《免疫学》（英文版） Chapter 04 Antibodies Structure and Function

Antibodies chapter 4 Structure and function NTIBODIES ARE THE ANTIGEN-BINDING PROTEINS present on the B-cell membrane and secreted by plasma cells. Membrane-bound antibody con- fers antigenic specificity on B cells; antigen-specific prolifer- ation of B-cell clones is elicted by the interaction of membrane antibody with antigen Secreted antibodies cir- culate in the blood, where they serve as the effectors of hu- moral immunity by searching out and neutralizing antigens or marking them for elimination. All antibodies share struc- tural features, bind to antigen, and participate in a limite number of effector functions IgM, the First Responder The antibodies produced in response to a particular anti- gen are heterogeneous. Most antigens are complex and con- Basic structure of antibodies different antigenic determinants, and the immune system usually responds by producing antibodies to several a Obstacles to Antibody Sequencing epitopes on the antigen. This response requires the recruit- a Immunoglobulin Fine Structure ment of several clones of B cells. Their outputs are mono- a Antibody-Mediated Effector Functions antigenic determinant. Together, these monoclonal antibod Antibody Classes and Biological Activities ies make up the polyclonal and heterogeneous serum anti- The B-Cell Recepto The Immunoglobulin Superfamily Basic structure of antibodies Monoclonal Antibodies Blood can be separated in a centrifuge into a fluid and a cel- ular fraction. The fluid fraction is the plasma and the cellu lar fraction contains red blood cells, leukocytes, and platelets. Plasma contains all of the soluble small molecules peak in the aliquot that had been reacted with antigen(Fig and macromolecules of blood, including fibrin and other ure 4-1). Thus, the y-globulin fraction was identified as con- proteins required for the formation of blood clots. If the taining serum antibodies, which were called immunoglob- blood or plasma is allowed to clot, the fluid phase that re- ulins, to distinguish them from any other proteins that might mains is called serum. It has been known since the turn of be contained in the Y-globulin fraction. The early experi the century that antibodies reside in the serum. The first ments of Kabat and Tiselius resolved serum proteins into evidence that antibodies were contained in particular three major nonalbumin peaks-o, B and Y We now know serum protein fractions came from a classic experiment by that although immunoglobulin G(lgG), the main class of Tiselius and E.A. Kabat, in 1939. They immunized rabbits antibody molecules, is indeed mostly found in the y-glob bulin nt amounts of it and oth then divided the immunized rabbits serum into two classes of antibody molecules are found in the a and the B peaks corresponding to albumin and the alpha (a), beta(B), and gamma(y) globulins. The other serum aliquot was re- Antibodies are heterodimers removed; the remaining serum proteins, which did not react Antibody molecules have a common structure of four oth the antigen, were then electrophoresed. A comparison peptide chains(Figure 4-2). This structure consists of two the electrophoretic profiles of these two serum aliquots identical light(L)chains, polypeptides of about 25,000 revealed that there was a significant drop in the y-globulin molecular weight, and two identical heavy(H)chains, larger

peak in the aliquot that had been reacted with antigen (Fig￾ure 4-1). Thus, the -globulin fraction was identified as con￾taining serum antibodies, which were called immunoglob￾ulins, to distinguish them from any other proteins that might be contained in the -globulin fraction. The early experi￾ments of Kabat and Tiselius resolved serum proteins into three major nonalbumin peaks—,  and . We now know that although immunoglobulin G (IgG), the main class of antibody molecules, is indeed mostly found in the -globulin fraction, significant amounts of it and other important classes of antibody molecules are found in the and the  fractions of serum. Antibodies Are Heterodimers Antibody molecules have a common structure of four peptide chains (Figure 4-2). This structure consists of two identical light (L) chains, polypeptides of about 25,000 molecular weight, and two identical heavy (H) chains, larger chapter 4 ■ Basic Structure of Antibodies ■ Obstacles to Antibody Sequencing ■ Immunoglobulin Fine