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    145. The problem of Molecular Recognition by a Selective System- Edelman

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    The Problem of Molecular Recognition by A Selective System - Edelman

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    Camilo Olaya

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    4. The Problem of Molecular Recognition by a Selective System GERALD M. EDELMAN Immunology has been profoundly altered in the last decade by two major developments: the theory of clonal selection and the chemical analysis of antibody structure. As a result of these developments, it has become clear that the central problem of immunology is to understand the mechanisms of selective molecular recognition in a quantitative fashion. The molecules and cells mediating selection in the immune response are known or can be known and, above all, the time scale of the selective events is well within that required for direct observation and experimentation. Aside from evolution itself, there are few such well-analysed examples of selective systems in biology, or in other fields for that matter. For this reason, the immune system provides a unique opportunity to analyse the problem of selection under defined and experimentally measurable conditions which have so far been hard to achieve elsewhere. In this paper, I shall briefly outline the idea of clonal selection as it operates in immunology, compare some of its features to those of natural selection and attempt to show how progress in molecular immunology (the reduc- tionist approach) has clarified and sharpened the notion of clonal selection and its relation to the specificity of molecular recognition. My purpose is not epistemological, nor shall I attempt an analysis of the reductionist or holist Position, for I believe that in their most extreme forms there is a fruitless opposition implied by these terms. Nevertheless, a description of the interplay between the phenomenological approach of the clonal selection theory and the mechanistic approach implicit in the analysis of antibody structure may Provide a useful example of the importance and limitations of reductionism in @ particular biological system. ‘The work of the author was supported by USPHS Grants AM 04256 and Al 09273. 46 Studies in the Philosophy of Biology Antibodies and the idea of molecular recognition The most striking feature of the immune response is not just its specificity but its specificity over such an enormous range of chemical structures. Anti- bodies may be generated to proteins, carbohydrates, nucleic acids and various artificially synthesised organic compounds or haptens, Indeed, antibodies may be generated to antibodies themselves in both the same and different animal species. It is the combination of the range of recognition with the exquisiteness of the specificity that gave rise to the early paradoxes in immunological thinking and led to an important idea as well as an erroneous theory. The important idea, which emerged from Ehrlich’s early studies, from Landsteiner’s epoch-making experiments and from Pauting’s analysis of chemical bonding, was that of molecular complementarity (Landsteiner, 1945). A thermodynamic analysis of antigen-antibody interactions indicated that the free energies of binding were of the order of 5-10 kcal/mole. From a knowledge of the chemistry of non-covalent interactions and a comparison of cross-reacting haptenic homologues which differed slightly in shape, it was concluded that antigens and antibodies interacted over very short distances. This implied that there was a very close fit between some portion of the anti- genic determinant and the antigen-combining site of the antibody molecule. Given this notion, it was reasonable for Pauling to clarify earlier ideas of Breinl, Mudd and Haurowitz (Jerne, 1967) into an instructive theory of antibody formation. This theory states that the complementarity is imposed upon the antibody by the antigen which acts as a template for the combining site. At first glance, this seems to be the only reasonable solution to the problem of molecular recognition, for the range of antigens is so great and some of them are so obviously unable to act during evolution of the immune system, that only a direct information transfer from antigen to antibody would seem to account for the specificity." ‘This theory has turned out to be incorrect. It is not appropriate here to trace the origin of the error, but it is important to realise that it was a reduc- tionist theory and that its failure rested not in its reductionism but in approaching the analysis of the system at only one level, that of the molecule rather than that of the cell. It was clear, for example, quite early that the instructionist theory could not easily explain immunological memory; nonetheless, the instructionist position held sway for a long time. ‘The paper that finally opened the way to modern theories of selection was that of Jerne entitled ‘The natural selection theory of antibody formation’ (erne, 1955). Jerne approached the problem from an entirely different point of view. He assumed that there was (1) a random mechanism for ensuring the limited synthesis of antibody molecules possessing all possible combining 1 It is interesting to note the analogy to Lamarckian thinking: the environment (antigen) actually alters the biological information system (antibody-forming apparatus). Molecular Recognition by Selective Systems 47 sites in the absence of antigen, (2) a purging mechanism for repressing anti- body synthesis against autoantigens, (3) a selective mechanism for promoting synthesis of those antibodies that fit a particular antigen best. This selective theory (the origins of which have been charmingly described by Jerne, 1966) still made the curious assumption that it was the antibodies that were circulating in the blood that brought the antigen to the appropriate cell. This failure to analyse the site at which recognition takes place led to a variety of objections. But it must be stated that even though Ehrlich (1900) had formulated a ‘selective’ theory very early, Jerne was the first to see truly the fundamental nature of the problem as one of selection. Burnet (1959) modified Jerne’s theory and shifted the object of selection from free antibody molecules to cells which were committed to making antibodies of one specificity. These