Issue February 2006

category image Volume 23
No. 4 (p 357-484)
February 2006
ISSN 0739-110
Open Access

The Increased Flexibility of CDR Loops Generated in Antibodies by Congo Red Complexation Favors Antigen Binding (p. 407-416)

The dye Congo red and related self-assembling compounds were found to stabilize immune complexes by binding to antibodies currently engaged in complexation to antigen. In our simulations, it was shown that the site that becomes accessible for binding the supramolecular dye ligand is located in the V domain, and is normally occupied by the N-terminal polypeptide chain fragment. The binding of the ligand disrupts the β-structure in the domain, increasing the plasticity of the antigen-binding site. The higher fluctuation of CDR-bearing loops enhances antigen binding, and allows even low-affinity antibodies to be engaged in immune complexes. Experimental observations of the enhancement effect were supported by theoretical studies using L λ chain (4BJL-PDB identification) and the L chain from the complex of IgM-rheumatoid factor bound to the CH3 domain of the Fc fragment (1ADQ-PDB identification) as the initial structures for theoretical studies of dye-induced changes. Commercial IgM-type rheumatoid factor (human) and sheep red blood cells with coupled IgG (human) were used for experimental tests aimed to reveal the dye-enhancement effect in this system. The specificity of antigen-antibody interaction enhanced by dye binding was studied using rabbit anti-sheep red cell antibodies to agglutinate red cells of different species. Red blood cells of hoofed mammals (horse, goat) showed weak enhancement of agglutination in the presence of Congo red. Neither agglutination nor enhancement were observed in the case of human red cells. The dye-enhancement capability in the SRBC-antiSRBC system was lost after pepsin-digestion of antibodies producing (Fab)2 fragments still agglutinating red cells. Monoclonal (myeloma) IgG, L λ chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.

Marcin Krol1
Irena Roterman1
Anna Drozd2
Leszek Konieczny2,*
Barbara Piekarska2
Janina Rybarska2
Pawel Spolnik2
Barbara Stopa2

1Dept. of Bioinformatics and Telemedicine
Medical College
Jagiellonian University
Kopernika 17
31-501 Krakow, Poland
2Institute of Medical Biochemistry
Medical College
Jagiellonian University
Kopernika 7
31-034 Kraków, Poland
*mbkoniec@cyf-kr.edu.pl

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Introduction

Antigen binding by antibodies has been found to be significantly enhanced in the presence of Congo red and also other related strongly self-assembling dyes (1, 2). This is the effect of dye complexation by antibodies currently engaged in interaction with the antigen (2). The supramolecular character of the dyes seems to represent a key binding structure, enabling complexation to proteins (3, 4).

The enhancement was studied and basically quantified in the standard SRBC-antiSRBC agglutination system, and also with the use of radio-labeled specific polyclonal IgG (1). Enhancement of immune complexation was most convincingly evidenced by the increased ligation of low-affinity antibodies, that is, antibodies with not enough affinity to form stable immune complexes with red cells in the absence of Congo red (1, 5). The number of antibodies bound to red cells increases with increasing Congo red concentrations, until the resulting enhancement of agglutination reaches a plateau. This is likely due to the expiry of low-affinity antibodies still capable of agglutinating red cells in the presence of dye, and as well the decreased influence of the size of agglutinates, growing with increasing dye concentrations (1, 2, 3). The enhancement capability of different Congo red-related dyes depends on their self-assembling property. It increases with the increasing tendency for self-assembly, reaches optimum, and then decays (3). Most likely the decay is caused by the excessively increased rigidity of the supramolecular dye ligands, reducing their plasticity. The high rigidity of the dye ligand hinders its sufficiently rapid attachment and fitting to the sites in the antibody molecule that become transiently accessible during the brief complexation of low-affinity antibodies with the antigen.

