Data
obtained with the biosensor were transferred directly to the Fast Fit
program (Fisons Applied Sensor Technology). This program uses an
iterative curve-fitting to derive the observed rate constant and the
maximum response at equilibrium due to ligand binding at the particular
ligand concentration. The association of a soluble ligand with an
immobilized macromolecule can be described by the pseudo first order
equation Rt = R0 + E(1 e
k obst), when only
one binding site is available for the ligand (24). In this equation,
Rt is the IAsys response at time t in
units of arc s (this is proportional to the concentration of ligand-protein complex at time t, Rt [ligand-protein]t), R0 is the IAsys
response at time t = 0 in units of arc s induced by the
addition of the ligand solution to the buffer in the cuvette (this
represents a net displacement of the biosensor signal, and its value is
determined by the Fast Fit program for each binding curve analyzed),
R0 [ligand-protein]0, E is
the maximum IAsys response in units of arc s due to bound ligand at
equilibrium (E [ligand-protein]0
), and
Kobs is the observed rate constant (termed
kon in Fast Fit) given by
Kobs = kon[ligand] + koff (27). For two binding sites the Fast Fit
program uses the equation Rt = R0 + E1(1 ekobs1t)+
E2(1 ekobs2t) to derive
the kinetic constants (24). In this equation E1 and E2 are the maximum response at equilibrium
due to binding of the ligand to the high and low affinity binding site,
respectively; and kobs1 and
kobs2 represent the observed rate constants for the high and low affinity binding site, respectively. This equation assumes that, at equilibrium, the ligand-protein complex is stable; thus, the equilibrium line must be parallel to the starting base line
([ligand-protein]), and at least 80-90% of the data must be taken into account when fitting data to a curve for either single or double exponential binding.
Kinetic Constants for HRG-IgG and HRG-IgM Interactions--
The
binding of HRG to L-chain containing IgG subclasses (IgG) and
IgM could only be fitted to a single exponential and not to a double
exponential. A linear relationship was obtained by plotting
kobs versus ligand concentration for
the interaction of HRG with these IgG subclasses (IgG) and IgM. In
contrast, no linear relationship was obtained by plotting
kobs versus ligand concentration for
the interaction of HRG with immobilized human IgG subclasses containing
L-chain (IgG), indicating that the data does not fit an
exponential curve. Therefore, in these instances, similar to other
kinetic analyses (27), the first region of the progress curve that best
fits the single exponential term (assuming that thermodynamic
equilibrium is reached) was used to evaluate the parameters for the
highest affinity interaction. This was done by plotting
ln((E (Rt R0))/E) versus time and
selecting the linear region of this plot.
In some instances the Fast Fit program was used to extrapolate the
dissociation data to the base line for determination of the
koff. Values of
Kd values obtained by using the
relationship Kd = koff/kon were in good
approximation with those obtained by Scatchard analysis of the extent
of association (not shown).
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RESULTS |
Binding of HRG to IgG Subclasses Possessing L-
Chain--
We have previously shown that HRG binds to immobilized
human IgG (from pooled human serum) with high affinity
(Kd = 85 ± 15 nM) (16). To
determine whether HRG binds with a similar affinity to each different
IgG subclass, preliminary experiments were carried out to examine the
binding of biotinylated IgG1, IgG2, IgG3, and IgG4 to
immobilized HRG in an ELISA assay. In these studies, which involved the
incubation of HRP-STP conjugate followed by color development as
described previously (16), the binding of each IgG subclass to the
immobilized HRG could be detected. The experiments indicated that each
of the four different subclasses of IgG binds to immobilized HRG in a
concentration-dependent manner. Moreover, the dissociation
constants (Kd) for the binding of IgG() to
immobilized HRG, as determined by Scatchard analysis after plotting
(1/maximum bound) versus (1/IgG concentration), was found to
follow the relationship IgG2 > IgG1 >>> IgG3 > IgG4 (data not shown).
To further characterize the interaction of HRG with IgG subclasses, the
binding of native human HRG to each of the four IgG subclasses
possessing the L-chain also was examined using the IAsys biosensor.
