©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Observations on the Binding of Four Anti-carbohydrate Monoclonal Antibodies to Their Homologous Ligands (*)

(Received for publication, November 3, 1995; and in revised form, December 27, 1995)

Eugenia M. Nashed C. P. J. Glaudemans

From the From NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The binding of four monoclonal immunoglobulins, two with specificity for beta(16)-linked D-galactopyranans (IgA X24 and IgA J539) and two with specificity for the chain terminus of alpha(16)-linked D-glucopyranans (IgA W3129 and IgA 16.4.12E), was measured with a number of their homologous oligosaccharide ligands at different temperatures. The results show a linear relationship between lnK and 1/T, where K is the affinity constant and T is the absolute temperature. The unitary free energy of binding, DeltaG(u), is virtually independent of T, and the DeltaS(u) is small when compared with DeltaG(u). The enthalpy changes derived from van't Hoff plots are large and negative, indicating an exothermic binding effect, whereas the entropy changes are small and negative, indicating minor overall hydrophobic contributions. Measurements of the free energies of binding, in low and high salt buffers, of methyl beta-D-galactopyranoside and the methyl glycoside of beta(16)-D-galactopyranotetraose with anti-galactan IgA X24 indicate that the monosaccharide has no hydrophobic interaction with the highest affinity subsite of IgA, whereas the tetraoside might have a modest hydrophobic interaction with the three other hapten-binding subsites of IgA. The standard entropy change of binding of the two groups (galactosyl and glucosyl) of oligosaccharides to the two respective sets (anti-galactan and anti-dextran) of antibodies shows a distinct, differing correlation with the hapten chain length within each set. This correlation agrees with the type of association previously established between the antibodies and either the interior determinants of the antigen (in the case of the anti-galactans) or the chain terminus (in the case of the anti-dextrans).


INTRODUCTION

We have shown in the past how murine monoclonal antibodies of the v(H)441 gene-family (Hartman and Rudikoff, 1984) can bind to the interior stretches of their homologous antigenic linear beta(16)-linked galactan polysaccharide (Glaudemans, 1987; Glaudemans and Kovác, 1988; Ziegler et al., 1992). Each of the two (per monomeric IgA) antibody combining areas made up of the interface of the N-terminal ends of the heavy and the light chain is filled by binding maximally to a stretch of four sequential galactosyl residues in the homologous, antigenic polysaccharide chain. It must be kept in mind that we showed (Glaudemans, 1991; Glaudemans and Kovác, 1988) that for this family of galactan-specific antibodies, each of the four galactosyl residues of the determinant has its own subsite on the antibody surface, and these four antibody subsites have differing affinities for ``their'' galactosyl residue.

In the case of the binding of murine monoclonal antibodies to alpha(16)-linked glucan polysaccharide (dextrans), other work (Cisar et al., 1974; Weigert et al., 1974), as well as our own work (Bennett and Glaudemans, 1979a; Nashed et al., 1990), has shown that there is a special group of these antibodies that bind only to the terminal, upstream end of the alpha(16)-linked glucan polysaccharide chain and that these antibodies are incapable of binding interior segments of the dextran polysaccharide chain. These latter antibodies also have combining areas that can optimally accommodate a stretch of some four sequential glucosyl residues in this case. However, here that stretch must include the one and only terminal glucosyl residue that occurs at the nonreducing (upstream) end of the dextran polysaccharide antigen. Again, we showed that similar to the galactan/anti-galactan system, each glucosyl residue has its own subsite, this time the terminal sugar residue having maximal affinity for its subsite and being bound inside a cavity that is part of the antibody combining area (Nashed, et al., 1990). Thus, we see that in the case of the anti-galactans, antibodies can bind all along the galactan polysaccharide chain, ``loading up'' the polysaccharide chain with antibody molecules. On the other hand, for the group of anti-dextran antibodies discussed above, they only engage the nonreducing tetrasaccharidic terminus of the dextran polysaccharide chain, whereas the rest of that antibody-bound polysaccharide chain exits from the antibody combing area into the solution, incapable of binding additional antibody molecules.

