It had been determined by several means that the proteins with antibody activity (immunoglobulins) had a molecular weight of approximately 150 kda.

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1 Immunology Dr. John J. Haddad Chapter 4 Antibody Structure and Function In 1890, Emil von Behring and Shibasaburo Kitasato showed that serum (the straw-colored liquid remaining after blood clots and the clot is centrifuged away) from animals previously immunized to idiphtheriai could transfer resistance to unimmunized animals. Throughout the first part of the 20 th century, scientists tried to identify the active component in serum. In the 1930 s, Elvin Kabat and others showed that proteins in a fraction of serum called γ-globulin had activities of antitoxins, precipitins and agglutinins that had previously been thought to be separate activities. The active molecules in the γ-globulin fraction were given the name, antibodies. Much work all over the world followed to determine the identity and structure on antibody molecules. A major mystery was how proteins in the γ-globulin fraction could be so specific recognizing and neutralizing only the organism against which an individual was immunized. Even Linus Pauling, a Nobel laureate for his work on the nature of the chemical bond, weighed in with a theory. He suggested that immunoglobulin molecules the class of proteins to which antibodies belong were malleable proteins that could fold themselves around antigens and then, having taken a shape complementary to the antigen, persist to recognize only that antigen whenever they encounter it. (He was wrong.) The answer came in the 1950 s and 1960 s as a result of work by Gerald Edelman at the Rockefeller University and Rodney Porter (at Oxford University in England). They shared the Nobel Prize in Medicine in Also contribution by Alfred Nisonoff at Brandeis University. It had been determined by several means that the proteins with antibody activity (immunoglobulins) had a molecular weight of approximately 150 kda. Porter added the proteolytic enzyme, papain, to the fraction of serum that contained the antibody activity. He then fractionated the resulting fragments by ion exchange chromatography. He showed that the immunoglobulins were cleaved into 3 large fragments 2 of them identical ((45 kda in molecular weight) and able to bind to the antigen at a single site, and one of approximately the same size that did not bind antigen and which crystallized spontaneously. He called these Fab (for fragment antigen-binding) and Fc (for fragment crystallizable). Nisonoff used an approach similar to Porter s, except that he used the enzyme pepsin. He obtained a fragment somewhat smaller than immunoglobulin that, like immunoglobulin, had 2 antigen-binding sites. He called this (Fab ) 2, and then found that if he added an agent that disrupts disulfide bonds (i.e., a reducing agent such as 2- mercaptoethanol or dithiothreitol), the (Fab ) 2 broke down into two identical fragments (called Fab ), each of which bound antigen. Edelman studied the γ-globulin structure by treating it with reducing agent, rendering the reduced cysteines unreactive with an alkylating agent, followed by transfer to a denaturing solvent a solvent such as 6 M urea that disrupts van der Waals interactions that help hold protein structure in place. By electrophoresing the resulting solution on a gel, he saw chains of two sizes. When he used a column that separates proteins on the basis of size, he could account for his input of protein by concluding that it broke down into 2 chains 50 kda in molecular weight (heavy chains) and 2 chains of 25 kda (light chains). This information was reconciled by both Edelman and Porter into the first real model of the immunoglobulin molecule (Figure 4-2). These studies gave the overall structure of immunoglobulin molecules, but could not give details about the actual sequences of the heavy and light chains. The main reason was that immunoglobulins isolated from serum are highly heterogeneous in amino acid sequence. None of the protein chemical techniques available could shed light on the regions of the molecule responsible for antigen binding what everyone wanted to know. 15

