7
CROSSLINKING OF ANTIBODY MOLECULES BY BIFUNCTIONAL ANTIGENS
Danute E. Nitecki, Virgil Woods and Joel W. Goodman Department of Microbiology, University of California, San Francisco, San Francisco, California 94143 ABSTRACT A requirement for at least two antigenic determinants to induce humoral antibody responses has been demonstrated using derivatives of a small molecule, L-tyrosine-p-azobenzenearsonate (RAT). This molecule itself induces only cellular immunity in guinea pigs. Assymetric bifunctional antigens composed of one RAT moiety and one haptenic determinant, such as DNP, with or without a spacer, induce cellular immunity to RAT and anti-DNP antibody. A symmetrical bifunctional antigen comprised of two RAT determinants separated by a rigid spacer, (PRO)lO' induces cellular and humoral responses, but the same two funcE10ns separated by a flexible spacer (6-aminocaproyl) gives cellular responses only. The bifunctionality of the latter antigen is probably compromised by intramolecular stacking of azoarsonate groups, since this molecule exhibits extensive hypochromism in physiological solution. The simplest hypothetical model of cell cooperation leading to a humoral response, i.e., bridging of T and B cells by antigen, requires the congregation of two lymphocytes on a molecule as small as DNP-RAT. Since the means of direct demonstration of such an interaction are not available, the capacity of such bifunctional molecules to bridge the receptors of specific antibody molecules was examined here. Thin layer gel chromatography was used to assess the crosslinking of the specific antibodies. It was found that all the bifunctional molecules examined were able to polymerize specific antibodies, regardless of whether they were effective mediators of cell cooperation in vivo.
139
M. Friedman (ed.), Protein Crosslinking © Plenum Press, New York 1977
140
D.E. NITECKI ET AL.
The immune response in vertebrate animals can be roughly divided into two compartments: namely, the cellular immune response and the humoral immune response (circulating antibody). The cells reacting in both of these responses are lymphocytes. Somewhere in the maturation pathway of these cells, those involved in cellular immune reactions are influenced by the thymus and are hence designated as T cells. The cells involved in the humoral response are designated B cells and are ultimately responsible for synthesis and secretion of circulating antibody (Greaves, Owen, and Raff, 1974). In order to produce a full immune response, that is, humoral as well as cellular immunity, the antigen must possess at least two functional structures or determinants (Alkan et al., 1972). One determinant provides the immunogenicity and stimulates the T cell compartment. The second determinant, frequently called a hapten, provides the structure against which the antibody specificity is directed. Complex protein molecules usually contain multiple determinants and even small proteins, such as lysozyme, has been shown to present several antigenic sites (Atassi, Lee, and Pai, 1976). It has been possible to show in our laboratory that a peptide hormone, glucagon, composed of twenty-nine amino acids (MW 3647), contains the immunogenic determinant in its C-terminal dodecapeptide and the haptenic determinant (against which most of the antibody was produced) in its N-terminal heptadecapeptide chain (Senyk, Nitecki, and Goodman, 1971; Nitecki et al., 1971). The sequence of events leading to cellular and antibody responses is unknown and subject to much speculation. It is possible, although by no means certain, that a bifunctional antigen such as glucagon bridges the T and the B cells at some point in time through their appropriate receptors. We have shown earlier that the small molecule, L-tyrosine-Eazobenzenearsonate (RAT), induces in guinea pigs cellular immunity only, i.e., no significant levels of humoral antibody can be demonstrated (Alkan, Nitecki, and Goodman, 1971). Thus, under these conditions the RAT molecule is a monofunctional immunogen and does not carry a haptenic determinant. Haptenic determinants, such as the dinitrophenyl moiety, can be attached to the a-amino group of RAT with or without a spacer. Immunization with such bifunctional assymetric antigens, e.g., dinitrophenyl-(6-aminocaproyl)O_3-RAT ~NP-(SAC)O_3-RAT], resulted in cellular immunity to tyrosine-azobenzenearsonafe group and antibody with anti-dinitrophenyl specificity (Alkan et al., 1972).