Evidence has been presented previously that the key interactions of both the beta(16)-linked galactans and the alpha(16)-linked dextrans with their respective homologous antibodies are mediated by hydrogen bonds (Glaudemans, 1987, 1991; Glaudemans and Kovác, 1988; Nashed et al., 1990). We wished to investigate if the binding of these two quite differing anti-carbohydrate antibody systems with their homologous antigen (galactan binding by two monoclonal intrachain binding anti-galactans and dextran binding by two monoclonal chain end-binding anti-dextrans) could also involve significant hydrophobic contributions. Thus, the enthalpy change (DeltaH°) of binding of these two sets of immunoglobulins with a series of their homologous oligosaccharide determinants was measured by using van't Hoff plots. The interactions were found to be associated with large enthalpy changes and with a small negative opposing entropy change.

Three possible modes of noncovalent interaction between proteins and ligands can a priori take place: electrostatic (i.e. charge interaction), hydrophilic (i.e. involving hydrogen bonding), and hydrophobic bonding (driven by the tendency of water to self-associate). It was shown before that interactions due to charge differences between a monoclonal immunoglobulin and several antigens are greatly diminished or even eliminated with increased salt concentration of the medium (Manjula et al., 1982). In order to evaluate this approach for a neutral ligand carrying charge separation, we here report on the affinity of a small zwitterion ligand, phosphorylcholine, for its monoclonal antibody HOPC8 (Bennett and Glaudemans, 1979b) in medium of high salt concentration.

Reaction rates between organic hydrophobic components, when occurring in aqueous solution, have been shown to be accelerated by higher salt concentrations (Breslow, 1991). Recently, it was reported that peptide hydrolysis catalyzed by retroviral proteases is accelerated by high salt concentration of the medium, and the effect is suggested to be due to hydrophobic substrate-protein interaction (Tropea et al., 1992). These observations led us to so evaluate a possible hydrophobic contribution to the binding of anti-galactan IgA X24 with galactosyl saccharides.

In addition, we report herein our correlation of thermodynamic parameters with the differing nature of the binding of the galactan/anti-galactan and the dextran/anti-dextran systems. As pointed out above, we had previously demonstrated that the two monoclonal anti-galactan antibodies are capable of binding any interior segment of the antigenic galactan chain (Glaudemans et al., 1993; Ziegler et al., 1992). On the other hand, the two monoclonal anti-dextran antibodies studied here bind exclusively to the chain terminus of their homologous dextran polysaccharide antigen (Glaudemans et al., 1989; Nashed et al., 1990). This fundamental difference is also reflected in the thermodynamic binding behavior of these two sets of antibodies with their homologous haptens, as we shall here demonstrate.


MATERIALS AND METHODS

Affinity constants for the association between ligands and antibodies were measured by monitoring the ligand-induced tryptophanyl fluorescence of the antibody as a function of ligand concentration (Jolley and Glaudemans, 1974) using a Perkin-Elmer LS 50 Luminescence Spectrophotometer. These were then expressed as the free energy of binding (-DeltaG° = RTlnK) and are listed in Table 1, Table 2, III, IV, and V. Affinity measurements, including those in solutions of varying NaCl concentrations, were conducted in buffered (pH 7.4-7.6) solutions. When affinity measurements were conducted at differing temperatures, the temperatures were monitored by thermocouple probes in the cuvettes during measurement. The isolation of the various antibodies has been described (Bennett and Glaudemans, 1979a, 1979b; Glaudemans et al., 1989; Nashed et al., 1990; Potter and Glaudemans, 1972). We are grateful to Drs. Tsukasa Matsuda and Elvin A. Kabat for a gift of IgA 16.4.12E. We thank Dr. Pavol Kovác for samples of the synthetic oligosaccharides used in this work (Kovác, 1988; Kovác and Lerner, 1988). Phosphorylcholine was used as its NaCl salt (Bennett and Glaudemans, 1979b).