As noted above, these studies required the immobilization of the IgG
subclasses rather than the HRG, as the immobilization of the HRG to the
biosensor surface resulted in the HRG losing its ability to interact
with any of the immunoglobulins, probably due to steric effects.
Therefore, for these studies 20-60 ng of biotinylated human IgG (of
the indicated subclass possessing L-chain) was immobilized via STP
coupling onto the surface of a dextran cuvette, and the binding of
soluble HRG to the immobilized IgG subclass was carried out following
equilibration of the cuvette in PBS-BSA-T. Thus, HRG was added in
PBS-BSA-T, and the association phase for the binding of native human
HRG to the immobilized IgG1 was monitored for 5 min. Subsequently,
the cuvette was washed three times with PBS-BSA-T buffer (to bring the
liquid phase HRG concentration to zero), and the dissociation phase was
monitored for 5 min. The biosensor profiles for the binding of
different concentrations of HRG to immobilized human IgG1 are shown
in Fig. 1A. The data show that
the binding of HRG to IgG1 is dependent on the HRG concentration in
the range of 10-400 nM and that within this concentration
range the binding of HRG is saturable with near-maximal binding (~500
arc s) occurring at a HRG concentration of 400 nM. The
binding of HRG was significantly inhibited (>80%) when the HRG was
preincubated before the addition to the cuvette with soluble IgG1
(IgG1/HRG molar ratio > 4, data not shown), suggesting that
the HRG interacted specifically with IgG1. The observed rate
constant for each binding curve, at the indicated concentrations of
HRG, was obtained by fitting the curve to the single exponential
expression using the Fast Fit program as described previously (16). A
plot of the observed rate constant (kobs, s1) against the concentration of HRG (nM)
approximated a straight line (see Fig. 1B), with the line of
best fit revealing that HRG binds to immobilized IgG1 with an
on-rate of 1.73 ± 0.04 × 105
M1 s1 (slope of the plot) and
an off-rate of 0.53 ± 0.02 × 103
s1 (intercept with the y axis) (see Table
I). The dissociation constant for this
interaction was determined either using the relationship
Kd = koff/kon or by Scatchard
analysis of the maximum amount of bound HRG at equilibrium; both
methods gave a Kd of 3.0 ± 0.1 nM
(see Table I).
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Fig. 1.
Optical biosensor determination of binding
constant for the interaction of HRG with immobilized
IgG1. For each indicated concentration of
HRG, the association or binding of HRG to the immobilized IgG1 was
monitored for 5 min. The overlay plots representing the binding of HRG
to the immobilized IgG1 are shown in A. Subsequently, the
contents of the cuvette were removed, and the cuvette was washed three
times with PBS-BSA-T before monitoring the dissociation phase for 5 min
(not shown). The value of kobs for the binding
curve for each HRG concentration was determined using the linearization
method (Fast Fit program), and each value ( ) was plotted against the
concentration of HRG (B). The plot of
kobs against HRG concentration gives a straight
line; the slope represents kon, and the
y-intercept represents koff for this
interaction. Error bars represent the ±S.E. obtained from
three separate experiments for each concentration of HRG.
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Table I
Rate and dissociation constants for the interaction of HRG with various
and light chains containing ligands (IgG subclasses, BJ
proteins, and IgM) in the presence and absence of Zn2+
Kinetic constants for the interactions indicated were determined by
applying the Fast Fit program to the binding data obtained using the
IAsys biosensor. All experiments were performed by the addition of HRG
to a dextran cuvette containing the immobilized protein in PBS-T-BSA
buffer (pH 7.4) in the absence (control) or presence of 20 µM Zn2+. Each constant represents the mean ± S.E. of three independent experiments. Footnotes
d-f indicate that changing the light chain type
from to for a particular IgG subclass significantly affected
the HRG-ligand binding constant. Footnotes a-c
indicate the presence of Zn2+ significantly affected the
HRG-ligand binding constant.