cells were supposed to generate the types by somatic mutation and were then selected by antigen to divide and produce progeny which could synthesise more of the same kind of antibody (Figure 1). This theory accounted for a variety of biological phenomena in immunity including memory, the increase in avidity of antibodies with time of immun- isation, and the problem of self-recognition. ‘This last difficulty was fully recognised by Jerne, Burnet, and particularly by Medawar (1957). According to the theory, self-recognition could be stated in terms of repression or death of the appropriate clones of cells. Lederberg made a clear and incisive analysis of the genetic requirements of clonal selection which included a considera- tion of the problem of tolerance and self-recognition (Lederberg, 1959). Clonal Setection Theory Fertilized egg multiplies ond differentiates to form 0 large number af immuna- togicolly committed celis Antigenié stimutation ©) Figure 1. The clonal selection theory. 48 Studies in the Philosophy of Biology ‘The appearance of Burnet’s theory was pivotal in that it shifted the level of reduction from antibodies per se to cells and spoke in terms that biologists could understand. It thus was a clear challenge to instructive theories and, as shown by the subsequent facts, it was pointed in the right direction. On one point the theory was triumphantly clear: one cell makes only one kind of antibody, and antigen in the appropriate form can interact with that antibody and stimulate cell maturation and division. Both points have been amply confirmed since by experiment. It must not be assumed, however, that the molecular approach was useless. Indeed, the analysis of antibody chain structure (Edelman, 1959), the finding that different antibodies appeared to have different amino acid compositions and the finding that no antigen could be demonstrated in cells actively synthesising antibody (Nossal, 1965) all quite independently dealt severe blows to instructive theories. The mortal blow came when it was shown that antibodies could be denatured and renatured in vitro in the absence of antigen and yet regain their specificity (Haber, 1964). The incompleteness of the theory of clonal selection ‘The difficulties of the theory of clonal selection as originally formulated centred around its failure to analyse exactly how antibody specificity was generated over a sufficient range. It also failed to present a framework for the quantitative analysis of the number of different antibodies required, of the thresholds of cellular response and of the population dynamics of lymphoid cells. Thus, for example, the theory came under strong attack when Simonsen (1967) showed that a smafl number of transplanted cells in a graft versus host reaction could be specific for a relatively Jarge number of antigens. The specificity problem can be stated in many ways, but perhaps the simplest way to pose it is to ask the following questions: (1) how many antigens are there? 2) how many antibodies are there? (3) how many antigen-binding cells are there? Before considering some experimental data which shed light on the problems of specificity and range in clonal selection, provisional answers may serve to set some limits. (1) The number of distinct antigens cannot be measured but must be very large. This conclusion derives from the fact that non-cross-reacting anti- bodies may be generated against proteins from a great variety of species as well as against antibodies themselves. Proteins in general have aperiodic structures and possess more than one kind of antigenic determinant on each polypeptide chain. Moreover, different antibodies may be made to denatured and native forms of the same protein. (2) If one cell makes one particular antibody, the number of different antibodies cannot exceed the number of antigen-binding cells. As I will show Molecular Recognition by Selective Systems 49 later, however, structural analysis indicates that the number of possible antibodies is legion. (3) The total number of lymphoid cells varies in different organisms capable of producing antibodies. It can be as small as 100 000 in a tadpole or as large as 10'! or 10'? ina man. The main point deserving analysis is this: the clonal selection theory implies that there must be very many more ways in which an antigen can be bound by a variety of antibodies than there are antigens to be recognised. Another way of putting this is to assert that the antibody forming system must be degenerate with regard to a given antigen—that is, many different anti- bodies must be capable of binding that antigen more or less well. Were this not the case, and were there a best or even a unique antibody for each antigen, then either each antibody must have been selected specifically by its antigen during evolution, or the clonal selection theory would have to assume a miraculous 1:1 correspondence between each antibody and its correspond- ing antigen. In either case, the theory would be vitiated. The facts are in accord with the idea of degeneracy: each antigen in general elicits the synthesis of a heterogeneous collection of antibodies, But this degeneracy points up the problem quite sharply, for it must imply that at some level there is a lack of specificity or an extensive cross-reactivity among different antibodies. At what level does this occur, and how is it that one does not see more evidence of it in circulating antibodies? To answer this question even provisionally we must examine the require- ments of the clonal selection theory in greater detail, particularly in the light of modern findings on antibody structure. The requirements of clonal selection The theory has three main requirements: (1) A source of diversity amongst different antibodies and a means of committing a cell or phenotypically restricting it to one of each possible kind of antibody (see figure 1). (2) A means of trapping antigen or favouring encounter with antigen binding cells. (3) A means by which this encounter is amplified by triggering the cells to divide and produce more antibodies of the same type. From analyses of the structure of antibodies (Edelman and Gall, 1969; Edelman, 1970a; Gally and Edelman, 1972), it has become clear that the basis for antibody diversity is variation in the amino acid sequences of approximately the first 110 residues (variable regions) of the light and heavy Polypeptide chains of which the antibody