Dye-induced enhancement of immune complexation has been thought to arise from the lessening of the V-C interdomain linkage, allowing more independent rotation of the V domains and hence easier fitting of bivalent antibodies to randomly distributed antigenic determinants (6, 7, 8). The accessibility of the V domain for dye penetration and binding is caused directly (according to previous studies concerning the L chain structure) by the increased mobility or displacement of the N-terminal chain fragment. It is likely a consequence of the rotational restrictions and subsequent constraints generated in domains of IgG upon binding of the antibody to the antigen (7, 9, 10, 11, 12). The constraints become diminished as an effect of dye ligation, which stabilizes the altered structure, making antigen binding easier. According to this interpretation, the enhancement effect results from the facilitated rotation of V domains versus C domains (7, 8).

There is another mechanism however, which may be predicted to explain the enhanced antigen-antibody interaction in the presence of supramolecular ligands: namely, structural changes induced by dye binding may lead to some domain instability, and the subsequent increase of the rotational freedom of CDR-bearing loops, facilitating antigen binding by making a better fit of the antibody binding site to the antigen determinants. The increased plasticity of the binding area may thus favor antigen-antibody complexation (8, 14, 15, 16). However, the share of the light chain in the observed enhancement effect seems most significant, because it is usually less engaged than the heavy chain in immune complexation (17, 18). Its CDR2 loop is withdrawn into the binding site, and naturally its engagement in antigen binding is rather limited (19, 20). Hence, the light chain should have a higher potential than the heavy chain to influence antigen binding upon alteration and relaxation of its CDR loops.

The present paper shows the probable mechanism of the enhancement effect. The nature and ligation properties of self-assembling compounds (rigid, planar, polyaromatic rings) seem useful for pharmacological applications. The effect on antigen binding and complement fixation is not the only prospective area of interest (5, 21, 22, 23).

Also promising is the use of supramolecular structures as carriers in immunotargeting technique, based on their ability to intercalate many drugs and simultaneously bind specifically to immune complexes (22, 23).

Methods

Theoretical Analysis

Two distinct protein structures were analyzed in this paper in respect to the enhancement effect. The initial setup of the immunoglobulin light chain dimer molecule (PDB code 4BJL) (24) is described in detail elsewhere (7, 13). The crystal structure of the (IgMRF) Fab-Fc (IgG) complex (PDB code-1ADQ) (24, 25) was obtained from the PDB. The CL and CH domains of the Fab fragment were deleted to decrease the system?s size and save computation time. Supramolecular Congo red complexes composed of four dye molecules were then manually introduced into cavities formed in the VL and VH domains by the removal of N-terminal polypeptide chain structures, as described elsewhere (13). This was followed by Monte Carlo (MC) simulations of the ligands and the binding pocket to relieve any steric overlaps and to allow the ligand and the protein to fit each other. During the MC simulations the antigen molecule was kept fixed.

The resulting Fv-antigen-dye complex and also the Fv-antigen complex were solvated in rectangular boxes.

All MD and MC simulations were performed using the CHARMM 27b2 program (26). The protein was represented by the charmm22 all-atom force field, extended to incorporate the ligand molecule (27), and the solvent was represented by the TIP3P model (28). The dielectric constant was fixed at 1.0. All bond lengths between hydrogens and heavy atoms were fixed using the SHAKE algorithm (29), which allowed a time step of 2 fs. The particle Mesh Ewald (PME) method was used to calculate nonbonding interactions.

Periodic boundary conditions were used to avoid edge effects during the simulation. The pressure was kept fixed at 1 atm, using the Nose-Hoover algorithm implemented in CHARMM.

The grid density was ∼1 point/Å, κ=0.32, and the sixth interpolation order was used. A discrete space cutoff of 10Å was used.

Materials and Methods ? Experimental

The azo dye Congo red was purchased from Aldrich Chemical Co. Inc. (Milwaukee, U.S.A.). All reagents used were of analytical grade.

