Supramolecular Congo Red as Specific Ligand of Antibodies Engaged in Immune Complex
Supramolecular Congo red has been used to validate long-lasting theories regarding intramolecular signaling in antibodies and its relation to activation of the complement system. Strong enhancement of antigen-antibody complexation resulting from the binding of supramolecular ligands enables also polyclonal antibodies having intermediate affinity to trigger complement cascade apart of high affinity antibody fraction. This would not have been possible in the absence of Congo red. The property of antibodies provides specifically their ability to trigger the complement system allowed when sufficient structural strain is produced by antigen complexation provides an evidence of intramolecular signaling.
The selective complexation of supramolecular ligands with antibodies engaged in immune complexes enables their using as carriers of drugs in immunotargeting system.
KeywordsIntramolecular immunological signal Complement activation IgG V domain stability N-terminal fragment Enhancement of antigen binding Congo red as carrier of drugs Immunotargeting system Congo red selective complexation of antibodies in immune complexes
Self-associating organic molecules which form ribbonlike micellar structures may, owing to their structural characteristics, penetrate inside proteins and form stable complexes. Such penetration is possible in areas of the protein which have been destabilized, either temporarily or permanently – such as antibody /antigen complexes. Since Congo red (CR ) has been used in research as the most typical supramolecular protein ligand, the presented experiments and analysis also focus on this particular dye. CR binds strongly to antibodies, enabling us to study (among others) intramolecular signaling related to complement system activation. What is more, the mutual affinity of CR and immune complexes paves the way towards immunotargeting, i.e. targeted delivery of drugs. This is due to the fact that supramolecular CR – a micelle-like structure – may intercalate foreign bodies, including drug molecules. Congo red does not react with free antibodies – it is only capable of binding to antibody/antigen complexes where structure of antibody undergoes some alteration due to interaction with the antigen. Any potential drug intercalated into the CR micelle can thus be delivered to an area where the antigen is plentiful, ensuring targeted action. This chapter discusses the presented topics in detail.
2.1 Looking for Evidence of Postulated Intra-molecular Immunological Signaling
Once the structure of immunoglobulins has been divined, it soon became clear that their Fab and Fc fragments play differing roles in the process of triggering immunological response. While the Fab fragment selectively binds to the antigen, the Fc fragment – separated by a hinge – appears to be involved in triggering complement system activation through complexation of the C1q subcomponent. Notably, the Fab -antigen interaction is independent of Fc and proceeds even when the Fc fragment has been removed by digestion [1, 2].
Nevertheless, despite significant effort by many leading researchers, the specifics of this mechanism have proven exceedingly difficult to elucidate and some uncertainties persist. In attempting to explain intramolecular signaling, analysts initially focused on the hinge region which links Fab and Fc. Experimental data indicates that subclasses of immunoglobulins which differ with respect to the composition of this hinge region also exhibit variable efficiency of Fab -to-Fc signal transmission, and moreover that reduction of the disulfide bond in the hinge region prevents successful activation of the complement system [3, 4, 5]. In turn, some attention was directed towards structural strain in the antibody molecule, produced by antigen binding and regarded as a possible signal carrier. This view is embodied in the so-called distortive mechanism theory. Another competing theory proposed an “all or none” switching mechanism, i.e. an allosteric model based on the assumption that immunoglobulins are, in fact, allosteric .
Since none of the presented models succeeded in providing a satisfactory explanation, further analysis was needed. Some researchers noted the fact that, under ordinary circumstances, the formation of an active immune complex involves many different antibodies, and that complement activation requires local concentration of Fc fragments. This so-called associative model appeared to explain the signaling puzzle to a sufficient degree, particularly given the lack of evidence favoring intramolecular signaling [7, 8]. Earlier theories were swept aside and the issue appeared solved. This situation persisted for many years, until scientists learned how to produce monoclonal antibodies via crystallization of Fab fragments cleaved from the IgG molecule, and formulated new analysis protocols based on the use of small antigens (haptens) [9, 10, 11]. Surprisingly, these studies produced little in the way of useful results. Structural changes appeared small, even negligible – again suggesting that intramolecular signaling must somehow involve torsional effects, which emerge only when the antigen is bound to a complete, two -arm antibody .
Spectacular progress in genetics achieved in the 1980s, particularly the ability to synthesize arbitrarily modified antibodies, brought new hope of understanding the purported intramolecular signaling mechanism. Still however, despite some focus on the interaction of CH1 and CH2 domains , the problem of signal remained practically unsolved [12, 13, 14].
While the “full” two-arm molecule constructed from free Fab and Fc fragments exhibited complement activation potential (to a limited degree), its one-arm equivalent proved entirely inert. This suggested that even a deficient antibody may transmit the signal, if only suitable conditions exist for structural strain to emerge. Nevertheless, the participation of the hinge region in signal transmission remained a mystery [3, 15, 16].
2.2 Evidences of Intramolecular Signaling Supplied by Using Congo Red
A whole new approach to the problem was enabled by the use of CR , based on our team’s original concept. While CR had long been known as a useful amyloid stain , its interaction with amyloids was explained as individual molecules attaching themselves to specific binding sites which recognize the dye. In contrast, our study revealed that CR may form complexes with a wide variety of proteins and that it does so as a supramolecular ligand – i.e. a distinct structure consisting of many associated dye molecules acting as a single unit [17, 18, 19, 20, 21, 22, 23, 24, 25].