CROSSLINKING OF ANTIBODY MOLECULES
141
H2N-fH-COOH CH 2
~I N~N-@-As°3H2 -
OH
RAT
H2N-CH-COOH
I
,
CH 2
H20 3 AS-@-N=N-
~-
N=N-@- AS0 3H2
OH Bis-RAT
DNP-RAT
Symmetrical bifunctional antigens composed of two identical RAT determinants, i.e., molecules containing two azobenzenearsonate tyrosines separated by various spacers,were synthesized and used as antigens (Bush et al., 1972). These were found to induce only cellular immunity if the two determinants were separated by flexible spacers; that is, they behaved as monofunctional antigens. However, replacement of the flexible spacers by a rigid decaproline chain provided an antigen which was able to provoke cellular as well as humoral anti-RAT responses.
142
D.E. NITECKI ET AL.
We have hypothesized that the inability of, for example, flexible RAT-6-aminocaproy1-RAT molecule. to behave like a bifunctional antigen and to induce antibody responses is due to intramolecular stacking of the two azobenzenearsonate groups, which could compromise its bifunctiona1ity as an antigen. This was supported by an observed hypochromic decrease in the extinction coefficient of the RAT-6-aminocaproy1-RAT compound as compared with rigid RAT-decapro1y1RAT compound (in physiological solution). These observations raise intriguing questions in terms of simple models of T and B cell cooperation leading to the humoral antibody response. The requirement for at least two antigenic determinants is well established; mechanisms involving bridging of T and B cells have been postulated (Goodman, 1975). It is difficult, however, to visualize how two lymphocytes could congregate on a molecule as small as dinitropheny1-RAT. Since direct binding between T and B cells is impossible to test experimentally at present, we attempted to assess the capacity of these bifunctional antigens to bridge specific antibody molecules (Woods, Nitecki, and Goodman, 1975). While antibodies cannot be equated with cells, the antigen receptors on cell surfaces are probably akin to antibody, at least in the case of the B cell, and this approach represents a first approximation. Anti-dinitropheny1 and anti-RAT antibodies were obtained in rabbits by conventional immunization methods with appropriately conjugated proteins. These antibodies were purified by affinity chromatography on cyanogen bromidetteated Sepharose columns (Porath et a1., 1973) conjugated with either 6-aminocaproy1-RAT or DNP-ova1bumin; the purified antibodies were quantitated by specific precipitin reactions. The cross1inking of the antibodies by various antigens was investigated by thin layer gel chromatography (TLG) on glass plates coated with Sephadex G-200 superfine (Klaus, Nitecki, and Goodman, 1972). The antigens used were: RAT; DNP-RAT; acety1RAT-(6-aminocaproy1)1_3-RAT; cyc10-L-RAT-L-RAT (L-tyrosine-diketopiperazine conjugatea with one azobenzenearsonate group on each phenol ring); cyc10-L-RAT-D-RAT; acety1-RAT-(Pro1y1)10-RAT; Bis-RAT (L-tyrosine conjugated with two azobenzenearsonate groups on the same phenol ring); N,N'-bis-DNP-1,5 pentanediamine. Preparations of antibody and bifunctional molecules were mixed in equimo1ar ratios considered to favor cross1inking, based on the bivalency of antibodies. After brief incubation at room temperature, the mixtures were assayed by TLG, the developed plates printed off on paper and the paper stained by Coomassie BB R250 dye for visualization. TLG on Sephadex G-200 allows rapid and simultaneous analysis of antibody polymerization in multiple samples. Honomer rabbit IgG and a human dimer IgA myeloma protein (provided by Dr. A.-C. Wang) were readily resolved by this technique, but pentameric IgM and dimeric IgA were not distinguishable from each other. Hence, in these experiments dimers and higher order polymers were resolved from monomeric IgG but not from each other.