Antibodies: IgG anti-SRBC was isolated from the pooled sera of immunized rabbits by chromatography on DE-52 cellulose (Whatman International Ltd., England) and further purified on Sephacryl S-300 (Pharmacia, Sweden). IgM rheumatoid factor was isolated from commercial high rheumatoid factor serum (Sigma) by salting out with ammonium sulfate. Before use, its activity was estimated by standard tests, using latex microgranules coated with human IgG (Emapol, Poland). Polyclonal human IgG was purchased from Sandoz Pharma Ltd. (Switzerland) or prepared from the serum of a healthy donor. The (Fab)2 fragments were obtained by pepsin proteolysis of the respective IgG.

Immunoglobulins: Monoclonal IgG was isolated from myeloma serum. Light-chain λ dimer (An) was salted out from the urine of a patient suffering from multiple myeloma, and further purified as described earlier (30).

Agglutination: Standard agglutination tests were carried out at room temperature in barbital or phosphate-buffered saline. To estimate the agglutinating activity of rheumatoid factor, polyclonal human IgG was coupled to sheep red blood red cells and used as an antigen (31). Coupling was checked by agglutination of red cells with specific anti-IgG antiserum.

Results

Previous studies of Congo red complexation by L λ chain, aimed at identifying the dye ligation site, indicated that a supramolecular dye ligand composed of a few Congo red molecules binds to the V domain and occupies the cavity that becomes available for dye penetration and binding as an effect of transitory dislocation of the N-terminal chain fragment from its packing locus (7, 9). The flexibility of the N-terminal chain fragment, which may increase when the antibody binds to the antigen, probably favors this particular localization of the dye (6, 9, 32). The unstable packing of the N-terminal chain fragment facilitates its replacement by the dye ligand, which strongly competes for the same locus in the V domain in a way similar to dye binding by α-1-proteinase inhibitor (7, 9, 33). The liberated chain may then be cut off by proteolysis (9). However, replacement of the N-terminal polypeptide chain by the supramolecular dye ligand results in domain destabilization, involving in particular the region stabilized mainly by the upper core (7, 9, 17, 34).

In consequence, CDR-bearing loops become more relaxed. Their fluctuation and their engagement in antigen binding may increase. Figures 1 and 2 show (based on L λ chain ? 4BJL as a model protein), that the increased relaxation of CDR-bearing loops arises directly from the dye-induced disruption of β-pleated sheets.


Figure 1: The increased relaxation of CDR2- and CDR1-bearing loops of the L chain (4BJL) upon Congo red binding (five-dye-molecule ligand), resulting from the interruption of stabilizing hydrogen bonds (post-dynamics forms). Left, without Congo red; right, with Congo red bound. Arrows indicate the positions of Cα atoms of residues involved in the network of hydrogen bonds. CDR2 bearing loop Cα-Cα distance Arg62-Ser77 (4.41 Å-before and 10.89 Å-after dye complexation). Cα-Cα distance Ser64-Ala75 (5.11 Å-before and 9.18 Å-after dye complexation). CDR1 Cα-Cα distance Gly24-Thr70 (4.72 Å-before and 11.63 Å-after dye complexation).

Figure 2: Changes in the number of hydrogen bonds stabilizing (A) CDR2- and (B) CDR1-bearing loops in L chain (4BJL) upon binding of dye ligands containing 3, 4 and, 5 dye molecules.
Simulation of the corresponding dye-derived structural changes in the L chain following complexation of the antigen to the antibody molecule were based on rheumatoid factor Fc complex as a model (25). Rheumatoid factors (RF) seem to represent a low-affinity form of antibody suitable for analysis. RF was selected for studies also because of the relatively high similarity of its light chain to the previously studied L λ chain (4BJL), and because this model may be available for experimental studies independently. The analysis employed both theoretical and experimental approaches. The heavy chain from the Fc fragment and V domains H and L (Fv) from IgMRF were taken for calculation of the complex structure.