Structure ■ Antibody-Mediated Effector Functions ■ Antibody Classes and Biological Activities ■ Antigenic Determinants on Immunoglobulins ■ The B-Cell Receptor ■ The Immunoglobulin Superfamily ■ Monoclonal Antibodies Antibodies: Structure and Function A   -  present on the B-cell membrane and secreted by plasma cells. Membrane-bound antibody con￾fers antigenic specificity on B cells; antigen-specific prolifer￾ation of B-cell clones is elicted by the interaction of membrane antibody with antigen. Secreted antibodies cir￾culate in the blood, where they serve as the effectors of hu￾moral immunity by searching out and neutralizing antigens or marking them for elimination. All antibodies share struc￾tural features, bind to antigen, and participate in a limited number of effector functions. The antibodies produced in response to a particular anti￾gen are heterogeneous. Most antigens are complex and con￾tain many different antigenic determinants, and the immune system usually responds by producing antibodies to several epitopes on the antigen. This response requires the recruit￾ment of several clones of B cells. Their outputs are mono￾clonal antibodies, each of which specifically binds a single antigenic determinant. Together, these monoclonal antibod￾ies make up the polyclonal and heterogeneous serum anti￾body response to an immunizing antigen. Basic Structure of Antibodies Blood can be separated in a centrifuge into a fluid and a cel￾lular fraction. The fluid fraction is the plasma and the cellu￾lar fraction contains red blood cells, leukocytes, and platelets. Plasma contains all of the soluble small molecules and macromolecules of blood, including fibrin and other proteins required for the formation of blood clots. If the blood or plasma is allowed to clot, the fluid phase that re￾mains is called serum. It has been known since the turn of the century that antibodies reside in the serum. The first evidence that antibodies were contained in particular serum protein fractions came from a classic experiment by A. Tiselius and E. A. Kabat, in 1939. They immunized rabbits with the protein ovalbumin (the albumin of egg whites) and then divided the immunized rabbits’ serum into two aliquots. Electrophoresis of one serum aliquot revealed four peaks corresponding to albumin and the alpha (), beta (), and gamma () globulins. The other serum aliquot was re￾acted with ovalbumin, and the precipitate that formed was removed; the remaining serum proteins, which did not react with the antigen, were then electrophoresed. A comparison of the electrophoretic profiles of these two serum aliquots revealed that there was a significant drop in the -globulin IgM, the First Responder 8536d_ch04_076 9/9/02 12:03 PM Page 76 mac76 mac76:385_reb:

8536d_ch04_076-1049/6/02 9: 02 PM Page 77 macas Mac 85: 365_smm pldsby et al./ Immunology Se Antibodies: Structure and Function chapter 4 CHon the heavy chain Antibodies are glycoproteins; with few Albumin exceptions, the sites of attachment for carbohydrates are re- stricted to the constant region. We do not completely under- stand the role played by glycosylation of antibodies, but it probably increases the solubility of the molecules. Inappro priate glycosylation, or its absence, affects the rate at which antibodies are cleared from the serum and decreases the effi- ciency of interaction between antibody and the complement system and between antibodies and Fc receptors Chemical and Enzymatic Methods Revealed Basic Antibody Structure Our knowledge of basic antibody structure was derived from a variety of experimental observations. When the Y-globulin Migration distance fraction of serum is separated into high-and low-molecular- weight fractions, antibodies of around 150,000-MW, des- IGURE 4-1 Experimental demonstration that most antibodies are ignated as immunoglobulin G(IgG)are found in the low- in the y-globulin fraction of serum proteins. After rabbits were im- molecular-weight fraction In a key experiment, brief diges munized with ovalbumin(OVA), their antisera were pooled and elec- tion of IgG with the enzyme papain produced three frag trophoresed, which separated the serum proteins according to their ments, two of which were identical fragments and a third that electric charge and mass. The blue line shows the electrophoretic was quite different(Figure 4-3). The two identical fragments pattern of untreated antiserum. The