CR /light chain complexation may be accelerated by heating, which further perturbs the N-terminal fragment. In contrast, when dealing with immune complexes, complexation appears to be induced by structural strain resulting from antigen binding. This phenomenon, however, only emerges in complete two -arm antibodies, attaching themselves to antigen determinants located randomly on the cell surface. Neither isolated Fab fragments nor their dimers are capable of binding CR , even in their complexed state. This proves that the antibody -antigen reaction is not directly responsible for the affinity to CR , and that dye complexation requires structural strain in the antibody molecule [26, 29, 30, 31].
CR is complexed by whole antibodies in complex with haptens, but only when they are fixed on a solid surface, i.e. under conditions which lead to structural strain in bivalent antibodies .
At this point it would be useful to determine the mechanism which drives increased complexation capabilities in the presence of CR , and also to find out whether such upregulation is accompanied by the corresponding increase in complement system activity (which would prove the existence of an intramolecular signal).
The assumption that, by binding its natural biological ligand, the protein undergoes structural rearrangement which favors penetration and complexation of supramolecular dye would explain why the ligand cannot be easily released once the protein-dye complex has formed. This phenomenon appears actual in situations where irreversibility is finally expected – immune complexation, C1q binding etc. On the other hand, the same phenomenon would tend to inhibit the action of enzymes where the ligand must be released following catalysis. Indeed, such inhibition has been confirmed in the scope of complement activation which depends on the action of convertases .
The observed enhancement of antibody /antigen complexation capabilities may also be due to another factor: increased flexibility of the V domain, caused by penetration of a large noncovalently stabilized ligand to packing cavity of the replaced N-terminal fragment, bestowing greater internal mobility upon the domain (particularly its CDR loops) and therefore enabling them to align themselves to the antigen with greater accuracy .
The discovery and subsequent theoretical study of antigen complexation enhancement triggered by CR creates new possibilities with regard to analysis of intramolecular signal leading to complement system activation – assuming that such signal exists. Due to inhibition of convertase (and therefore of the complement system) by CR , measured as the efficiency of hemolysis, the signal transfer stage (immune complex/C1q) has been separated from the remainder of the activation cascade, including convertase. This reveals activation potential, since both the immune complex and the subsequent complex with C1q are insoluble and may therefore be separated from excess CR by washing, then combined with the remaining components of the complement system, thus preventing undesirable inhibition. To this end we have employed a commercial-grade C1q reagent (QUIDEL USA) and, separately, a serum containing complement system components but deprived of C1q (QUIDEL USA).
The immune complex (agglutinate ) binds antibodies with varying affinity for red cells which participate in the immune response (SRBC/anti-SRBC). Weak (low-affinity) antibodies are quickly washed out in the absence of CR . The remaining complexes contain antibodies with strong or moderate affinity. The introduction of CR stabilizes the immune complexes formed by weak antibodies, allowing them to remain in the agglutinate . Nevertheless, such antibodies remain incapable of triggering hemolysis even when CR is present. Their properties may be studied by analyzing wash-out samples. Of course, antibodies which resist washing out are also characterized by variable affinity: the group includes strong (high-affinity) antibodies which do not require CR to form stable complexes and trigger hemolysis, but also weak (low-affinity) antibodies stabilized by CR but still unable to trigger hemolysis. The specific affinity threshold established by the wash-out procedure is somewhat arbitrary and depends on a number of conditions. It is assumed that CR account for approximately 50% of the washout-resistant pool. Interestingly, this group contains also antibodies which are unable to trigger complement activation, but which gain this ability by interacting with CR . This suggests that a sufficiently powerful intramolecular signal may only be generated by “strong” antibodies which incur significant structural strain when binding the antigen. This natural threshold appears evolutionarily conditioned to prevent accidental thus potentially dangerous activation of the complement system [35, 36, 37, 38].
In this way, the use of a supramolecular dye approaches understanding both the function and the purpose of intramolecular signaling.
2.3 Application of Congo Red for Immunotargeting
It should be noted that in vitro test results do not always translate into similar outcomes in vivo. Successful use of supramolecular drug carriers in living organisms remains a complex problem due to undesirable reactions with serum proteins, particularly albumin. To determine whether the presented method is feasible, an Arthus reaction has been triggered in a rabbit host – i.e. the TNP antigen (ghosts of rabbit red blood cells conjugated with human IgG) was injected into the earlobe of an sensitized rabbit, producing local inflammation caused by aggregation of immune complexes. The other ear was subsequently injected with 2.5 ml 5 mg/ml) isotonic steryl CR solution. The ear where the Arthus reaction had originally been triggered was then backlit for photographic documentation (the rabbit’s thin earlobe is easily penetrated by visible light, simplifying the process). Unfortunately, the unaided human eye is unable to distinguish between CR and hemoglobin – the blood present in the vessels in earlobe produces an color image similar to CR . Enhanced visibility of small blood vessels suggests that an inflammatory process is ongoing, which further hampers attempts to visualize dye accumulations. Effective analysis therefore required the use of specially prepared spectroscopic filters (the spectra of CR and hemoglobin differ somewhat).
2.4 Toxicity of CR and Its Applicability for Immunotargeting
In addition to its immune complexation potential, CR is also being studied in the context of amyloidaffinity, although the presented applications of supramolecular ligands are based on laboratory experiments. Practical medical applications would require further, independent research.
We acknowledge the financial support from the National Science Centre, Poland (grant no. 2016/21/D/NZ1/02763) and from the project Interdisciplinary PhD Studies “Molecular sciences for medicine” (co-financed by the European Social Fund within the Human Capital Operational Programme) and Ministry of Science and Higher Education (grant no. K/DSC/001370).
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