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Similar biosensor experiments carried out to study the interaction of
soluble HRG with immobilized IgG2, IgG3, and IgG4 (each
immobilized on to separate biosensor cuvettes) also indicated that the
binding of HRG to each of these subclasses was saturable and dependent
on the HRG concentration. As previously, the biosensor profiles for the
binding of HRG (at each concentration) to each IgG subclass was fitted
to a single exponential to derive the kobs. As
shown in Fig. 2B and Table I,
the on-rate, off-rate, and Kd for the interaction of
HRG with IgG2 were found to be 2.8 ± 0.3 × 105 M1 s1, 1.4 ± 0.2 × 103 s1, and 5.0 ± 1.3 nM, respectively. Interestingly, the on-rate for the
interaction of HRG with IgG3 was 0.13 ± 0.01 × 105 M1 s1 (see Fig.
2C) and that for HRG with IgG4 was 0.11 ± 0.01 × 105 M1 s1 (see
Fig. 2D); these values are ~15-30-fold slower than those observed for IgG1 or IgG2. Further analysis showed that the off-rate and Kd for the interaction of HRG with
immobilized IgG3 were 1.9 ± 0.1 × 103
s1 and 148 ± 13 nM, respectively. The
corresponding values for IgG4 were different: off-rate = 2.9 ± 0.2 × 103 s1, and
Kd = 268 ± 27 nM. Since the
half-life (t1/2) corresponding to the off-rates for
the interaction of HRG with these -containing IgG subclasses are
relatively long (t1/2 ~ 8-20 min for IgG1 and
IgG2; and t1/2 ~ 4-6 min for IgG3 and
IgG4), the calculated values for the on-rate are unlikely to be
significantly affected by dissociation of the respective ligand,
especially since only the initial part of the binding curve was used in
each of the analyses. The on-rates for the binding of HRG to the four
different IgG subclasses therefore follows the series IgG2 > IgG1 >>> IgG3 > IgG4 (see Fig. 2 and Table I). These
studies show that the kinetics of the binding of HRG to IgG depends on
the IgG subclass, with HRG having a faster binding rate and higher
affinity for IgG1 and IgG2 than for IgG3 and IgG4.
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Fig. 2.
Determination of the binding constants for
the interaction of HRG with immobilized IgG1, IgG2, IgG3, and
IgG4. The binding of human HRG to each of eight different IgG
subclasses (IgG1, IgG1, IgG2, IgG2, IgG3, IgG3,
IgG4, and IgG4) was examined using the IAsys biosensor. Each
human IgG subclass was immobilized onto a separate biosensor cuvette,
and then different concentrations of HRG were added, and the
association and dissociation of HRG were monitored exactly as described
in the legend to Fig. 1. The kobs values for the
binding of HRG to each IgG subclass was determined by fitting the
biosensor data to a single exponential. The kobs
was plotted against the HRG concentration for each condition.
Panel A shows the interaction of HRG with IgG1 () and
IgG1 ( ); panel B shows the interaction of HRG with
IgG2() and IgG2 (); panel C shows the
interaction of HRG with IgG3 () and IgG3 (); and
panel D shows the interaction of HRG with IgG4 () and
IgG4 (). Some binding experiments also were carried out in
binding buffer containing 20 µM added Zn2+;
the kobs values, plotted against the HRG
concentration for the HRG-IgG1 ( ), and the HRG-IgG1 ( ])
interactions, carried out in PBS-BSA-T containing 20 µM
added Zn2+, also are shown in A. Error
bars represent ±S.E. obtained from three separate experiments for
each concentration of HRG.
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Binding of HRG to IgG Subclasses Possessing L-Chains--
The
finding that HRG differs in its ability to interact with different
subclasses of IgG containing the L-chain suggested that differences
also may exist in the ability of HRG to interact with subclasses of IgG
containing the L-chain. Therefore, to study the binding of HRG to
different L-chain-containing IgG subclasses, each subclass
(IgG1, IgG2, IgG3, and IgG4) was immobilized onto a
separate biosensor cuvette, and the binding of soluble HRG to each
subclass was analyzed using the biosensor. The binding of HRG to each
different IgG subclass was carried out separately at a range of
different concentrations of HRG (10-600 nM) in PBS-BSA-T.
The binding of HRG to each IgG subclass was found to be saturable
and dependent on HRG concentration (data not shown). The biosensor read
out for each concentration of HRG was fitted to a single exponential to
obtain the average kobs for the interaction, and
the average kobs was then plotted against the
HRG concentration.