is made (figure 2). In addition, the variability is increased by the possibility of random assortment among different light and heavy chains. The number of different sequences and combinations possible is of the order of 10'¢ or greater. There are 10" or so lymphoid cells, and it is obvious that the diversity possible in the system is 50 Studies in the Philosophy of Biology Antigen binding ~ site (Carbohydrote) Figure 2. A model of the structure of a human IgG antibody molecule. The variable regions of heavy and light chains (Vir and Vz), the constant region of the light chain (C,) and the homology regions in the constant region of the heavy chain (C1, Cx? and C3) are thought to fold into compact domains (delineated by dotted fines), but the exact conformation of the polypeptide chains has not been determined. The vertical arrow represents the twofold rotation axis through the two disulphide bonds linking the heavy chains. A single interchain disulphide bond is present in each domain. Carbohydrate prosthetic groups are attached to the Cyx2 regions. sufficient for the binding of a large number of antigens of different shape. The intriguing problem of the genetic origin of sequence diversity remains unsolved. It has been critically examined elsewhere (Edelman and Gall, 1969; Gally and Edelman, 1972), and for this reason it will only be considered here as it bears upon the problem of specificity. Commitment of a cell to one of the possible light-heavy chain pairs is not fully understood but may be related to the unusual arrangement of the genes that specify antibody chains in a series of clusters. Each of these clusters contains separate genes for variable regions (V genes) and for constant regions (C genes) of antibody molecules. This arrangement has been called a translocon (Gally and Edelman, 1972), to emphasise the requirement that information from a V gene must be translocated and linked with that from a C gene to make a single VC gene for an antibody chain. Whatever the mechanism of such an event, it would serve to restrict the cell so that it would make only one kind of light chain (say Vi22C,) and one heavy chain (say VussCy) Although additional mechanisms of gene contro! must be invoked (as in any differentiated cell), it is not difficult to see how the require- ment for phenotypic restriction might be met by such an arrangement. Molecular Recognition by Selective Systems St It appears that the second requirement of clonal selection theories, that for antigen trapping, is mediated by a subtle interaction between two types of cells both with antibody receptors. These are respectively the thymus- derived lymphocyte (T cell) and the bone marrow-derived lymphocyte (@B cell). Primary immunisation by protein antigens requires cooperation between a T cell and a B cell; one of the more likely hypotheses is that T cells locally concentrate the antigen for presentation to B cells (Gowans, Humphrey and Mitchison, 1971), possibly by attachment of the antigen- antibody complex from a T cell to a third cell which then presents the complex to the B cell. In this way, the B cells are triggered to mature and divide (Feldman, 1972). This triggering phenomenon is the third requirement of clonal selection and here practically nothing is known of either the threshold or the mechanism of stimulation. This may be the major factor, however, in providing a solution to the central dilemma of specificity in clonal selection. As originally propesed, the clonal selection theory was noncommittal about the strength of binding of an antigen required for triggering or about whether triggering was a function of the avidity for antigen of the receptor antibody. Because an analysis of this problem is central to the relationship between specificity and selection, and because the problem can be attacked on logical as well as experimental grounds, it deserves detailed analysis. A two-factor theory for the specificity of clonal selection It has been observed that Darwin’s theory of evolution differed from previous theories in that they considered only one factor as the driving force for evolution. Darwin's theory and its more sophisticated successors considered the interplay between variation among organisms and natural selection. A similar situation may obtain in clonal selection. Diversification of antibodies may to a certain extent be under selective pressure by groups or classes of chemical substances during evolution. But according to the theory of clonal selection, it is impossible for each cell receptor to have been selected for or against during evolution. Instead, a great number of antibody variants have been generated (by whatever process; see Edelman and Gall, 1969; Gally and Edelman, 1972; Edelman, 19705), many of which will never be selected during the lifetime of the organism. The central question with which this paper has been concerned is: under these circumstances alone, how specific can the system be? In order to approach this question, it is important to distinguish several levels (Gally and Edelman, 1972) of potential diversity in the clonal system (figure 3). Almost all antibody-producing cells whose products have been characterised are derived from precursor cells that have been stimulated to proliferate (by antigen or otherwise) to form a clone of cells, each committed to the production of the same antibody. The phenotypic expression of the antibody genes is therefore mediated in the animal by the somatic division of 52 ‘Studies in the Philosophy of Biology Different Ig N genes: (Genotype! Zygole ‘Somotic praliferotien Tronslocation Generation of diversity? Immunacompatent Different Za's small monocytes Gadhdhdddadadad produced foveal Crimor ype) Antigen triggered clonal growin requires Tees ond LR, gene function) Af Different tgs 1g sesratng or COCODOORN In memory cells (Clonotype) Figure 3. A model of the somatic differentiation of antibody-producing cells according to the clonal selection theory. The number of Ig genes, which equals N in the zygote, may increase during somatic growth so that in the immunologically mature animal K different cells are formed, each committed to the synthesis of a structurally distinct Ig (indicated by the arabic number). A small proportion of these cells proliferate upon antigenic stimulation to form J different clones of cells, each clone producing a different antibody. N

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