CROSSLINKING OF ANTIBODY MOLECULES
143
The results are shown in Figures 1 and 2. The purified antibody preparations migrated as single components on Sephadex G-200 TLG (Fig. 1) with mobility identical to that of an authentic sample of rabbit IgG (not shown). N-DNP-C,HlO-N-DNP did not change the chromatographic pattern of anti-RAT but ed to the appearance of a spot of more rapid mobility with anti-DNP, indicating the formation of antibody oligomers. Conversely, the cyclic bifunctional RAT molecule, cyclo-L-RAT-D-RAT, in which the arsonate groups extend from opposite sides of the diketopiperazine ring plane, had no effect on the pattern of anti-DNP but produced an oligomeric component with anti-RAT. Hence, specific cross-linking of antibody molecules took place in the presence of an appropriate symmetrical bifunctional antigen. The asymmetric bifunctional antigen, DNP-RAT, in which the DNP group was substituted directly on the amino group in the side chain of tyrosine, did not crosslink either anti-DNP or anti-RAT antibodies but did produce polymers with a 1:1 mixture of the two preparations (Fig. 1). As seen in Figure I, the two antibodies did not interact in the absence of the crosslinking antigen. Symmetrical bifunctional RAT antigens with flexible SAC spacers or rigid decaproline spacers crosslinked anti-RAT antibodies to varying degrees, based on relative intensities of the monomeric and oligomeric spots (Fig. 2). Increasing the size of the flexible spacer beyond a single SAC unit appeared to enhance polymerization (compare G, I and J in Fig. 2), although quantitative comparisons may be somewhat questionable. Even Bis-RAT, with two azobenzenearsonate groups on a single phenol ring, produced some polymerization, whereas RAT itself did not. Bifunctional RAT did not polymerize anti-DNP antibody. The present study attempts to determine if a correlation exists between the bridging capability and the capacity of bifunctional molecules to implement the (presumed) cooperation between T and B cells in the in vivo induction of a humoral antibody response. The rationale is based on the premise that a molecule which is unable to bridge antibodies should likewise be incapable of mediating cell cooperation, although the converse does not necessarily follow. A comparison of the capacities of bifunctional antigens to bridge antibody molecules and to induce antibody responses in vivo, presumably through the mediation of cell cooperation, is-Shown in Table 1. All these compounds induce a cellular type of response (delayed hypersensitivity) in guinea pigs. All the bifunctional molecules were able to crosslink antibody although only DNP-RAT and Ac-RAT-(PRO)lORAT have been effective inducers of humoral immune responses and, therefore, agents presumably able to mediate cell cooperation.
®
A
~
~
B
____
C
D
E
___L_____L____L___
F
~L_
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___ L
H
~
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~
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~
Figure 1. TLG patterns of A: anti-RAT + N-DNP-C SH10-N-DNP; B: anti-DNP + cyc1o-L-RAT-D-RAT; n C: anti-RAT + cyc1o-L-RAT-D-RAT; D: anti-DNP + N-DNP-C SH1 -N-DNP; E: anti-DNP + DNP-RAT; F: anti- A RAT + DNP-RAT; G: anti-DNP + anti-RAT + DNP-RAT; H: anti-BNP + anti-RAT. ~ ~ r
- -__
@®®~®®~ @
~ @
.... t
~~ ~ ~ ~ ~
B C
D
E
F
G
H
J
Figure 2. TLG patterns of A: anti-RAT; B: anti-RAT + Bis-RAT; C: anti-RAT + arsanilic acid; D: anti-RAT + RAT; E: anti-RAT + Ac-RAT-(PRO)l -RAT; F: anti-RAT + cyclo-L-RAT-L-RAT; G: anti-RAT + Ac-RAT-SAC-RAT; H: anti-RAT + cyclo-L-RAT-D-RAT; I: anti-RAT + Ac-RAT-(SAC)3RAT; J: anti-RAT + Ac-RAT-(SAC)2-RAT.
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146
D.E. NITECKI ET AL.