To facilitate comparison of the results from IgG subclasses containing
either or L- chains, the data for each -containing subclass
was plotted on the same graph as the corresponding -containing subclass; the results for IgG1 and IgG2 are shown in Fig. 2,
A and B, and those for IgG3 and IgG4 are shown in
Fig. 2, C and D. From the line of best fit it was
calculated (Table I) that the on-rate for the interaction of HRG with
IgG1 and IgG2 was 0.44 ± 0.02 × 105
M1 s1 and 0.27 ± 0.01 × 105 M1
s1, respectively; these values are 5-10-fold slower than
for the binding of HRG to IgG1 and IgG2 (Fig. 2, A and
B). The off-rates and Kd were found to be
8.1 ± 2.4 × 103 s1 and 189 ± 43 nM for the HRG-IgG1 interaction and 3.1 ± 0.2 × 103 s1 and 112 ± 3 nM for the HRG-IgG2 interaction, respectively.
Interestingly, the dissociation constants were 20-60-fold higher than
those observed for IgG1 and IgG2 (see Fig. 2 and Table I).
As shown in Fig. 2 and Table I, similar studies indicated that the
binding of HRG to immobilized IgG3 and IgG4 is faster than the
binding of HRG to IgG1 and IgG2. The on-rate, off-rate, and
Kd for the HRG-IgG3 interaction were 1.16 ± 0.09 × 105 M1
s1, 12.2 ± 2.1 × 103
s1, and 109 ± 29 nM, respectively (Fig.
2C, Table I). The corresponding values for the HRG-IgG4
interaction were 2.16 ± 0.03 × 105
M1 s1, 20.8 ± 0.8 × 103 s1, and 96 ± 5 nM,
respectively (Fig. 2D, Table I). The t1/2 corresponding to the off-rates ranged from 1.4-3.8 min and 0.6-1.0 min for the interaction of HRG with IgG1/IgG2 and
IgG3/IgG4, respectively; the effect of dissociation on the
measurement of the on-rates for these interactions were minimized by
using the initial (<30 s) part of the binding curve. The data thus
indicate that although the on-rate of these interactions are similar to those observed for the interaction of HRG with IgG1 and IgG2, the
off-rates are much higher, resulting in a higher Kd (approximately 20-fold higher) than for the interaction of HRG with IgG1 and IgG2 (compare data in Table I). In summary, the on-rates for the binding of HRG to IgG subclasses containing the L-chain followed the relationship IgG4 > IgG3 >>>
IgG1 > IgG2 (see Fig. 2 and Table I).
Effect of Zn2+ on the Binding of HRG to IgG
Subclasses--
Previously it was shown that the presence of
Zn2+ potentiates the binding of HRG to human IgG (from
pooled human serum) but inhibits the binding of HRG to the complement
component C1q (16). To determine the effect of Zn2+ on the
binding of HRG to IgG subclasses, biosensor experiments were performed
using separate cuvettes containing each immobilized IgG subclass (as
described above). The binding of HRG to each different IgG subclass was
carried out in PBS-BSA-T containing 20 µM added
Zn2+ (PBS-BSA-T-Zn). As shown in Fig. 2A and
Table I, in the presence of Zn2+ the on-rate of the
HRG-IgG1 interaction was increased from 1.73 ± 0.04 × 105 to 6.21 ± 0.13 × 105
M1 s1, whereas the off-rate was
decreased from 0.53 ± 0.02 × 103 to 0.37 ± 0.17 × 103 s1. The dissociation
constant was decreased ~5-fold, changing from 3.0 ± 0.1 to
0.60 ± 0.01 nM (see Table I).
Experiments also were performed to examine the effect of
Zn2+ on the binding of HRG to immobilized IgG1. In
contrast to the HRG-IgG1 interaction, the presence of
Zn2+ slightly, but not significantly, decreased the on-rate
of the HRG-IgG1 interaction from 0.44 ± 0.02 × 105 to 0.34 ± 0.02 × 105
M1 s1, (without affecting the
off-rate of this interaction). As previously, under the conditions used
in the analyses the measured on-rates are unlikely to be significantly
affected by dissociation. Thus the Kd was increased
slightly from 189 ± 43 to 266 ± 34 nM (see
Table I). Similarly, the effect of Zn2+ on the binding of
HRG to other IgG subclasses was studied as described above. The results
show that although the presence of Zn2+ generally increases
the affinity of the binding of HRG to all IgG subclasses possessing L-chains, it tends to slightly decrease the affinity of the binding of
HRG to IgG subclasses possessing L-chains (see Table I). However,
except for IgG1, the effects of Zn2+ on the HRG-IgG
interaction were small and, in most cases, not statistically significant.
