Who started library? It’s Nature. Nobody
knows when and how it started. However, before human intelligence was improved
or even before the first appearance of the human race, nature has utilized
libraries though a function called, evolution.
A good example is immune response of antibodies.
Antibody immune responses are only found in higher animals of vertebrata. It
is one of the most elaborate defensive systems ever found to protect the body
from foreign substances. The invading substance is called
antigen (immunogen). Because any substances
as well as proteins and carbohydrates can become antigens, types of antigens are
unlimited. An immunoglobulin molecule, antibody, which reacts selectively with
antigens, a sort of protein with only consisted of 20 amino acids, and has
determining regions of the heavy- and light-chain V domains. Considering that
genetic information in DNA controls protein synthesis, does a living organism
have to have all the information about each antibody? Is there billions of
genetic information to synthesize billions of antibodies to fight against
billions of antigens? Tens of thousands of total human genes cannot manage this
huge number.
Antibody
<http://www.sdix.com/tsd/, http://www.accessexcellence.org/AB/GG/Antibody.html>
To solve the problem, a living organism uses a library method. Among the entire antibody structure, diversity of antigen binding sites is the most important. The binding site is consisted of at least five fragments of random peptides including the heavy- and light-chains. It’s not encoded by one gene. If there are 10 fragments, total combination is 100,000 and this method enables very diverse structures to form. Immune system actually uses only several hundreds genes to build up billions of antibodies to fight against foreign substances.
Antibody Attack
(http://www.gcarlson.com/method_atc.htm,
www.biotech.ufl.edu/~hcl/
antibody_apps.htm)
Let’s talk about how a library method is applied to antibody formation. An antibody is a protein called immunoglobulin (Ig) and categorized into G, A, M, E, D, and etc. The most general antibody is IgG, which has 3-D Y structure of two sets of the heavy- and light-chains and protein mass is about 150 kD. Fab (antigen binding fragment), antigen recognition site, is at the end of an antibody and a bridge is called Fc (crystalizable fragment). The antigen binding site is composed of 108 amino acids and forms by combinations of three fragments, V (Variable), D (Diversity), and J (joining). In other word, it is a V-D-J combination of one among various V’s (V1, V2, V3...V250), one among various D’s (D1, D2, D3...D15), and one among various J’s (J1, J2, J3...J5). Therefore, the number of total combinations is 250 x 15 x 5 = 18,750, and it only applies to the heavy chain. The light chain has a combination of V-J without D region, which gives 250 (V) x 4 (J) = 1,000 choices. So the number of the total combinations of the heavy and light chains is 18,750 x 1,000 = 18,750,000. In addition, alternative splicing produces additional diversity to the chains because linkage bridges of D-J parts and V-J parts often do not match. The alternative splicing then multiplies the possible number of antibodies to 3 times greater. This is relevant to library production.
Antibody Production
<http://nongae.gsnu.ac.kr/~sykim/body239.jpg>
The antibody with this combination formed based on inherited genetic
information of V-D-K is called a
Germline
Antibody. Even though various kinds of antigens invade, there will be
several antibodies that bind to the antigens with relatively high affinity.
However, 18,750,000 x 3 (by alternative splicing) are not the total of possible
combinations. There are more! Because there are 108 amino acids consisting of
the binding site and 20 kinds of amino acids, theoretically 20108 antibodies can
be made.
Nature performs another magic at this point. Among germline antibodies a
selected antibody (lead antibody) with high affinity toward a certain antigen is
produced by a B lymphocyte, a sort of somatic cells. The B
lymphocyte keeps creating new modified
antibody groups by partial mutations. It is called somatic mutation. New
diversity is created by the somatic
mutation, and new optimized antibodies with increased affinity toward
antigens are selected. This step is relevant to Library
Optimization process.
There are two competitive models about antibody-antigen binding or enzyme and
substrate binding, which are lock-and-key
model and induced-fit model.
Lock-and-key is a model that explains the basis of specificity as the exact fit
of antigen to a site on the antibody that is complementary in shape and
electronic charge, and induced-fit is a model relatively large antibody binds
preferentially with several conformers of the antigen by changing its shape of
binding site to fit. According to recent research on germline antibody and
somatic mutation antibody, it was found that germline antibody changes its shape
to bind to antigens by following induced-fit theory. On the other hand, antibody
of somatic mutation follows lock-and-key theory. In this case, mutated amino
acids take roles to support a certain shape of the antibody at non-binding
regions. Therefore, at the beginning when antigen is first recognized, germline
antibody, which flexibly grasps the antigen, is selected, and then somatic
mutation improves antigen by fixing the grasp conformation and increasing the
affinity toward the antigen.