2 Myeloma Important information on this question came from the study of pure proteins (i.e., homogeneous) that had all of the properties of normal serum immunoglobulins but were produced by certain cancer patients in gram quantities. These patients all had plasma cell tumors, referred to as myelomas tumors of a single cells that normally produce antibody that secreted large quantities of what appeared to be a single pure protein (called a myeloma protein) into their serum. Many of the patients also excreted a protein in their urine that turned out to be dimers of the light chain contained in their myeloma protein. These were called Bence-Jones proteins after their discoverer. In humans, the antigen bound by a patient s myeloma protein was not known. The plasma cell that became a cancer cell apparently did so at random. It was discovered, however, that myelomas could be induced in a particular strain of mice (BALB/c) by intraperitoneal injection of mineral oil. When this was done to mice that had been immunized, some antigen-specific myelomas could be obtained. Amino acid sequence analysis of the chains of myeloma proteins and Bence-Jones proteins provided important clues to the basis for: The antigen specificity of individual antibody molecules The specificity of antibody responses for the immunizing antigen Light chains Amino acid sequence analysis showed that light chains are of two kinds called kappa (κ) and lambda (λ). Both types of L chain are about 214 amino acids (aa) in length. Beginning at residue 108, all kappa chains have one sequence and lambda chains have a different shared sequence. Now let s compare lots of kappa chains from different myeloma and Bence-Jones proteins. All have the same sequence of their C-terminal 107 aa (called the C region for constant). However, each has a different sequence in its N-terminal 107 aa. The N-terminal sequences (called the V regions (for variable) are not all wildly different some positions are the same, others have one or two conservative alternatives, and some are highly variable. (Fig. 4-8) Similar variability is seen in the N-terminal region of myeloma lambda chains. If you look carefully, the amino acid sequences of V-regions of kappa chains are more similar to each other than they are to lambda V-regions. Similarly, lambda chain V-regions are more similar to each other than they are to kappa chain V-regions. As we will see, kappa and lambda light chains are encoded by different genetic loci residing on different chromosomes. Different species have different ratios of kappa and lambda chains: Heavy chains Mouse about 95% kappa Human about 60% kappa, 40% lambda Chicken 100% lambda Sequencing of the myeloma heavy chains gave a similar story. The sequences of the N-terminal approximately 110 aa varied from one chain to another, However, 5 classes of C region sequences were observed called mu (μ), delta (δ), gamma (γ), alpha (α) and epsilon (ε). Interestingly, unlike the case for kappa and lambda light chains, looking at the V region sequences, you could not tell which class of C-region sequence it came from. As we shall see, all heavy chains share the same set of V regions. 16

3 The degree of variability of each position of the V H regions varies in a manner similar to kappa light chains. Some residues vary very little from H chain to H chain, and some are highly variable (or hypervariable). The genetic locus encoding the heavy chains is located on a chromosome different from either kappa or lambda light chains. In fact, the gamma C-terminal sequences could be subdivided into four subclasses, and the alphas into two subclasses. Not of primary importance for our discussions. The five basic classes of antibodies take their name from the class of heavy chain they contain: IgM contains μ H chains IgG contains γ H chains IgA contains α H chains IgD contains δ H chains IgE contains ε H chains We will see below that another name for an H chain or immunoglobulin class is isotype. Overall structure of the immunoglobulin molecules All antibodies are made up of the basic four chain unit two heavy chains linked to each other by disulfide bond(s), and each heavy chain bound to a light chain (see Figures 4-2 and 4-7). If you trace the polypeptide backbone of the light chain (Figure 4-5), you observe what is called the immunoglobulin fold that is observed in each domain of all antibody chains. The C H region has 4 strands making one anti-parallel β pleated sheet and 3 strands making a second anti-parallel β pleated sheet. These are folded over and an internal disulfide bond runs between them. This overall pattern is seen in every C domain of heavy and light chains. The structure of the V L domain and the V H domain is similar to C domains but not identical. Very important: the most variable positions of the light and heavy chains are located at the ends of the regions that would form the Fab. This is the location of the antigen-binding site. This is why so many different antibodies that bind so many different antigens are possible. The positions of highest variable form the antigen binding sites. Since the hypervariable regions contribute directly to the shape and other properties of the antigen-binding site that make it complementary to the epitope bound, the hypervariable regions of L and H chains are referred to as the complementarity-determining regions or CDRs of the antibody chains. Antibody structure and function Antibodies are at least bivalent a four-chain unit has two identical antigen-binding sites. These sites bind antigen non-covalently, with interactions between antibody and antigen mediated by ionic forces, hydrogen bonds and van der Waals interactions. Domains are specialized for function: V regions for antigen binding; C L and C H 1 to stabilize interactions of L and H chains of V regions can interact to form antigen binding site; Hinge for moderate flexibility; and last two C H regions to carry out effector functions of the antibody molecule. The μ and ε chains do not have a hinge region, but have an extra C domain in its place. In IgM and IgE molecules, this extra C domain serves a similar function to the hinge region of the other immunoglobulin isotypes. Effector functions are defined as the special attributes and functions of a particular immunoglobulin isotype such as: Its ability to cross the placenta (IgG) 17