Binding of HRG to BJ Proteins--
The above studies indicated
that the L-chain isotype may be a major factor in determining the
kinetics of the interaction between HRG and the different IgG
subclasses. To further characterize the interaction of HRG with IgG,
studies were therefore carried out using and L-chain-containing
BJ proteins. For these studies, lyophilized BJ proteins were dissolved
in PBS and purified by gel filtration on fast protein liquid
chromatography using a Superose 12 column. Two protein peaks were
obtained with the BJ preparation, and a single higher molecular
weight peak was obtained with BJ. Subsequently, SDS-polyacrylamide
gel electrophoresis analysis of the protein peaks showed that BJ
consisted of a lower molecular weight monomeric and a higher molecular
weight dimeric form, whereas only a single peak of the dimeric form of
BJ could be detected (not shown). The dimeric forms of both BJ
and BJ were biotinylated as described under "Materials and
Methods" and then used in ELISA assays to assess their ability to
bind immobilized HRG. Preliminary experiments indicated that both
biotinylated and BJ proteins bound to immobilized HRG in an
ELISA assay, that the binding was concentration-dependent,
and that HRG bound more strongly to the than to the L-chains
(data not shown).
The interaction of HRG with BJ proteins also was studied using the
biosensor. For these studies each BJ protein was immobilized onto a
separate biosensor cuvette to permit an analysis of the binding of HRG
in solution. The biosensor data indicated that the binding of HRG to
both BJ and BJ proteins was saturable and dependent on the HRG
concentration. Similar to the analysis of other biosensor data
described above, for each concentration of HRG, the observed rate
constant (kobs) was obtained by fitting the
binding curve to a single exponential using the Fast Fit program. Binding studies showed that the change of the
kobs for the interaction of HRG with both BJ
and the BJ was dependent on the HRG concentration. The results
(Table I) indicate that the on-rate, off-rate, and the
Kd for the binding of HRG to immobilized BJ are 0.77 ± 0.04 × 105 M1
s1, 2.6 ± 0.3 × 103
s1 and 35.2 ± 4.9 nM, respectively, and
that the corresponding values for the binding of HRG to immobilized
BJ are 0.38 ± 0.02 × 105
M1 s1, 2.90 ± 0.04 × 103 s1, and 76 ± 6 nM,
respectively. As outlined above the measured on-rates are unlikely to
be significantly affected by dissociation (t1/2 ~ 4 min for the interaction of HRG with BJ and BJ). The results
indicate, therefore, that the affinity for the binding of HRG to BJ
is significantly higher than for the binding of HRG to BJ.
To determine whether Zn2+ affects the ability of HRG to
interact with BJ and BJ experiments were carried out as described above, with the exception that the HRG was added to the cuvette in
PBS-BSA-T-Zn. As shown in Table I, in the presence of Zn2+
the on-rate of the HRG-BJ interaction was increased from 0.77 ± 0.04 × 105 to 1.30 ± 0.04 × 105 M1 s1, and the
off-rate was decreased from 2.6 ± 0.3 × 103
to 1.4 ± 0.7 × 103 s1. The
Kd was significantly reduced from 35.2 ± 4.9 to 10.4 ± 1.2 nM. In contrast, an investigation of
the effect of Zn2+ on the HRG-BJ interaction indicated
that the presence of Zn2+ only slightly decreased the
on-rate of the interaction from 0.38 ± 0.02 × 105 to 0.32 ± 0.05 × 105
M1 s1 but significantly
increased the off-rate of the interaction from 2.90 ± 0.04 × 103 to 4.5 ± 0.1 × 103
s1 (see Table I). The Kd was thus
significantly increased from 76 ± 6 to 140 ± 5 nM. These results show that Zn2+ potentiates
the binding of HRG to BJ but inhibits the binding of HRG to BJ
(see Table I).
