ieee enna ee eee TT ANTIBODY STRUCTURE, 93 General Features of Antibody Structure, 98 Structural Features of Antibody Variable Regions, 101 Structural Features of Antibody Constant Regions, 103 Monoclonal Antibodies, 106 ‘SYNTHESIS, ASSEMBLY, AND EXPRESSION OF IMMUNOGLOBULIN MOLECULES, 107 Holl-Life of Antibodies, 109 ANTIBODY BINDING OF ANTIGENS, 110 Features of Biologic Antigens, 110 Structural and Chemical Basis of Antigen Binding. 111 ‘STRUCTURE-FUNCTION RELATIONSHIPS IN ANTIBODY MOLECULES, 113 Features Related to Antigen Recognition, 113 Features Related to Effector Functions, 114 SUMMARY, 115 Inbibodies | Prnte- loxins ‘Antibodies are circulating proteins that are produced in vertebrates in response to exposure to foreign structures known as antigens, and are the mediators of humoral immunity against all classes of microbes./Amtibodies are ‘extremely diverse and specificin their ability to recognize foreign molecular structures” Because these proteins were discovered as serum molecules that provided pro- tection against diphtheria toxin, they were initially called antitoxins. When it was appreciated that similar proteins Balt be generated against many substances, not just jerobial toxins, they were given the general name antibodies, The substances that stimulated production ‘of or were recognized by antibodies were then called antigens, Antibodies and T cell antigen receptors (see Chapter 7) are the two classes of molecules used by the adaptive immune system to specifically recognize and respond to antigens (Table 5.1). Major histocompatibili complex (MHC) molecules,also bind ata antigens, Sc her She ese S very different and their function is to display the peptides to T cells, not respond to the Antibodies and Antigens antigens (see Chapter 6). Antibodies were the first typ of antigen binding molecule to be discovered, recogniz the widest range of antigenic structures, have the greates ability to discriminate between different antigens, an bind antigens with the greatest strength, In this chapte we describe the structure and antigen-binding propertie of antibodies. Antibodies are synthesized only by cells of the B lym phocyte lineage and exist in two forms: membrane-boun antibodies on the surface of B lymphocytes function a antigen receptors, and secreted antibodies function protect against microbes. The recognition of antigens b membrane-bound antibodies on naive B cells activate these lymphocytes and initiates a humoral immun response. The activated B cells differentiate into plasm cells that secrete antibodies of the same specificity as th antigen receptor. Secreted forms of antibodies are presen in the plasma (the fluid portion of the blood), in mucos. secretions, and in the interstitial fluid of tissues. In th effector phase of humoral immunity, these secrete antibodies neutralize microbial toxins, prevent the ent and spread of pathogens, and trigger several effect mechanisms that climinate the microbes. The elimination of antigens often requires interactio of antibodies with other components of the immur system, including molecules such as compleme: proteins and cells such as phagocytes and mast cell Antibody-mediated effector functions include neutraliz Saat tion of microbes or toxic microbial products: of the complement. system, ops oer enhai col ts ago eee inediated cytotoxicity, by which antibodies target infec Tims, We will describe thes detail in Chapter 13. When blood or plasma removed from an individu forms a clot, antibodies remain in the residual. flui Which is called serum. Serum lacks coagulation facto (which are consumed during clot formation) but contai all the other proteins found in plasma. Any serum samp that contains detectable antibody molecules that bind: a particular antigen is commonly called an antiserur ‘The study of antibodies on reactions with antige is therefore called serologypThe concentration of an body molecules in serum spfcific for a particular antigser structurally diverse antigens. In every individual, there are millions of different clones of B cells, each producing antibody molecules with identical antigen-binding sites but which differ from the antigen-binding sites of anti- ‘he effector functions foperties of antibodies bodies produced by other clone: are associated with the non-antigen-binding portions, and common physicochemical ® Secreted IgG, Heavy chain & Antigen- binding site Light: chain Fab Fe receptor/ region complement Ra binding sites eo Tailpiece“c C Disulfide bond Ig domain ) FIGURE 5.1 Structure of an antibody molec cule, The antigen regions end intl pieces. The locations of complement: constant regions are approximations. B, Schematic di surface of a B lymphocyte. The IgM molecule hes on form of the antibody has C-terminal tensmembrane the plasma membrane. C, Structure of a human IgG riobon diagram of a secreted IgG molecule, the identi can be easly visualized, although they ate identical, ate shown in gray. (Courtesy of Dr. Alex McPherson, ical ding sites are formed by the juxtaposition of molecule as revealed by ‘and the light chains are University of Calitornia, Antibody Structure which exhibit relatively few variations among different angibodies. CC antibody molecule has a symmetric core structure imposed of two identical light chains and two identi cal heavy chains (Fig. 5.1). Both the light chains and heavy chains contain a series of repeating homologous structural units, each about 110 amino acid residues in ‘8 Membrane IgM Antigen- binding site membrane of B cells oy erystollagraphy. ) A, Sche rematic diagram of a secreted IgG mo! VY. and V4 domains. The heavy chain C and Fe receptor-binding sites within the heavy chain lagram of a membrane-bound igM molecule on the: 'e More Cy, domain than lgG has, and the membrane {and cytoplasmic portions that anchor the molecule in y Xray crystallography. In this heavy chains are colored blue and red 0 that they Colored green; caroohydrates Irvine.)