TABLE 1 Capacity of Mono- and Bifunctional Antigens to Bridge Antibody Molecules and to Induce Humoral Antibody Responses Bridging capacity
Antigen
Humoral response Ppting antibody jlg/ml < 2
RAT
52 ± 17
DNP-RAT
+
cyclo-L-RAT-D-RAT
+
< 2
Ac-RAT-SAC-RAT
+
< 2
Ac-RAT-(SAC) -RAT 2
+
< 2
Ac-RAT-(SAC) -RAT 3
+
< 2
Ac-RAT-(PRO)
+
135 ± 18
10
-RAT
Monofunctional RAT induces a pure cellular immune response, apparently does not mediate T cell-B cell cooperation, and does not crosslink anti-RAT antibodies, as expected. The smallest asymmetric bifunctional antigen, DNP-RAT, induces anti-DNP antibody responses in guinea pigs and crosslinks a mixture of anti-DNP and anti-RAT antibodies. The bond distances between the one carbon of DNP and the carbon ortho to the hydroxyl group of the phenol ring in DNPRAT total 8.181, a maximum value since it does not take bond angles or conformational folding into account. The fact that antibody molecules can be bridged by this bifunctional antigen does not, of course, prove that it can serve as a cellular bridge, but the observation that it induces an antibody response suggests that this may be so. Most interesting and surprising is the finding that Bis-~~T, a molecule in which the two functional groups (azobenzenearsonates) are separated by one carbpn atom, is also able to crosslink two antibody molecules and, moreover, crosslink them firmly enough to survive the rigors of thin layer gel filtration. The antibody combining sites have been generally estimated to accomodate 5-7 hexose
CROSSLINKING OF ANTIBODY MOLECULES
147
units or 3-6 amino acids (Goodman, 1975). The crosslinking of antibody by Bis-RAT is tantamount to a paperclip holding two cars together. Symmetrical bifunctional RAT molecules with flexible spacers do not induce significant humoral responses but polymerize anti-RAT antibody at least as effectively as the rigidly spaced RAT bifunctional (Fig. 2), which does induce anti-RAT responses. Intramolecular stacking has been proposed as an explanation of the inability of RAT-(SAC)1_3-RAT compounds to implement cell cooperation. This hypothesis was supported by hypochromism in the absorption spectra of the compounds, a predictable consequence of stacking. If this is the correct explanation, then stacking is an effective barrier for cell cooperation but not for antibody crosslinking. In any event, it is clear that the capacity of bifunctional antigens to crosslink antibody molecules is not a reliable measure of their ability to implement cell cooperation. ACKNOWLEDGMENTS We thank Miss Inge Stoltenberg for her skillful technical assistance. This work was supported by a grant from the United States Public Health Service (AI 05664). REFERENCES Alkan, S.S., Nitecki, D.E. and Goodman, J.W. (1971). Antigen-recognition and the immune response. The capacity of L-tyrosineazobenzenearsonate to serve as a carrier for a macromolecular hapten. J. Immunol., 107, 353. Alkan, S.S., Williams, E.B.~itecki, D.E. and Goodman, J.W. (1972). Antigen recognition and the immune response. Humoral and cellular immune responses to small mono- and bifunctional antigen molecules. 1. Exp. Med., 135, 1228. Atassi, M.Z., Lee, C.L. and Pai, R.C. (1976). Enzymic and Immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site. Biochim. Biophys. Acta., 427, 745. Bush, M.E., Alkan, S.S., Nitecki, D.E. and Goodman, J.W. (1972). Antigen recognition and the immune response: "Self-help" with symmetrical bifunctional antigen molecules. 1. Exp. Med., 136, 1478. Goodman, J.W. (1975). "Antigenic Determinants and Antibody Combining Sites," In The Antigens, M. Sela, Ed., Vol. 3, p. 127, Academic Press, New York, N.Y. Greaves, M.F., Owen, J.J.T. and Raff, M.C. (1974). T and B lymphocytes. Excerpta Med., Amsterdam.
148
D.E. NITECKI ET AL.
Klaus, G.G.B., Nitecki, D.E. and Goodman, J.W. (1972). Estimation of the molecular weights and stokes' radii of proteins of thinlayer gel filtration in guanidine hydrochloride. Anal. Biochem., 45, 286. Nitecki, D.E., Senyk, G., Williams, E.B. and Goodman, J.W. (1971). Immunologically active peptides of glucagon. Intrasci. Chern. Rep., 2, 295. Porath, J., Aspberg, K., Drevin, H. and Axen, R. (1973). Preparation of cyanogen bromide-activated agarose gels. J. Chromatog., 86, 53. Senyk~G., Nitecki, D.E. and Goodman, J.W. (1971). The functional dissection of an antigen molecule: Specificity of humoral and cellular immune responses to glucagon. 1. Exp. Med., 133, 1294. Woods, V., Nitecki, D.E., Goodman, J.W. (1975). The capacity of bifunctional antigens to bridge antibody molecules and to mediate cell cooperation. Immunochemistry, 12, 379.