Binding of HRG to IgM
The ability of HRG to interact with IgG
subclasses (particularly IgG1) with high affinity in a
Zn2+-dependent manner prompted an
examination of whether HRG also binds to other Igs such as IgM. For
these studies, experiments were conducted to determine whether human
b-IgM binds to immobilized HRG in an ELISA assay. A
concentration-dependent increase in the binding of b-IgM to the
immobilized HRG was detected. In subsequent studies the IAsys biosensor
was used to determine the kinetic constants for the interaction of HRG
with immobilized IgM. The b-IgM (20 ng/cuvette) was immobilized
onto the sensing surface of a biosensor cuvette by coupling it to
immobilized STP, and the association and dissociation of HRG in
PBS-BSA-T buffer was monitored using the biosensor. As shown in Fig.
3A, the binding of HRG to
immobilized IgM was saturable and was dependent on the concentration of
added HRG in the range of 0.5--
5 µM. For each
concentration of HRG the observed rate constant
(kobs) was determined from the biosensor data,
and the results are presented in Fig. 3B. It was calculated
(Table I) that the on-rate and off-rate for the binding of HRG to IgM
was 0.036 ± 0.005 × 105
M1 s1 and 7.1 ± 0.1 × 103 s1, respectively, and that the
Kd for this interaction was 1990 ± 50 nM.
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|
Fig. 3.
Determination of the binding constant for the
interaction of HRG with immobilized IgM.
To study the binding of HRG to the IgM, experiments similar to those
described in the legend to Fig. 1 were carried out with IgM
immobilized onto the biosensor cuvette. Overlay plots representing the
binding of HRG at 0.5-5 µM to the immobilized IgM are
shown in A. Overlay plots for similar experiments carried
out in buffer containing 20 µM Zn2+
(PBS-BSA-T-Zn) instead of PBS-BSA-T also were obtained (not shown). The
value of kobs for the binding curve at each
concentration of HRG in the presence or absence of added
Zn2+ was determined using the linearization method (Fast
Fit program). The results in B show plots of
kobs against HRG concentration for the HRG-IgM
interaction in PBS-BSA-T buffer (, control) and in PBS-BSA-T
containing 20 µM Zn2+ (). Error
bars represent ±S.E. obtained from three separate experiments for
each concentration of HRG.
|
|
Experiments also were carried out to assess the binding of HRG (1 µM) to immobilized IgM in the presence of different
concentrations of Zn2+ (5-20 µM). The
results showed a decrease in the binding of HRG to IgM with increasing
concentrations of Zn2+ (data not shown). Studies of the
binding of HRG at different concentrations (0.5-5 µM) to
immobilized IgM in the presence of 20 µM Zn2+
were carried out to determine the effect of Zn2+ on the on-
and off-rates of the HRG-IgM interaction (see Fig. 3B and
Table I). These experiments indicated that in the presence of
Zn2+ the on-rate of the interaction is decreased from
0.036 ± 0.005 × 105 to 0.016 ± 0.003 × 105 M1 s1
(~2.3-fold decrease), whereas the off-rate is increased from 7.1 ± 0.1 × 103 to 11.8 ± 1.6 × 103 s1 (~1.7-fold increase). From the
off-rates the t1/2 for the interaction of HRG with
IgM was ~1.6 min and ~1.0 min in the absence and presence of
Zn2+, respectively; with the analyses employed, these
values were not expected to significantly affect measurement of the
on-rates. The presence of Zn2+ therefore results in a
3.9-fold increase in the Kd for the HRG-IgM
interaction, increasing it from 1990 ± 50 to 7754 ± 1707 nM (see Fig. 3B and Table I).
| |
DISCUSSION |
It has been known for decades that in many species immunoglobulin
molecules can contain two types of L-chains, termed and , which
exhibit little amino acid sequence homology. The functional relevance
of these different L-chain isotypes, however, is not known. In this
paper it is shown that HRG, a relatively abundant plasma protein, binds
to all IgG subclasses, although with somewhat differing affinities.