(5~Antbodies and Antigens FIGURE 5.2 Structure of an Ig domain. Each domain is ‘composed of two antiparallel arrays off strands, colored yellow and red, to form two frpleated sheets held together by @ dsulfide bond. The alagram shows an Ig constant (C) domain containing three and four fh strands in the two adjacent sheets. Note that the loops connect strands that ate sometimes adjacent in the same fipleated shest, but the loops sometimes represent connections between the two cifferent shoets that make up an lg domain, length, that fold independently in a globular motif thatis called an 1g domain, which we introduced in Chapters 3 and 4. An Ig domain contains two layers of B-pleated sheet, each layer composed of three to five strands of antiparallel polypeptide chain (Fig. 5.2). The two layers are held together by a disulfide bridge, and adjacent strands of each B sheet are connected by short loops. Itis the amino acids in some of these loops that are the most variable and critical for antigen recognition, as discussed later in the chapter. Antibody heavy chains and light chains both consist of amino-terminal variable (V) regions that participate in antigen recognition and carboxy-terminal constant (C) regions; the C regions of the heavy chains help mediate some of the protective or effector functions of antibodies, In the heavy chains, the V region is composed of one Ig domain, and the C region is composed of three or four Ig domains, Each light chain is composed of one V region 1g domain and one C region Ig domain. Variable regions are so named because their amino acid sequences vary among antibodies made by different B cell clones. The V region of one heavy chain (Vy) and the adjoining V region of one light chain (¥;) form an antigen-binding site (see Fig. 5.1), Because the core structural unit of each antibody molecule contains two heavy chains and two light chains, every antibody molecule has at least two antigen-binding sites. The C region Ig domains are spatially separated from the antigen-binding sites and do not participate in antigen recognition. The heavy chain C regions interact with other molecules and cells of the immune system and therefore help mediate most of the biologic functions of antibodies, sometimes called “effector” functions. In addition, heavy chains exist in two forms that differ at their carboxy-terminal ends: one form of the heavy chain anchors membrane-bound antibodies in the plasma membranes of B lymphocytes, and the other form is found only in secreted antibodies, The C regions of light chains do not participate in effector functions and are not directly attached to cell membranes, Heavy and light chains are covaléntly linked by disul- fide bonds formed between cysteine residues in the carboxy terminus of the light chain and the Cyl domain of the heavy chain. Noncovalent interactions between the V, and Vy domains and between the C, and Cyl domains may also contribute to the association of heavy and light chains. The two heavy chains of each antibody molecule are also covalently linked by disulfide bonds. There are different kinds of antibodies, called classes or isotypes, which have different heavy chain structures, discussed in detail later in the chapter. In the IgG isotype, these disulfide bonds are formed between cysteine resi- dues in the C;2 domains, close to the region known as the hinge, described later in the chapter. In other isotypes, the disulfide bonds may be in different locations. Nonco- valent interactions (e.g., between the third Cy domains [G3]) also contribute to heavy chain pairing hese regions were by proteolysis of rabbit IgG molecules. In these molecules, the unfolded hinge region between the Cul and C,2 domains of the heavy chain is the segment most susceptible to proteolytic cleavage. If rabbit IgG is treated with the enzyme papain under conditions of limited proteolysis, the enzyme acts on the hinge region and cleaves the IgG into three separate pieces (Fig. 5.3A), ‘Two of the pieces are identical to each other and consist of the complete light chain (V, and C,) associated with @ Vu-Cul fragment of the heavy chain these F > bind 2 V4 domains, al disulfi "n pepsin (instead of papain) is used to cleave abl IgG under limiting conditions, proteolysis occurs distal to the hinge region, generating a F(ab’), frag. rent of Ig6 with the hinge and the interchain disulfide Figs amit and two identical antigen-binding sites (see The basic organization of the antibody molecule deduced from the rabbit IgG proteolysis experiments is common to all Ig molecules of all classes and all species, and the terms Fab, F(ab’),, and Fc are widely used to describe these different portions of human and mouse antibodies. In fact, these experiments provided the first evidence that the antigen recognition functions and the effector functions of Ig molecules are spatially separated, Many other proteins in the immune system, as well #8 numerous proteins with no known immunologic