Studies of post-dynamics structural changes were focused on the V domain of the light chain, because structural data were available from previous studies corresponding to the present analysis (13) and also because clearer effects connected with dye-modified antigen binding may be expected in the case of the light chain, as it is usually less engaged in specific immune complexation (19, 20).

The increased flexibility of the polypeptides in the V domain of the L chain (a component part of the RF-Fc complex) upon Congo red complexation is presented by an RMS-F plot (Fig. 3).


Figure 3: Fluctuation of V domains of L chain from the (IgMRF)Fv-CH3 (Fc domain) complex studied at room temperature with (black line) and without (gray line) dye bound. Shaded bars on abscissa represent CDR fragments. Insert shows the kinetics of RMS-D changes of a few amino acid peptides flanking CDR2 in the L chain with Congo red ligated.



Figure 4: Structural changes in the V domain of L chain from the (IgMRF)Fv-CH3 (Fc domain) complex upon ligation of supramolecular dye (four-Congo-red-molecule ligand) ? post-dynamics forms. A, left ? the complex without the dye. The interaction contact area of the CDR2 loop of the L chain is represented in a space-filling system. A, right ? the complex as seen according to the InsightII presentation. B ? the same complex after dye ligation in the InsightII presentation discloses the disruption of β-structure. Arrows indicate localization of dye (not shown) in the domain.

As may be concluded from Figure 4, the contacts between the CDR2 loop and the antigen become tighter upon complexation of the Congo red ligand, even allowing extra amino acids of this loop to approach and interact (Table I). This indicates the more active engagement of the L chain in antigen (Fc) binding.


The idea that the increased stabilization of the immune complex arises due to optimization of the induced fit within the binding site seems to offer a convincing explanation for the enhancement effect, which consequently remains a phenomenon specific in nature (14, 15, 16). The registered enhancement involves only low-affinity antibodies. In the employed technique there is no noticeable enhancement of agglutination by high-affinity antibodies that produce agglutination already without the dye. Hence the observed enhancement of agglutination caused by specific IgG is not much better than the enhancement found in the case of non-specific IgG, which is known to involve some low-affinity anti-red cell activity.

Specific and non-specific rabbit IgG, non-?specific? human polyclonal IgG (characterized by low-affinity anti-red cell activity) (35, 36, 37, 38, 39, 40), monoclonal human IgG, human polyclonal IgG fragmented by pepsin proteolysis, immunoglobulin light chain, and non-immunoglobulin protein (ovoalbumin) were used here for disclosure of any nonspecific protein effects. Figure 5 shows that the enhancement effect basically concerns only the specific antibodies. Enhancement of agglutination upon dye complexation was measured in the studied systems as the maximum possible extra (versus the control) dilution of antibodies still preserving agglutination capability.

Figure 5: Agglutination presented as the maximum dilution of antibodies with the agglutination capability of the red cells still preserved. Figure also includes other proteins of immunoglobulin and non-immunoglobulin origin used in place of antiSRBC antibody in agglutination tests to verify the specificity of enhancement. Thickened fragments at zero level represent zero agglutination found. The log scale was used to present the dilution-derived phenomena. Figures at the top of bars indicate the maximum dilution making agglutination of red cells still possible. Each bar represents the mean of three replicates. Insert shows the agglutination activity of commercial rheumatoid factor enhanced by Congo red. Sheep red cells with coupled human IgG were used as the antigen. IgGI, IgG from healthy donor; IgGII, pooled commercial IgG (human); and IgGIII, monoclonal myeloma IgG.

Experimental studies to reveal the expected enhancement in the RF-IgG system were performed using commercial polyclonal RF (IgM type) and sheep red cells coupled with human IgG. The red cells were chosen as the antigen carrier because of their neutrality to Congo red, ensuring higher reliability of the agglutination test. The enhancement of RF-Fc complexation is shown in Figure 5 (insert).