CROSSLINKING OF ANTIBODY MOLECULES BY BIFUNCTIONAL ANTIGENS
Danute E. Nitecki, Virgil Woods and Joel W. Goodman Department of Microbiology, University of California, San Francisco, San Francisco, California 94143 ABSTRACT A requirement for at least two antigenic determinants to induce humoral antibody responses has been demonstrated using derivatives of a small molecule, L-tyrosine-p-azobenzenearsonate (RAT). This molecule itself induces only cellular immunity in guinea pigs. Assymetric bifunctional antigens composed of one RAT moiety and one haptenic determinant, such as DNP, with or without a spacer, induce cellular immunity to RAT and anti-DNP antibody. A symmetrical bifunctional antigen comprised of two RAT determinants separated by a rigid spacer, (PRO)lO' induces cellular and humoral responses, but the same two funcE10ns separated by a flexible spacer (6-aminocaproyl) gives cellular responses only. The bifunctionality of the latter antigen is probably compromised by intramolecular stacking of azoarsonate groups, since this molecule exhibits extensive hypochromism in physiological solution. The simplest hypothetical model of cell cooperation leading to a humoral response, i.e., bridging of T and B cells by antigen, requires the congregation of two lymphocytes on a molecule as small as DNP-RAT. Since the means of direct demonstration of such an interaction are not available, the capacity of such bifunctional molecules to bridge the receptors of specific antibody molecules was examined here. Thin layer gel chromatography was used to assess the crosslinking of the specific antibodies. It was found that all the bifunctional molecules examined were able to polymerize specific antibodies, regardless of whether they were effective mediators of cell cooperation in vivo.
139
M. Friedman (ed.), Protein Crosslinking © Plenum Press, New York 1977
140
D.E. NITECKI ET AL.
The immune response in vertebrate animals can be roughly divided into two compartments: namely, the cellular immune response and the humoral immune response (circulating antibody). The cells reacting in both of these responses are lymphocytes. Somewhere in the maturation pathway of these cells, those involved in cellular immune reactions are influenced by the thymus and are hence designated as T cells. The cells involved in the humoral response are designated B cells and are ultimately responsible for synthesis and secretion of circulating antibody (Greaves, Owen, and Raff, 1974). In order to produce a full immune response, that is, humoral as well as cellular immunity, the antigen must possess at least two functional structures or determinants (Alkan et al., 1972). One determinant provides the immunogenicity and stimulates the T cell compartment. The second determinant, frequently called a hapten, provides the structure against which the antibody specificity is directed. Complex protein molecules usually contain multiple determinants and even small proteins, such as lysozyme, has been shown to present several antigenic sites (Atassi, Lee, and Pai, 1976). It has been possible to show in our laboratory that a peptide hormone, glucagon, composed of twenty-nine amino acids (MW 3647), contains the immunogenic determinant in its C-terminal dodecapeptide and the haptenic determinant (against which most of the antibody was produced) in its N-terminal heptadecapeptide chain (Senyk, Nitecki, and Goodman, 1971; Nitecki et al., 1971). The sequence of events leading to cellular and antibody responses is unknown and subject to much speculation. It is possible, although by no means certain, that a bifunctional antigen such as glucagon bridges the T and the B cells at some point in time through their appropriate receptors. We have shown earlier that the small molecule, L-tyrosine-Eazobenzenearsonate (RAT), induces in guinea pigs cellular immunity only, i.e., no significant levels of humoral antibody can be demonstrated (Alkan, Nitecki, and Goodman, 1971). Thus, under these conditions the RAT molecule is a monofunctional immunogen and does not carry a haptenic determinant. Haptenic determinants, such as the dinitrophenyl moiety, can be attached to the a-amino group of RAT with or without a spacer. Immunization with such bifunctional assymetric antigens, e.g., dinitrophenyl-(6-aminocaproyl)O_3-RAT ~NP-(SAC)O_3-RAT], resulted in cellular immunity to tyrosine-azobenzenearsonafe group and antibody with anti-dinitrophenyl specificity (Alkan et al., 1972).
