The Mysterious Absymes
The words “immunity” and “antibody” as well as the term “enzyme” are undoubtedly known to everybody. However, how many people have heard about the unique proteins produced by our immune system that are able to catalyze manifold reactions? Accompanying incurable autoimmune diseases, these multifunctional molecules can be our enemies, but may also become a true godsend for biotechnology and medicine…
What is immunity? This currently fashionable word denotes one of the major mechanisms defending humans from the adverse environmental factors (the Latin word immunis stands for “pure and free of something”). Of the numerous defense systems existing in multicellular organisms, only those that involve the specialized cells, lymphocytes, are related to the immunity. The unique characteristic feature of these cells is the ability to recognize a tremendous number (about 1018!) of diverse potentially harmful objects (bacteria, viruses, foreign molecules, and so on), including those life has never encountered during the billions years of its existence on the Earth.
The lymphocytic immunity is a rather new evolutionary gain: not more than 1.5 percent of multicellular species, starting from jawed fishes, possess it. The representatives of these species give a comparatively scanty offspring; therefore, the survival of each individual is essential for the preservation of species. The mere fact that each tenth cell in the body of an adult human is lymphocyte demonstrates the importance of immunity in our life!Only those species of multicellular organisms for which the survival of each individual is essential for species preservation possess the lymphocytic immunity
The role of immunity is to protect us not only from the external “interveners” of various types and calibers, but also from our own damaged cells. Any molecule that, having entered the body, is capable of inducing the immune response is called the antigen.
Overall, there are two types of immunity. The immune system of the first type provides the so-called humoral response. In this case, the body responds to an encounter with an antigen by producing specific protein molecules, antibodies (or immunoglobulins, Ig), which recognize the antigen, bind it, and neutralize it. The immune response of the second type is called the cell-mediated immunity and is provided by special killer lymphocytes, recognizing the foreign antigens upon their binding to their own specialized cells of the body.
The Genealogy of a Lymphocyte
Let us consider the immune system of the first type. Antibodies formed during the humoral response are produced by a special class of lymphocytes, B lymphocytes. Similarly to other blood cells, they originate from the stem cell, i. e., the undifferentiated hematopoietic cell of the bone marrow, as a result of a multistep “specialization”. The mature nonimmune lymphocytes (they are called naïve, because they are not yet specialized for fulfilling specific functions) circulate with the blood between various organs and tissues of the body.
During the differentiation of lymphocytes, the entire tremendous diversity of genetic codes for variable (dissimilar in different lymphocytes) regions of the future antibodies responsible for the recognition and binding of antigens is formed: the genetic material inherited from the stem cell is rearranged in each lymphocyte in its own unique manner.
The naïve lymphocytes “make acquaintance” with various antigens in the peripheral lymphoid organs (lymph nodes, the spleen, and lymphoid tissues of the digestive, respiratory, and urogenital systems). Upon encountering a particular antigen, the lymphocytes that have succeeded in recognizing it commence an active reproduction and additional structural rearrangement of the genes encoding the variable part of antibodies. Thus, a new clone of B lymphocytes is formed that synthesizes antibodies capable of binding this particular antigen in a highly specific manner and eventually degrading it.
The immune system is able in principle to produce up to 106 variants of differentiated lymphocytes (and the corresponding variants of antibodies) to one and the same antigen; however, their actual number is smaller. Finally, only several percent of the cells that comprise the most efficiently acting antibodies remain from this multitude, whereas the initial overproduction is the necessary price for the accuracy and efficiency of the future immune response.
By the way, if a naïve lymphocyte accidentally “gets acquainted” not with a foreign antigen, but with a protein of its own organism, it usually dies. That is why our immunity does not kill ourselves.
Failures in the Immunity
The ability to distinguish between “one’s own” and “somebody’s” is the most important feature of the immune system. Unfortunately, failures of this system can occur, and the “prohibited” clones of lymphocytes start propagating in the body. Such clones are true “subversives”, as they produce antibodies to molecules of their own organism. Several dozens of autoimmune diseases that arise due to such failures present an important medical problem of the modern society, as they affect many people, including those of young and middle ages.
How do these diseases appear? It is difficult to find the cause in each particular case; however, it is known that genetic predisposition, hormonal disbalance, adverse environmental conditions (radiation, infections, toxins, unhealthy climate, etc.) are among contributory factors. Exposure to these factors results in the accumulation of autoantibodies, the antibodies to the organism’s own antigens. In case of the well-known bronchial asthma, the antigen is a small protein, vasoactive intestinal peptide; in case of multiple sclerosis, the antigen is the protein of myelin (the protein-lipid envelope of nervous fibers). Several viral diseases, such as AIDS and viral hepatitis, which affect the immunity, are also accompanied by autoimmune reactions.
It is generally assumed that the antigens are mainly proteins and polysaccharides; however, other molecules, in particular, DNA, the main carrier of hereditary information, are also able to display antigenic properties. Interestingly, the concentrations of both DNA itself and the antibodies to it are increased in the blood of many autoimmune cases. Systemic lupus erythematosus is particularly noteworthy in this respect.