4 To be secreted into the gut (IgA) To survive in acidic environments such as the gut (IgA) To bind to cell surface receptors and mediate allergy (IgE) The ability to activate the complement system (certain IgG subclasses, IgM) The ability to polymerize into multiples of the basic four-chain unit (IgM, IgA) These effector functions are mediated by the portion of the H chains that would be found in the Fc fragment after protease digestion. Briefly reviewed the structures and functions of the five immunoglobulin isotypes (Figure 4-13) including the involvement of J chain in polymerization of basic four-chain units. Also, the involvement of the poly-ig receptor in secretion of IgA across the gut wall and stabilization of the molecule in the hostile environment of the gut. Brief discussion of the terms isotype, allotype and Idiotype: The use of immunoglobulins as immunogens results in antibody responses of differing strength and specificity, depending upon the donor and recipient. The epitopes recognized are predictable, and the antibodies produced can be used for various purposes. An antiserum produced by immunization of one species (rabbit) with IgG from a different species (human) contains anti-igg isotype antibodies. These antibodies recognize epitopes in the constant regions of the H and L chains, and can be used to detect human IgG antibodies and distinguish them from other isotypes (IgA, IgM, IgE, IgD). We will use antibodies like this in Chapter 6. An antiserum produced by immunizing one individual of a species with immunoglobulins from another individual of the same species may contain anti-allotype antibodies. The epitopes recognized may be in the C region of the L or H chain, and generally correspond to single amino acid substitutions that represent polymorphisms in the genes encoding the antibody chains. These antisera can be used in genetic typing. An antiserum produced by immunizing one individual of a species or inbred strain with a monoclonal antibody from the same species is likely to contain anti-idiotype antibodies. If the individuals do not differ in allotype, all of the antibodies will recognize epitopes unique to the V H and V L present in the monoclonal antibody. Most antibodies will recognize the antigen-binding site of the monoclonal antibody. The epitopes that define the antigen-binding site of the monoclonal antibody are collectively referred to as the idiotype of the antibody. Individual epitopes of the antigen-binding site are referred to as idiotopes. B cell receptor for antigen B cells contain surface-bound IgM (Figure 4-18). The carboxy-terminus of the μ chain of the IgM is different from what it is in secreted IgM. It contains a hydrophobid transmembrane segment that anchors the molecule in the plasma membrane. Only a very few amino acids extend into the cytoplasm far too little to act in signal transduction. The IgM molecule is therefore associated with a heterodimeric molecule, Ig-αIg-β, that caries out the necessary signaling (discussed Chapter 11). Recent evidence suggests that only one Ig-αIg-β heterodimer is associated with each membrane IgM molecule. Monoclonal antibodies Antibodies isolated from serum of an individual immunized with a complex immunogen consist of a collection of different antibodies that recognize different epitopes. These antibodies represent the products of many different clones of plasma cells, each of which makes a single antibody with characteristic V H and V L sequence (and therefore a characteristic antigen-binding site). If you could grow pure B cells clones in culture, you could isopate pure antibody against a single epitope. Such antibodies would be useful for diagnostic assays to be described later (Chapter 6). This goal can be accomplished by 18

5 physically fusing spleen B cells from an immunized a individual with a mutant of a myeloma tumor cell that grows in culture (is immortal). The resulting hybrid cell, or hybridoma, is able to grow in culture like the original tumor cell but produces the antibody specified by the B cell. To do this, you must use a mutant of the myeloma tumor cell that fails to produce its own immunoglobulin molecule and that also lacks the ability to produce an enzyme, HGPRTase. By culturing the fused cells in HAT medium, you establish a situation in which only hybrid cells consisting of a tumor cell and a B cell can grow. These cells are plated in tiny wells, and the medium from each well is tested for the desired antibody. Cells from producing the desired pure monoclonal antibody are isolated and stored frozen in liquid nitrogen. When antibody is needed, these can be returned to culture and the antibody purified from the culture fluids (see Figures 4-20, 4-21 and 4-22). Uses of these antibodies will be discussed in Chapter 6. One use we already know of monoclonal antibodies are used to define many of the CD antigens such as CD4 and CD8. 19