CROSSLINKING OF ANTIBODY MOLECULES
141
H2N-fH-COOH CH 2
~I N~N-@-As°3H2 -
OH
RAT
H2N-CH-COOH
I
,
CH 2
H20 3 AS-@-N=N-
~-
N=N-@- AS0 3H2
OH Bis-RAT
DNP-RAT
Symmetrical bifunctional antigens composed of two identical RAT determinants, i.e., molecules containing two azobenzenearsonate tyrosines separated by various spacers,were synthesized and used as antigens (Bush et al., 1972). These were found to induce only cellular immunity if the two determinants were separated by flexible spacers; that is, they behaved as monofunctional antigens. However, replacement of the flexible spacers by a rigid decaproline chain provided an antigen which was able to provoke cellular as well as humoral anti-RAT responses.
142
D.E. NITECKI ET AL.
We have hypothesized that the inability of, for example, flexible RAT-6-aminocaproy1-RAT molecule. to behave like a bifunctional antigen and to induce antibody responses is due to intramolecular stacking of the two azobenzenearsonate groups, which could compromise its bifunctiona1ity as an antigen. This was supported by an observed hypochromic decrease in the extinction coefficient of the RAT-6-aminocaproy1-RAT compound as compared with rigid RAT-decapro1y1RAT compound (in physiological solution). These observations raise intriguing questions in terms of simple models of T and B cell cooperation leading to the humoral antibody response. The requirement for at least two antigenic determinants is well established; mechanisms involving bridging of T and B cells have been postulated (Goodman, 1975). It is difficult, however, to visualize how two lymphocytes could congregate on a molecule as small as dinitropheny1-RAT. Since direct binding between T and B cells is impossible to test experimentally at present, we attempted to assess the capacity of these bifunctional antigens to bridge specific antibody molecules (Woods, Nitecki, and Goodman, 1975). While antibodies cannot be equated with cells, the antigen receptors on cell surfaces are probably akin to antibody, at least in the case of the B cell, and this approach represents a first approximation. Anti-dinitropheny1 and anti-RAT antibodies were obtained in rabbits by conventional immunization methods with appropriately conjugated proteins. These antibodies were purified by affinity chromatography on cyanogen bromidetteated Sepharose columns (Porath et a1., 1973) conjugated with either 6-aminocaproy1-RAT or DNP-ova1bumin; the purified antibodies were quantitated by specific precipitin reactions. The cross1inking of the antibodies by various antigens was investigated by thin layer gel chromatography (TLG) on glass plates coated with Sephadex G-200 superfine (Klaus, Nitecki, and Goodman, 1972). The antigens used were: RAT; DNP-RAT; acety1RAT-(6-aminocaproy1)1_3-RAT; cyc10-L-RAT-L-RAT (L-tyrosine-diketopiperazine conjugatea with one azobenzenearsonate group on each phenol ring); cyc10-L-RAT-D-RAT; acety1-RAT-(Pro1y1)10-RAT; Bis-RAT (L-tyrosine conjugated with two azobenzenearsonate groups on the same phenol ring); N,N'-bis-DNP-1,5 pentanediamine. Preparations of antibody and bifunctional molecules were mixed in equimo1ar ratios considered to favor cross1inking, based on the bivalency of antibodies. After brief incubation at room temperature, the mixtures were assayed by TLG, the developed plates printed off on paper and the paper stained by Coomassie BB R250 dye for visualization. TLG on Sephadex G-200 allows rapid and simultaneous analysis of antibody polymerization in multiple samples. Honomer rabbit IgG and a human dimer IgA myeloma protein (provided by Dr. A.-C. Wang) were readily resolved by this technique, but pentameric IgM and dimeric IgA were not distinguishable from each other. Hence, in these experiments dimers and higher order polymers were resolved from monomeric IgG but not from each other.