It was assumed for a long time that antibodies can neutralize antigens only via a stable binding followed by their discharge from the body. However, it appears that this is not the only possible route. Thus, it is high time for the digression on other proteins — enzymes.
A New Science: Abzymology
Everybody has known from the school years that enzymes are proteins catalyzing a diversity of chemical reactions. The molecule of an enzyme binds the molecule of a substance (substrate) to transform it into another substance (product); the reaction takes place in a specialized region of the enzyme, its active center.
It was assumed at the dawn of biochemistry that the structure of the enzyme active center corresponded precisely to the structure of the substrate, i.e., fitted it as the key fits the lock. However, L. Pauling, a future Nobel Prize winner, demonstrated in the 1940s that the active centers were complementary not to substrates and products, but to certain intermediate structures, the so-called transient states formed during the corresponding reaction.
This hypothesis suggested Pauling a rather interesting inference: if the immune system could produce antibodies to the transient states, then the resulting antibodies would display the properties of enzymes. As we have seen, the immune system produces an incredible number of various antibodies, and among them an antibody that would match even such an exotic antigen can always be found.
However, it is technically impossible to realize Pauling’s scheme just as it is, as the transient states exist for a very short time. Therefore, W. Jencks later proposed to use stable molecules with the structure similar to a transient state to obtain the corresponding antibodies.
This idea was first confirmed experimentally in 1986. The antibodies that possessed a catalytic activity were named abzymes (antibody enzyme). The abzymes known now catalyze over 100 various reactions, including reactions for which natural enzymes are unknown.
Thus, we may assume that a new branch of biochemistry, abzymology, has come into being. Its latest advances include the designing of new drugs, for example, the abzymes cleaving narcotics in the blood. This direction opens prospects for producing new protein catalysts with manifold properties for research and, what is no less important, for biotechnology and medicine.
However, these remarkable achievements do not close the history of abzymology: successful experiments on designing various artificial abzymes stimulated a new turn in the study of antibodies in the organism, which allowed natural abzymes to be discovered.
The first natural abzymes, discovered by the US team of S. Paul in the blood of bronchial asthma patients, not only efficiently bound the vasoactive intestinal peptide, but also catalyzed its hydrolysis (the breakdown by water). Soon after this event, the Moscow team headed by A. G. Gabibov isolated the antibodies hydrolyzing DNA from the blood of systemic lupus erythematosus cases.
Then, a number of natural antibodies capable of cleaving DNA, RNA, ATP, proteins, and polysaccharides were discovered at the Laboratory of Repair Enzymes, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences. Such enzymes are found in the blood of the patients suffering from systemic lupus erythematosus, autoimmune thyroiditis, polyarthritis, multiple sclerosis, viral hepatitis, and AIDS and are absent in the blood of both healthy donors and those suffering from other diseases of nonautoimmune nature.
How do abzymes appear in autoimmune patients? As is known, during these diseases cells die actively; as a result, concentrations of all cell components (proteins, DNA, etc.) in the blood as well as of various complexes between them increase. Thus, abzymes can be formed via two routes. First, they may be represented by the antibodies produced directly to the products of cell breakdown, i. e., the body’s own antigens whose structure in a complex with other blood components may mimic the structures of transient states.
The other route is more intricate. According to the theory of N. Jerne, the primary (idiotypic) antibodies are first produced against an antigen. These antibodies, being proteins themselves, may play the role of antigens, thereby inducing production of the secondary (antiidiotypic) antibodies, and so on. Similarly to the initial enzyme, all antibodies of the higher orders may display a catalytic activity. According to the data obtained by Novosibirsk scientists, the blood of autoimmune patients usually contains a “cocktail” of abzymes with various properties and histories.
The appearance of abzymes is frequently the earliest sign of autoimmune processes; therefore, testing for abzyme activities is an exclusively promising approach to early diagnostics, prediction of the acute attack of a chronic disease, and assessment of treatment efficiency of particular drugs.
For example, it is possible to diagnose multiple sclerosis only at the late stages according to characteristic plaques detectable by tomography in the patient’s brain. The most reasonable theory explaining the origin of this disease assigns the main role to the autoimmune inflammatory processes impairing the transduction of nerve impulses. We detected the antibodies that destroy the major myelin protein in the patients with multiple sclerosis. The assay for this abzyme activity can detect the disease at its early stage.
There is yet no general recipe for treating the autoimmune diseases: it is possible to maintain the life of such patients for a long time; however, nobody has succeeded in curing them, most likely, since the precise cause of these diseases is still vague. Many data suggest that the development of autoimmune diseases is connected with abnormalities in the growth rate and differentiation of stem cells in the bone marrow.
Our Laboratory conducts research using laboratory mice of a special strain, which develop spontaneously a disease analogous to systemic lupus erythematosus in humans. Before any outward manifestations become visible, the division pattern and specialization of the stem cells in these animals change noticeably. At this stage of the disease, it is already possible to “see” the abzymes in the blood. The other parameters that are conventionally used for diagnosing change considerably later, when the disease converts into pathology.