RESULTS AND DISCUSSION

In the ensuing discussion, two things must be kept in mind. The first is that antibody binding of small ligands, be they the complete determinant part of the antigen or only part thereof, is the same when measured with whole antibody, Fab fragment, oligomer, or monomer as we have shown previously (Jolley et al., 1973; Pavliak et al., 1993). Thus, results obtained here can be discussed strictly in terms of the combining area of any given antibody, irrespective of the nature of the monomeric or dimeric nature of the affinity purified antibody in question. Secondly, it should be kept in mind that the N-terminal ends of the heavy and light chains that together make up the general combining area of an antibody molecule measure some 40 Å across (Suh et al., 1986). The extended size of a (16)-linked glycosyl group is approximately 5 Å. The largest ligands here used are hexomeric glycosides, i.e. their optimal length cannot exceed 30 Å. Therefore, none of the ligands here used in binding measurements can ever occupy more than one antibody combining area at any one time.

The free energies of binding of phosphoryl choline, methyl beta-D-galactopyranoside (mebetaGal(1)), (^1)phenyl beta-D-galactopyranoside (phenylbetaGal), and the methyl beta-glycoside of beta(16)D-galactotetraose (mebetaGal(4)) with their respective homologous monoclonal antibodies as a function of NaCl concentration in the buffer medium at near neutral pH are given in Table 1. For IgA HOPC8 (Potter, 1977), it can be seen that phosphorylcholine shows a reduction in affinity in buffer solutions having higher NaCl concentrations. Charge interactions between specific protein amino acid residues and the hapten's zwitterion have been observed in the crystal structure of phosphorylcholine bound to IgA McPC603 (Padlan et al., 1985), which belongs to the same family as does IgA HOPC8. In other binding studies on IgA HOPC8 using phosphorylcholine and several of its analogs, evidence was presented for charge interactions in solution between ligand and immunoglobulin (Bennett and Glaudemans, 1979b). It was previously shown also that the interaction between charged antigens and an oppositely charged immunoglobulin were greatly diminished or eliminated in solution by the presence of high salt concentrations in the medium (Manjula et al., 1982). Table 1shows that in contrast to the phosphorylcholine/HOPC8 system, the influence of high NaCl concentration in the buffer on the affinity of either mebetaGal(1) or phenylbetaGal with IgA X24 is essentially nil. Others have shown that hydrophobic interactions between either small molecules (Breslow, 1991) or proteins and small molecules (Tropea et al., 1992) are emphasized by the presence of high salt concentration in the medium. Thus, our observation on the binding of mebetaGal(1) or phenylbetaGal with IgA X24 indicates the absence of hydrophobic contributions to the binding of a galactosyl residue in the highest affinity subsite of that antibody, one of the four subsites that together can bind the tetrasaccharide immunodeterminant specified by that immunoglobulin (Glaudemans, 1987). It can also be seen in Table 1that mebetaGal(4), which is essentially the complete immunodeterminant, in contrast to mebetaGal(1) shows a small increase in affinity for IgA X24 when measured in phosphate buffer containing increasing salt concentration. Because mebetaGal(1) does not show this increase, this indicates that for mebetaGal(4), any or all of the second, third, and fourth residues possibly have some hydrophobic interaction with their respective subsites on the antibody's surface. Thus, our observations on the binding of these haptens as a function of salt concentration in the medium tend to validate this method to distinguish between charge-, neutral-, and hydrophobic-related interactions.

The free energies (-DeltaG°= RTlnK) of association of the methyl beta-glycoside of beta(16)-D-galacto(oligo)saccharides (mebetaGal where n = 1, 2, 3, 4, 5, or 6) with the anti-galactans IgA X24 and IgA J539 and of the methyl alpha-glycoside of alpha(16)-D-gluco(oligo)saccharides (mealphaGlcwhere n = 1, 2, 3, 4, 5, or 6) with the anti-dextrans IgA 16.4.12E and IgA W3129 measured at several temperatures are given in Table 2, Table 3, IV, and V, respectively.