<http://www.biology.arizona.edu/immunology/tutorials/immunology/09t.html>
Somatic Mutation
<http://www.srl.cam.ac.uk/tcrg/stem.html>
Since antibody has been explained, let’s talk about several important concepts of immunology. The word “immunology” is derived from Latin word “immune” meaning “safety.” When a living organism is attacked by foreign substance, defensive functions immediately react against it. Immune response is one of the defensive functions. As the first defensive line, there are skin, stomach (gastric juice: pH 2), mucosa, tears, and saliva (lysozyme, IgA) against foreign substances such as virus, bacteria, and mold. When the first line is broken through, immune response is the next. There are innate immunity and acquired immunity. Complement, macrophage, lysozime, and interferon take parts in the innate immunity by nonspecific reaction. Therefore, lysozime and interferon are called natural antibiotics. Acquired immunity is a form of cellular defense which identifies certain foreign substances as harmful to the body. It can recognize various antigens, and such a powerful response can be possible due to evolution of three kinds of cell surface receptors. Each has high diversities, which are T cell, antibody, and MHC molecules. Considering the similarities of their protein structures, it is assumed that their origin is the same gene. Because it is difficult to classify if a certain foreign stimulus is harmful or harmless, our body always responses to an unknown substance as a harmful intruder. If immune response succeeds, the body recovers and a specific memory remains, acquired immunity, so that it can deal with the same substance next time. However, immune response does not always function in good ways. Extreme immune response can cause autoimmunity, graft-versus-host disease, and hypersensitivity. Therefore, immune response has to be inhibited in cases of autoimmunity, hypersensitivity, and transplantation surgery. Especially, if an organ acceptor has been transfused blood from an organ donor before surgery, transplantation will cause acute rejection because the acceptor is already immunized. For reference, house mice have unusually strong immune system and Syrian hamsters do not show any transplantation rejection. Human has almost the same organ systems as mice. Since the most four causes of human death are injury, infection, cancer, and degenerative diseases, it seems like they are all related to immune responses.
Antigen
processing
T,
B cells,
macrophages (called monocytes when it is
in blood),
dendritic cells, Langerhans cells,
mastcells, and
granulocytes are immune cells. In T
lymphocytes, there are helper T cells
(containing CD4 and bind to
MHC II) and
killer T cells (containing
CD8 and bind to
MHC I), and B cells express
antibodies and bind to antigens. Immune response can be divided into
humoral immunity through antibody
and cell mediated immunity
through killer T cell. Main recognition systems are T lymphocyte, B lymphocyte,
and MHC in immune system, and it is guessed that T cell immunity occurred before
the first appearance of antibodies. In B cell where T cell acceptor and antibody
are produced, DNA recombination and diversity is increased. Recombinant process
includes DNA restriction, and through additional restriction at mRNA step, only
one acceptor or antibody is produced among numerous possibilities.
Immunology Reference
Playfair, J. H. L., Immunology at a Glance, 5th ed.(1992)
Travers, J., Immunobiology, 2nd ed. (1996)
Roitt, I. Essential Immunology, 7th ed. (1991)
Kimball, J. W. Introduction to Immunology, 3rd ed. (1990)
▲ top
1798: Jenner gave inoculation method a trial.
Beginning of Immunology
1881-1885: Pasteur’s Vaccine (used cholera,
anthracnose, and hydrophobia)
1882: Mechnikov found macrophage’s phagocyte
activity
1890: Behring tried to cure tetanus with passive immunotherapy
1900: Landsteiner found ABO blood types. Red Cross
1906: Pirquet found allergy.
1910: Dale found histamine and established antihistamine industry.
1922: Fleming found lysozime and Penicillin.
1944: Medawar tried skin transplantation. (rejection occurred lol)
1947: Owen found twins do not show rejection to each other.
1957: Isaacs and Lindemann, found interferon.
1959: Gowans found lymphocyte cilculation
1960: Lymphocyte modification
1961: Discovered relation of immune response and thyroid
1966: Found T-B cell associated reaction
1971: Found inhibition by T cells
1974: Jerne proposed Network theory for immune control.
1975: Milstein & Kohler made monoclonal antibody
1980: Official extermination of Smallpox, but…
1981: AIDS appeared instead of smallpox
1984: Structure of T cell receptor found
1987: Structure of MHC I found
▲ top
Development of efficient and selective catalytic antibody is very important for the entire chemical reactions. The most outstanding bio catalyst in nature is enzyme, and enzymes are used in various chemical reactions. However, basically enzyme works under bio conditions and its reactions are limited to bio relevant reactions. Therefore, as a method to overcome such limitations of bio catalysts, Schultz and Lerner developed catalytic antibody by designing artificial antibody in 1986, which can catalyze certain reactions. The catalytic antibody is used for diverse chemical reactions such as hydrolysis, pericyclic reaction, isomerization, and redox reaction.
If transition state is more stabilized by reducing activation energy, the rate of chemical reactions including transition state of starting materials will be much faster. Such a hypothesis and possible theories have been suggested since 1960’s, but it seemed almost impossible to inject antigen of unstable transition state into a living organism to induce antibody production. However, by designing analogues of chemically stable transition states, which have similar structures and electric distribution, catalytic antibody became a very powerful and valuable weapon.