In further work, which can be regarded as successful, we studied another disease — autoimmune thyroiditis, the most frequent illness of the thyroid gland. It is known that the antibodies found in such patients hydrolyze the protein thyroglobulin, used for production of the hormone thyroxin. In addition, the antibodies with a DNA-hydrolyzing activity were detected in 65 percent of the autoimmune thyroiditis cases, and this activity correlated with the diagnostic characteristics standard for this pathology.
Such patients usually receive a thyroxin therapy; however, this gives only a temporary improvement without any effect on the autoimmune processes themselves. We proposed that a group consisting of 30 patients undergo an experimental treatment with plaquenil. For a long time, this drug had been used for controlling malaria, and then its ability to suppress efficiently many autoimmune reactions was accidentally discovered. The plaquenil therapy decreased drastically the level of DNA-hydrolyzing abzymes in the blood with a concurrent normalization of other parameters: the concentrations of thyroglobulin in the blood and the antibodies to this protein dropped, whereas the concentration of thyroid hormones restored virtually to the norm (note that no hormone therapy was administered). This treatment normalized the function of the thyroid gland and improved the general state of the patients.
Abzymes as Enemies and Friends
Presumably, abzymes also play a negative role in other autoimmune diseases besides multiple sclerosis and thyroiditis. For example, the mice immunized with the abzymes hydrolyzing vasoactive intestinal peptide develop asthma, as the destruction of this protein hormone causes breathing problems.
Abzymes from the blood of systemic lupus erythematosus cases, which hydrolyze DNA, appear cytotoxic, i. e., hinder cell growth. Several proteins of the cytokine family, natural growth regulators of many cell types (one of these proteins, the tumor necrosis factor, is very important for protection of the organism against cancer) display the same ability. Moreover, the cytotoxicity of abzymes in certain cases was even higher compared with that of cytokines. Unlike conventional antibodies, the abzymes in question are able to penetrate somehow into the cell nucleus through all the membranes, bind to DNA, cleave it, and thereby kill the cell. There are grounds to believe that the appearance of abzymes at the early stages of autoimmune diseases may stimulate further development of the diseases.
However, it appeared that abzymes are not only enemies: in the 1990s, Novosibirsk scientists quite unexpectedly discovered abzymes in abundance in the blood of completely healthy persons — pregnant women. Moreover, as it was found, the abzymes were present in the breast milk after childbirth!
What is the reason of this phenomenon? The point is that the immune system of pregnant women is reorganized in a specific manner — the singular immune “memory” is switched on. This memory accumulates information about all the adverse environmental factors during pregnancy. In the case of mammals, the result is that the breast milk contains high concentrations of antibodies to all the antigens that immunized the future mother during 1—3 months (but no more) before the delivery. In this process, it is of no importance how a particular antigen has entered the mother’s body (via the blood or food) and what its nature is (be it a food component or bacterial or viral protein).
The immune processes are launched with the beginning of lactation according to the information that has been accumulated in the immune “memory” to guarantee that the breast milk will contain all the antibodies necessary for protecting the newborn. This confers a passive immunity on the newborn, as his/her own immune system starts to develop actively only at the age of 4—6 months.
Breast milk appeared a richest source of the abzymes that hydrolyze DNA, RNA, polysaccharides, the milk protein casein, and other substances. The catalytic activity of these antibodies proved to be even higher than the activity of antibodies from the blood of autoimmune patients. Moreover, it contains also the unique abzymes catalyzing not the degradation of substrates but their modification: namely, they are able to phosphorylate lipids, proteins, and polysaccharides, i. e., to attach the phosphate residue to these substances at certain positions.
Presumably, the abzymes of milk play a positive role by enhancing the protective effect of passive immunity via hydrolysis and modification of foreign molecules of viral, bacterial, food, and other origins. Of interest here is the fact that all the relevant data suggest that pregnancy stimulates processes in the woman’s body that are very similar to those observed in the autoimmune patients.
However, these processes halt with the end of lactation in the woman’s organism, whereas in sick persons they become chronic. The answer to the question of how the potentially dangerous autoimmune reactions are switched off in the woman’s body at the necessary moment may give a clue to the therapy of yet incurable autoimmune diseases.
Beat Swords into Plowshares
Abzymology is a young science encountering numerous problems. Perhaps, the most basic of them is to understand whether the abzymes of mammals are a standby system of enzymes that operates under extreme conditions with the activity lacking in the common enzymes or a byproduct of the immune system function.Mother’s breast milk contains all the antibodies necessary for protecting the newborn, including abzymes
Anyway, the concealed potential of the organism related to the production of various abzymes is undoubtedly very high. Thus, the first in the world monograph about the catalytic antibodies, published by Wiley-VCH in 2005, opens with the well-known words of Isaiah the Prophet “...they shall beat their swords into plowshares...”, which are most appropriate for this case.
Who knows, perhaps, further research into these unusual multifunctional proteins will actually lead to new amazing discoveries in medicine and pharmacology, and our yesterday’s enemies will turn into most efficient new-generation drugs. Who knows? After all, the history of abzymology is at its very beginning…