The van't Hoff equation lnK/T = DeltaH°/RT(^2), where K stands for the equilibrium constant at constant pressure, can be rearranged to lnK = (DeltaH°/R) (1/T), which means that lnK/(1/T) = -DeltaH°/R. Thus the slope of the plot of lnK measured at constant pressure versus 1/T is equal to -DeltaH°/R for any given temperature. Because [(DeltaH°)/T](p) = DeltaC(p)°, this means that if the change in the heat capacity (DeltaC(p)°) with temperature is 0, the plot of lnK versus 1/T would be linear. This is what we observe over the temperature range studied (shown only for IgAs X24 and 16.4.12E, Fig. 1and 2).^2


Figure 1: Van't Hoff plot for the binding of the methyl beta-glycoside of beta(16)-D-galacto-oligosaccharides with anti-galactan IgA X24.



The unitary entropy (DeltaS(u)) is independent of concentration units chosen for the standard state and is defined as DeltaS° = DeltaS(u) + R ln (M), where R is the gas constant (8.317 J) and (M) is the cratic contribution to entropy (resulting from the entropy of mixing). Because the (M) is the reciprocal of the molarity of water, we obtain DeltaS° = DeltaS(u) - 33.42. Thus, DeltaG° = DeltaH° - T(DeltaS(u) - 33.42) or DeltaG° - 33.42T = DeltaH° - TDeltaS(u). One defines DeltaG° - 33.42T = DeltaG(u), which is the unitary free energy (Gurney, 1953). We observe that the unitary free energy of binding for each oligosaccharide appears to be nearly independent of temperature ( Fig. 3and 4).


Figure 3: Temperature dependence of the unitary free energy of binding (DeltaG(u)) of the methyl beta-glycoside of beta(16)-D-galacto-oligosaccharides with anti-galactan IgA X24.



Because (DeltaS(u)/T) = DeltaC(p)°/T, becomes (DeltaG(u))/T = (DeltaH°)/T - T(DeltaC(p)°/T) - DeltaS(u). Because (DeltaH°)/T is also equal to DeltaC(p)°, we have (DeltaG(u))/T = -DeltaS(u). We find DeltaG(u) to be nearly invariant with temperature, because DeltaS(u) is very small compared with DeltaG(u) (see Fig. 3and Fig. 4).


Figure 4: Temperature dependence of the unitary free energy of binding (DeltaG(u)) of the methyl alpha-glycoside of alpha(16)-D-gluco-oligosaccharides with anti-dextran IgA 16.4.12E.



Table 6and Table 7list the values for DeltaH°, DeltaS°, and DeltaS(u) for the various oligosaccharides and their respective homologous monoclonal antibodies. It can be seen from those data that the observed ligand-antibody association results in a large enthalpy change. The standard entropy change is small and negative and thus disfavors the binding. A distinct difference can be seen on the one hand between the anti-galactan monoclonal antibodies binding to beta-(16)-galactosyl ligands and on the other hand the anti-dextran monoclonal antibodies binding to alpha-(16)-glucosyl ligands ( Table 6and Table 7and Fig. 5and Fig. 6). It is known that the latter antibodies (W3129 and 16.4.12E) bind only to the chain terminus of their antigenic polysaccharide (Glaudemans et al., 1989; Nashed et al., 1990), whereas the former antibodies (X24 and J539) bind their homologous polysaccharide anywhere along the antigenic chain (Glaudemans, 1987; Glaudemans et al., 1993). It can be seen in Fig. 5and Fig. 6that the standard entropy change associated with the binding of each of these two classes of antibodies shows a distinct correlation with the chain length of their ligands; the anti-dextran antibodies show a nearly constant negative standard entropy change of association (a factor opposing the binding of ligand to the antibody) after the ligand has reached the size of the disaccharide (i.e. methyl alpha-isomaltoside). For the anti-galactan antibodies on the other hand, the negative standard entropy change of association continues to increase up to the pentasaccharide level. For the former case it was previously shown that the upstream (Pavliak et al., 1993) terminal glucopyranosyl residue of the homologous alpha(16)-gluco-oligosaccharides fits into a tight cavity-like pocket of the antibody with the penultimate glucosyl residue protruding from that pocket into the solution (Glaudemans et al., 1989, 1994a, 1994b; Nashed et al., 1990). On the other hand, for the anti-galactans the oligosaccharide hapten was shown to bind on the surface of the antibody combining area, with larger haptens making increasing linear contact (Glaudemans, 1987, 1991; Glaudemans et al., 1984) with the antibody surface. Thus, in the former case, the loss of degrees of freedom may hinder most in haptens having up to two glucosyl residues, whereas in the latter case the entropic opposition to binding continues to increase up to the pentasaccharide level.