CROSSLINKING OF ANTIBODY MOLECULES
143
The results are shown in Figures 1 and 2. The purified antibody preparations migrated as single components on Sephadex G-200 TLG (Fig. 1) with mobility identical to that of an authentic sample of rabbit IgG (not shown). N-DNP-C,HlO-N-DNP did not change the chromatographic pattern of anti-RAT but ed to the appearance of a spot of more rapid mobility with anti-DNP, indicating the formation of antibody oligomers. Conversely, the cyclic bifunctional RAT molecule, cyclo-L-RAT-D-RAT, in which the arsonate groups extend from opposite sides of the diketopiperazine ring plane, had no effect on the pattern of anti-DNP but produced an oligomeric component with anti-RAT. Hence, specific cross-linking of antibody molecules took place in the presence of an appropriate symmetrical bifunctional antigen. The asymmetric bifunctional antigen, DNP-RAT, in which the DNP group was substituted directly on the amino group in the side chain of tyrosine, did not crosslink either anti-DNP or anti-RAT antibodies but did produce polymers with a 1:1 mixture of the two preparations (Fig. 1). As seen in Figure I, the two antibodies did not interact in the absence of the crosslinking antigen. Symmetrical bifunctional RAT antigens with flexible SAC spacers or rigid decaproline spacers crosslinked anti-RAT antibodies to varying degrees, based on relative intensities of the monomeric and oligomeric spots (Fig. 2). Increasing the size of the flexible spacer beyond a single SAC unit appeared to enhance polymerization (compare G, I and J in Fig. 2), although quantitative comparisons may be somewhat questionable. Even Bis-RAT, with two azobenzenearsonate groups on a single phenol ring, produced some polymerization, whereas RAT itself did not. Bifunctional RAT did not polymerize anti-DNP antibody. The present study attempts to determine if a correlation exists between the bridging capability and the capacity of bifunctional molecules to implement the (presumed) cooperation between T and B cells in the in vivo induction of a humoral antibody response. The rationale is based on the premise that a molecule which is unable to bridge antibodies should likewise be incapable of mediating cell cooperation, although the converse does not necessarily follow. A comparison of the capacities of bifunctional antigens to bridge antibody molecules and to induce antibody responses in vivo, presumably through the mediation of cell cooperation, is-Shown in Table 1. All these compounds induce a cellular type of response (delayed hypersensitivity) in guinea pigs. All the bifunctional molecules were able to crosslink antibody although only DNP-RAT and Ac-RAT-(PRO)lORAT have been effective inducers of humoral immune responses and, therefore, agents presumably able to mediate cell cooperation.
®
A
~
~
B
____
C
D
E
___L_____L____L___
F
~L_
G
___ L
H
~
m
~
z
~
Figure 1. TLG patterns of A: anti-RAT + N-DNP-C SH10-N-DNP; B: anti-DNP + cyc1o-L-RAT-D-RAT; n C: anti-RAT + cyc1o-L-RAT-D-RAT; D: anti-DNP + N-DNP-C SH1 -N-DNP; E: anti-DNP + DNP-RAT; F: anti- A RAT + DNP-RAT; G: anti-DNP + anti-RAT + DNP-RAT; H: anti-BNP + anti-RAT. ~ ~ r
- -__
@®®~®®~ @
~ @
.... t
~~ ~ ~ ~ ~
B C
D
E
F
G
H
J
Figure 2. TLG patterns of A: anti-RAT; B: anti-RAT + Bis-RAT; C: anti-RAT + arsanilic acid; D: anti-RAT + RAT; E: anti-RAT + Ac-RAT-(PRO)l -RAT; F: anti-RAT + cyclo-L-RAT-L-RAT; G: anti-RAT + Ac-RAT-SAC-RAT; H: anti-RAT + cyclo-L-RAT-D-RAT; I: anti-RAT + Ac-RAT-(SAC)3RAT; J: anti-RAT + Ac-RAT-(SAC)2-RAT.
A
1_ J
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146
D.E. NITECKI ET AL.