However, even though the analogue antigen is synthesized and injected into a living organism with transport proteins, sometimes antibody does not show enough catalytic activity or even do not form. Or the formed antibody with enough activity can be missed by inappropriate screening methods. In such cases, it’s not easy to find out the problems and the best solution is to design a new antigen again. Therefore, efficient antigen designs and efficient screening methods are required and focused for development of superior antibody.
In 1997, Scientists at Scripps Research Institute developed a new method to fish up antibodies of efficient activity by covalent bonding (Science, 1997, 275, 945). They obtained genes expressing binding sites of antibodies and inserted library of random arrangements of the genes into bacteriophage genome. Inserted genes are expressed as parts of phage’s surface proteins, and there are numerous same antibody proteins expressed on a phage. They invented a reaction to cut carbohydrate’s anomic region by fixed reaction substrates on solid phase. When the substrate is hydrolyzed by catalysts, highly active functional group is artificially designed to form. The reaction and covalent bonding occur at the same time and consequently the reacted phage is fixed to the solid phase. After washing left over phages and reaction materials, only antibodies of catalytic reaction are obtained. Even though some antibodies are structurally modified by covalent bonding, there is an advantage of amplifying the selected antibodies as pure as well by amplification of phages. This new method enables scientists to make antibodies of 100 times more active than antibodies from hybridoma method, and it seems like after several mutation cycle even superior antibodies can be made. This is outstanding practical advance, and moreover the research result realized a short term molecular evolution.
For last ten years, the catalytic antibody has become the most powerful candidate that can imitate enzyme’s catalytic activity. However, there have been criticisms refuting if the catalytic property of catalytic antibody is from a specific binding or from protein’s general properties. For instance, in 1996 Hollfelder and others refuted that albumin, a common protein, could be used as a catalyst and antibodies were not particularly needed (Nature, Sep. 5. 1996, 60). The protein’s enzymatic activity was never found before and it was only known as an ion-transporter. None the less, the reaction catalyzing rate of albumin is almost the same as that of the catalytic antibody. This suggestion recalled the controversy if making a system of similar enzymatic catalytic reaction is the best method.
Due to the attractive point of particular properties and million times fastened
reaction rate, there have been lots of attempts to copy enzymatic activity, not
only on antibodies. A typical example would be a design of intramolecular
reaction imitating enzymes and substrate complexes. Especially hydrolysis
reactions of amides and esters were studied by nucleophiles or various
functional groups that can be attacked by general acids and bases. There are two
ways to increase reaction rates in such experiments. One is when movements of
reaction materials are reduced and give high possibility to react, and the other
is to design reacting groups’ structural arrangements are profitable for
reactions. In some cases when distances and directions of reaction groups are
well controlled, reaction rates jumped up to 100 million times faster. But,
because catalysts and substrates have to be bonded covalently, it is not a real
catalyst reaction. Regeneration of catalysts does not happen after the reaction.
Enzymes have evolved to release its products to increase substrate affinity, to
achieve chemical reactions, and to perform the next reaction. Its imitating
artificial machinery is invented by Micell. It can control reaction rates by
static attractions. Also, polymers like polyvinyl imidazole which showed strong
properties at Michaelis-Menten kinetics study can be another good example. Even
though most of such systems could not copy active regions as elaborately as
cyclodextrine or macrocycle, their reaction rates were accelerated to 100~10,000
times faster.
A fundamental concept of enzyme’s catalytic activity is that enzyme’s active
sites selectively lower substrate’s transition energy. In the catalytic
antibody, heptene (immune reaction inducing molecule) is the very stable
molecule that similarly copied 3-D structures and electric distributions of
reactants’ transition states. Therefore, catalytic antibody’s reaction
acceleration was expected faster than 100~10,000 times suggested above.
Catalytic acceleration can be calculated by ratio of kcat of antibody-substrate
complex conversion to non-catalytic reaction rate. If antibody does not show a
similar acceleration to enzymes, it is because of insufficient imitation of the
transition state. Another reason would be antibody’s exposure to solvents.
Usually enzyme-substrate complexes are formed when they are blocked from
solvents, but antibody-antigen reactions occur on a surface of a protein exposed
to solvents. Antibodies cannot provide necessary amino acid branches for
chemical reactions and bring out reactant groups closer.
<http://www.scripps.edu/research/skaggs97/>
Why is the enzyme so special? The above explanation might focus too much on stabilities of reactants’ transition states. The free energy difference of reactants and transition states is important. How can it be reduced? Because polarities of substrates and enzymes are not so different, it is a little weird to find that affinity of enzymatic transition state is a billion times higher than transition state of substrates. If there are hidden clues, they must be arrangement inducing substrates activated before they get to transition states and surroundings to induce it easily. Structures of such surroundings then provide molds to chemical reactions by arrangement of 반응참여기 and fixed water molecules. In contrast, if the same reaction occurs in solvents, rearrangement by the solvents will interrupt it to go to its transition state.