Figure 5: Relationship between the standard entropy change of binding of both IgA W3129 and IgA X24 and the chain length of their respective homologous haptens.




Figure 6: Relationship between the standard entropy change of binding of both IgA 16.4.12E and IgA J539 and the chain length of their respective homologous haptens.



Hydrophobicity has been defined as the tendency of nonpolar substances to aggregate in aqueous solution so as to minimize their interaction with water molecules (Ben-Naim, 1980). It is to be expected that the binding of any ligand to its antibody, even those ligands that we consider as hydrophilic, for instance multiple hydroxyl-bearing haptens such as (oligo)saccharides, could have some hydrophobic component. Indeed, in a case where both hydrogen bonding and significant hydrophobic interactions had been found by x-ray diffraction analysis of the antibody having the immunodeterminant bound to its binding site (Cygler et al., 1991), it has been shown that antigen-antibody interactions can also have favorable entropy contributions (Sigurskjold and Bundle, 1992) to the binding. In another case, thermodynamic van't Hoff analysis of the interaction between di- and trinitrophenyl ligands with monoclonal antibody MOPC 315 revealed the interaction to have positive DeltaH° and DeltaS° values, and the interaction was thus deduced to be hydrophobically driven (Tanaka et al., 1986). Furthermore, study of the binding of D-glucose to yeast hexokinase showed the enthalpy change (DeltaH°) to be nearly zero over the temperature range studied, so that the change in heat capacity was also found to be essentially zero (Takahashi et al., 1981).

We have found that the interaction of mebetaGal(4) in binding to anti-galactan IgA X24 is moderately influenced by high salt concentration of the medium. Thus it appears unlikely that large hydrophobic contributions to the interaction play a role (Breslow, 1991; Tropea et al., 1992). Because the anti-galactan immunoglobulins of this family all show the same binding characteristics (Glaudemans, 1987), this would also hold for the interaction of IgA J539 with its homologous oligosaccharide determinant. This agrees in turn with our observation that this binding shows enthalpy and entropy changes that are comparable with those of IgA X24.

In the case of the dextran-anti-dextran system, due to the paucity of antibodies, we could not acquire independent information on the nature of the binding, such as the influence of high electrolyte concentration on the affinity. However, our finding that a high negative enthalpy change accompanies the association strongly indicates that hydrophobic effects are unlikely to dominate the interaction between antibody and bound ligand in this case as well.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: phenylbetaGal, phenyl beta-D-galactopyranoside; mebetaGal(1), methyl beta-D-galactopyranoside; mebetaGal(2), methyl beta-glycoside of beta(16)D-galactobiose. Higher oligosaccharides in the same series are denoted by mebetaGal(2), where n = 3, 4, 5, or 6. Similarly, mealphaGlc(1) is methyl alpha-D-glucopyranoside, mealphaGlc(2) is methyl alpha-glycoside of alpha (16)D-glucobiose, etc.

(^2)
Heat capacities at some of these temperatures will be measured using microcalorimetry. Quantities of antibodies required for that are large (7 times 10 mmol) compared with the requirement for the fluorescence method used here (1.5 times 10 mmol) i.e. some 400 times more per measurement. We will report in the future on the comparison of the results.


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