Thus, the kinetics of the HRG-IgG interaction is profoundly affected by
whether the L-chain isotype is or . For example, our results
show that the affinity for the binding of HRG to IgG1 is ~60-fold
greater than the affinity for the binding of HRG to IgG1, and the
affinity of the binding of HRG to IgG2 is ~ 20-fold greater
than that for binding to IgG2 (see Fig. 2 and Table I). In contrast,
the binding of HRG to IgG3 and IgG4 (when each contained the L-chain instead of ) showed a different pattern. In summary, the
results show that although human HRG can interact with all human IgG
subclasses, the kinetics of binding are dependent not only on the
particular subclass (or constituent H-chain) of the IgG molecules but
also on the type of its constituent L-chains. Since HRG has been shown
to inhibit the formation of IICs (16), these data provide the first
evidence for a functional difference between the role of and L-chains of immunoglobulin.
Recently we showed that the affinity of the binding of HRG to pooled
human IgG was potentiated 4-5-fold by the presence of physiological
concentrations (20 µM) of Zn2+ (16). In the
present study, an examination of the effect of Zn2+, added
in the form of ZnCl2, on the binding of HRG to different IgG subclasses revealed that only the HRG-IgG1 interaction was significantly affected, with the Kd approximately a
6-fold lower in the presence of Zn2+ (Table I). In contrast
to its effect on the HRG-IgG1 interaction, the presence of
Zn2+ had little or no effect on the HRG-IgG1
interaction. In fact, Zn2+ further accentuated the
influence of the L-chain isotype on HRG-IgG1 binding, with the
Kd of 0.6 nM and 266 nM (a
443-fold difference) for the binding of HRG to IgG1 and IgG1, respectively.
Zn2+ is an interesting regulator of the IgG1-HRG
interaction, and the level of Zn2+ is reported to vary in
tissues (28) with relatively large amounts of Zn2+ being
released locally by degranulating platelets (28, 29). The question
arises, therefore, as to whether the findings on the effects of
Zn2+ are physiologically relevant. The concentrations of
free Zn2+ in plasma is reported to be ~109
nM, but the real concentration is difficult to determine
(30). Evidence suggests that in plasma a large proportion (~98%) of the Zn2+ is bound to proteins like albumin (and HRG), and a
small proportion presumably exists complexed to other metabolites such
as amino acids and citrate, which can bind Zn2+ weakly
(26). The binding buffer used in all the studies described in this work
contained 1% BSA, and Zn2+ was always equilibrated in the
binding buffer before the analysis of protein interactions. As
Zn2+ interacts physiologically with the BSA and the molar
concentration of BSA was at least 10 times higher than that of the
added Zn2+, it could be expected that the free
Zn2+ concentration in the binding buffer used in the
experiments would be buffered to near physiological levels. Our
previous studies (16) showed that the binding of HRG to IgG is
decreased significantly (compared with the binding in the absence of
any added Zn2+) after adding 1 mM EDTA alone or
after adding 20 µM Zn2+ and 1 mM
EDTA (16). Similarly, in the present work, an effect of EDTA was
observed on the interaction of HRG with the L-chain and with IgG
even in the absence of added Zn2+, and essentially
identical binding data could be obtained using HEPES instead of
phosphate in the binding buffer (data not shown). These findings
indicate that an effect of Zn2+ is already apparent
(presumably due to trace amounts of Zn2+ in the buffer or
the BSA) in the absence of any added Zn2+ and, hence, that
the observed effects of Zn2+ occur at Zn2+
concentrations that are physiologically relevant.
The finding that HRG binds with different affinities to different IgG
subclasses depending upon whether or L-chains are present
raised the intriguing possibility that HRG binds directly to the or
L-chains of IgG. An analysis of the binding of HRG to BJ proteins
with or L-chains revealed that HRG binds to both BJ and
BJ but with different kinetics, resulting in the affinity of the
binding of HRG to immobilized BJ being ~2-fold higher than that
for the binding to immobilized BJ (see Table I). Interestingly, the
presence of Zn2+ increased the affinity of the binding of
HRG to BJ but decreased that for the binding of HRG to BJ,
resulting in the affinity for the binding of HRG to BJ being
~14-fold higher than that for the binding to BJ. Consistent with
an effect of Zn2+ on the HRG- L-chain interaction (see
above), these findings suggest that the presence of the L-chain in
IgG1 and IgG2 facilitates the interaction of HRG with these IgG subclasses.