However, the increased plasticity of the antigen-binding site resulting from dye ligation should be associated with some broadening of antibody specificity (15, 41).

The agglutination and the enhancement of agglutination of foreign red cells by rabbit anti-sheep-red cell antibodies were used to reveal this expected effect. In some studied cases the enhancement was found to differ slightly from what was predicted, but the effect does not seem significant (Fig. 6).

Figure 6: Agglutination test to verify the specificity of the enhancement effect. Rabbit anti-SRBC IgG was used to agglutinate red blood cells originating from different species. Conditions as in Figure 5. GRBC, Goat red blood cells; HsRBC, Horse red blood cells; SRBC, Sheep red blood cells; and HRBC, human red blood cells.

Discussion

The antigen binding site of antibodies is located between the V domains at the tip of Fab. It is composed of six hyper-variable loops that together form the region of interaction with the antigen. Antigen-binding specificity and affinity results from the particular combination of simple, individual amino acid side chain rotational movements and concerted movements of CDR loops (14, 42). Recently, however, it has become evident that CDR loop length is an important determinant of antigen-binding affinity (43, 44). Also important is VH-VL relative displacement (45, 46). The contribution of CDR loops in VH-VL surface contact is about 40%, based on analysis of the known Fab antigen complexes (42). Supramolecular dye ligands found to occupy the site in the domain that becomes accessible for dye binding after replacement of the N-terminal chain fragment cause significant disruption of the β-conformation of polypeptides, also involving the loops bearing CDRs (47). Hence, the increased mobility of CDR loops that occurs as a result of dye complexation may facilitate their engagement in antigen binding.

The enhancement of antigen complexation confirms that the higher fluctuation within the domain is counteracted by the increased enthalpic component resulting from better fitting and the possible involvement of extra polypeptide portion in the formation and stability of the complex (14).

Alteration of the binding site is not driven genetically in this case. It is simply an artificial effect resulting from the higher mobility of polypeptides within the region of the binding site, allowed by the complexation of the dye ligand. The whole domain enlarges upon dye binding, also influencing the VH-VL interface (45, 46) as found by changes of the coordinates of residues involved in interface interaction.

The present analysis was focused on the loops of the region stabilized by the upper core, and in particular the CDR2 loop in the V domain of the L chain, as it is usually the CDR least engaged in antigen-binding, so its dye-induced mobilization may be expected to become evident (19, 20, 48). The increased flexibility of individual CDR loops may also broaden antibody specificity as a side effect. While the enhancement effect caused by the relaxation of CDR loops due to dye complexation makes sense and was supported experimentally, the expected broadening of specificity requires further studies. The diverse intensification of enhancement in red cells originating from different hoofed mammals and human in the agglutination test suggests the expected blunting of antibody specificity upon dye binding. An alternative explanation is possible, however, based on some antigenic relationship revealed or augmented by the enhancement effect (49). Some agglutination of rabbit red cells by pooled human polyclonal IgG in the presence of dye also seems to be connected with increased antibody reactivity. In reality, polyclonal IgG is known to contain some anti-red cell activity which may also involve structurally related foreign antigens (35, 36, 37, 38, 39, 40). The enhancement capability is lost after pepsin digestion of antibodies, indicating that complete immunoglobulin with the preserved flexibility restrictions is needed for the dye to enhance agglutination (10, 11, 12, 50). Monoclonal myeloma IgG revealed no agglutination, with or without the dye. Thus, the specificity of the enhancement effect seems at least dominant.

Assuming that the dye-derived enhancement effect is correlated predominantly with the specific antibody interaction, it seems natural that immune complexation clears the way for dye binding, followed by the increased relaxation in the domain derived from the disruption of β-structure.

The ligation effects of self-assembling dyes on antibodies might be employed successfully in pharmacological procedures.

Acknowledgement

Contract grant sponsor: KBN; contract grant numbers: 6 P05F-012 and 4 T11F 015 22.

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