TABLE 1 Capacity of Mono- and Bifunctional Antigens to Bridge Antibody Molecules and to Induce Humoral Antibody Responses Bridging capacity
Antigen
Humoral response Ppting antibody jlg/ml < 2
RAT
52 ± 17
DNP-RAT
+
cyclo-L-RAT-D-RAT
+
< 2
Ac-RAT-SAC-RAT
+
< 2
Ac-RAT-(SAC) -RAT 2
+
< 2
Ac-RAT-(SAC) -RAT 3
+
< 2
Ac-RAT-(PRO)
+
135 ± 18
10
-RAT
Monofunctional RAT induces a pure cellular immune response, apparently does not mediate T cell-B cell cooperation, and does not crosslink anti-RAT antibodies, as expected. The smallest asymmetric bifunctional antigen, DNP-RAT, induces anti-DNP antibody responses in guinea pigs and crosslinks a mixture of anti-DNP and anti-RAT antibodies. The bond distances between the one carbon of DNP and the carbon ortho to the hydroxyl group of the phenol ring in DNPRAT total 8.181, a maximum value since it does not take bond angles or conformational folding into account. The fact that antibody molecules can be bridged by this bifunctional antigen does not, of course, prove that it can serve as a cellular bridge, but the observation that it induces an antibody response suggests that this may be so. Most interesting and surprising is the finding that Bis-~~T, a molecule in which the two functional groups (azobenzenearsonates) are separated by one carbpn atom, is also able to crosslink two antibody molecules and, moreover, crosslink them firmly enough to survive the rigors of thin layer gel filtration. The antibody combining sites have been generally estimated to accomodate 5-7 hexose
CROSSLINKING OF ANTIBODY MOLECULES
147
units or 3-6 amino acids (Goodman, 1975). The crosslinking of antibody by Bis-RAT is tantamount to a paperclip holding two cars together. Symmetrical bifunctional RAT molecules with flexible spacers do not induce significant humoral responses but polymerize anti-RAT antibody at least as effectively as the rigidly spaced RAT bifunctional (Fig. 2), which does induce anti-RAT responses. Intramolecular stacking has been proposed as an explanation of the inability of RAT-(SAC)1_3-RAT compounds to implement cell cooperation. This hypothesis was supported by hypochromism in the absorption spectra of the compounds, a predictable consequence of stacking. If this is the correct explanation, then stacking is an effective barrier for cell cooperation but not for antibody crosslinking. In any event, it is clear that the capacity of bifunctional antigens to crosslink antibody molecules is not a reliable measure of their ability to implement cell cooperation. ACKNOWLEDGMENTS We thank Miss Inge Stoltenberg for her skillful technical assistance. This work was supported by a grant from the United States Public Health Service (AI 05664). REFERENCES Alkan, S.S., Nitecki, D.E. and Goodman, J.W. (1971). Antigen-recognition and the immune response. The capacity of L-tyrosineazobenzenearsonate to serve as a carrier for a macromolecular hapten. J. Immunol., 107, 353. Alkan, S.S., Williams, E.B.~itecki, D.E. and Goodman, J.W. (1972). Antigen recognition and the immune response. Humoral and cellular immune responses to small mono- and bifunctional antigen molecules. 1. Exp. Med., 135, 1228. Atassi, M.Z., Lee, C.L. and Pai, R.C. (1976). Enzymic and Immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site. Biochim. Biophys. Acta., 427, 745. Bush, M.E., Alkan, S.S., Nitecki, D.E. and Goodman, J.W. (1972). Antigen recognition and the immune response: "Self-help" with symmetrical bifunctional antigen molecules. 1. Exp. Med., 136, 1478. Goodman, J.W. (1975). "Antigenic Determinants and Antibody Combining Sites," In The Antigens, M. Sela, Ed., Vol. 3, p. 127, Academic Press, New York, N.Y. Greaves, M.F., Owen, J.J.T. and Raff, M.C. (1974). T and B lymphocytes. Excerpta Med., Amsterdam.
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Klaus, G.G.B., Nitecki, D.E. and Goodman, J.W. (1972). Estimation of the molecular weights and stokes' radii of proteins of thinlayer gel filtration in guanidine hydrochloride. Anal. Biochem., 45, 286. Nitecki, D.E., Senyk, G., Williams, E.B. and Goodman, J.W. (1971). Immunologically active peptides of glucagon. Intrasci. Chern. Rep., 2, 295. Porath, J., Aspberg, K., Drevin, H. and Axen, R. (1973). Preparation of cyanogen bromide-activated agarose gels. J. Chromatog., 86, 53. Senyk~G., Nitecki, D.E. and Goodman, J.W. (1971). The functional dissection of an antigen molecule: Specificity of humoral and cellular immune responses to glucagon. 1. Exp. Med., 133, 1294. Woods, V., Nitecki, D.E., Goodman, J.W. (1975). The capacity of bifunctional antigens to bridge antibody molecules and to mediate cell cooperation. Immunochemistry, 12, 379.