Structure studies have shown that most of the amino acid sequence
differences between the different IgG subclasses are located in the
hinge region, which may give rise to differences in the length and
flexibility of this region of the IgG molecule and, hence, to possible
functional differences between the different IgG subclasses. Although
the amino acid sequence of the constant region of the L-chain is
different from that of the constant region of the L-chain, the two
regions are homologous and structurally related. Less pronounced is the
structural homology between the variable regions of the and L-chains (31). The fact that our data show the binding of HRG to IgG is
profoundly influenced by the L-chain isotype, but that the H-chain
isotype also modulates the interaction indicates that the constant
regions of H- and L-chains, rather than the variable regions, determine
HRG binding. This notion is also supported by our observation that HRG
interacted similarly in experiments using different myeloma sources of
the same IgG subclass and L-chain isotype (data not shown).
Furthermore, our earlier experiments indicate that HRG interacts with
F(ab')2 fragments of IgG (16) but does not interact with
the Fc portion of IgG. Evidence that the L-chain forms a key HRG
binding site on IgG comes from our observation that HRG interacts with
BJ L-chain dimers of either the or isotype with reasonable
affinity (Kd = 35 or 76 nM,
respectively), and that L-chains have a much higher affinity for
HRG (~13-fold) than L-chains in the presence of Zn2+
(data not shown). These findings are consistent with each molecule of
HRG interacting with both a H-chain (presumably hinge region or CH1
domain) and an L-chain (constant region) of the IgG molecule. Although
a precise determination of the stoichiometry of the interaction between
HRG and each IgG subclass was considered beyond the scope of the
present study, an estimate of the stoichiometry could be made from the
maximum binding at equilibrium of the HRG to the various
immunoglobulins. Thus, our biosensor data indicate that approximately
one HRG molecule can interact with each immobilized dimeric and
dimeric L-chains, and approximately four HRG molecules can interact
with each of the IgG subclasses containing the light chain. Of
course, these estimates are only approximate, as maximum binding levels
are affected by dissociation rates and possible inactivation of HRG
binding sites on immunoglobulins following their immobilization.
Clearly, the mechanism(s) and precise regions of these molecules
involved in the interaction of HRG with IgG and individual L-chains
remain to be determined and await further studies using techniques like
site-directed mutagenesis and x-ray crystallography.
BJ proteins are of pathological importance in diseases associated with
elevated levels of L-chains in the blood circulation, where the
deposition of BJ proteins can be associated with renal and/or systemic
L-chain deposition disease. Approximately 80% of patients with L-chain
deposition disease have L-chain deposits (renal tubular casts) due to
deposition of L-chain (32, 33). Evidence suggests that the
insolubilization and aggregation of the unfolded L-chain protein
may occur via intermolecular hydrophobic interactions (34), but
hitherto, no mechanism capable of protecting against the
insolubilization and deposition of these light chains in either the
kidney or the blood vessel wall has been reported. The present findings
that HRG binds strongly to BJ proteins, particularly BJ, suggests
that HRG may be an important factor in regulating the insolubilization,
aggregation, and pathological effects of BJ proteins in patients with
L-chain deposition disease.
The IgG form of rheumatoid factor is purported to be the more
pathogenic form of Ig in relation to rheumatoid arthritis and systemic
lupus erythematosus (35), despite the predominant form of rheumatoid
factor detected in the sera of patients with rheumatoid arthritis and
systemic lupus erythematosus being IgM. It was important, therefore, to
determine whether HRG also interacts with IgM. Kinetic studies of the
binding of soluble HRG to immobilized IgM showed that HRG binds to
the IgM with an affinity that is ~650-fold lower than for the binding
of HRG to IgG1 (see Fig. 3 and Table I). Interestingly, the presence
of 20 µM Zn2+ reduced the affinity of the
HRG-IgM interaction by ~3.9-fold. It is noteworthy that in these
experiments the binding of HRG
and 
Light
Chains*
,
TOP
ABSTRACT
INTRODUCTION