black line shows the pattern of antiserum that was incubated with ova to remove anti-ova anti body and then electrophoresed (Adapted from A. Tiselius and E.A Heavy chain Light chain Kabat, 1939, ). Exp. Med. 69: 119, with copyright permission of the u,ya,8,or Rockefeller University Press. polypeptides of molecular weight 50,000 or more. Like the tibody molecules they constitute, H and l chains are also called immunoglobulins. Each light chain is bound to a heavy chain by a disulfide bond, and by such noncovalent in teractions as salt linkages, hydrogen bonds, and hydrophobic bonds, to form a heterodimer(h-L). Similar noncovalent in- teractions and disulfide bridges link the two identical heavy and light(H-L)chain combinations to each other to form the Biological CHO auv basic four-chain(H-L)2 antibody structure, a dimer of dimers. As we shall see, the exact number and precise posi tions of these interchain disulfide bonds differs among anti- body classes and subclasses The first 110 or so amino acids of the amino-terminal re- COO- CO0- on of a light or heavy chain varies greatly among antibodies of different specificity. These segments of highly variable se- FIGURE 4-2 Schematic diagram of structure of immunoglobulin quence are called V regions: VL in light chains and VH in heavy. derived from amino acid sequencing studies. Each heavy and light All of the differences in specificity displayed by different anti- chain in an immunoglobulin molecule contains an amino-terminal bodies can be traced to differences in the amino acid se- variable() region(aqua and tan, respectively) that consists of 100- quences of V regions. In fact, most of the differences among 110 amino acids and differs from one antibody to the next. The re- antibodies fall within areas of the V regions called comple- mainder of each chain in the molecule-the constant (C) regions mentarity-determining regions( CDRs), and it is these CDRs,(purple and red)-exhibits limited variation that defines the two on both light and heavy chains, that constitute the antigen- light-chain subtypes and the five heavy-chain subclasses. Some binding site of the antibody molecule. By contrast, within the heavy chains(Y, 8, and a) also contain a proline-rich hinge region same antibody class, far fewer differences are seen when one (black). The amino-terminal portions, corresponding to the V re- compares sequences throughout the rest of the molecule. The gions, bind to antigen; effector functions are mediated by the other regions of relatively constant sequence beyond the variable re- domains. The u and e heavy chains, which lack a hinge region, con- gions have been dubbed C regions, CL on the light chain and tain an additional domain in the middle of the molecule Gotowww.whfreeman.com/immunology

polypeptides of molecular weight 50,000 or more. Like the antibody molecules they constitute, H and L chains are also called immunoglobulins. Each light chain is bound to a heavy chain by a disulfide bond, and by such noncovalent in￾teractions as salt linkages, hydrogen bonds, and hydrophobic bonds, to form a heterodimer (H-L). Similar noncovalent in￾teractions and disulfide bridges link the two identical heavy and light (H-L) chain combinations to each other to form the basic four-chain (H-L)2 antibody structure, a dimer of dimers. As we shall see, the exact number and precise posi￾tions of these interchain disulfide bonds differs among anti￾body classes and subclasses. The first 110 or so amino acids of the amino-terminal re￾gion of a light or heavy chain varies greatly among antibodies of different specificity. These segments of highly variable se￾quence are called V regions:VL in light chains and VH in heavy. All of the differences in specificity displayed by different anti￾bodies can be traced to differences in the amino acid se￾quences of V regions. In fact, most of the differences among antibodies fall within areas of the V regions called comple￾mentarity-determining regions (CDRs), and it is these CDRs, on both light and heavy chains, that constitute the antigen￾binding site of the antibody molecule. By contrast, within the same antibody class, far fewer differences are seen when one compares sequences throughout the rest of the molecule. The regions of relatively constant sequence beyond the variable re￾gions have been dubbed C regions, CL on the light chain and CH on the heavy chain. Antibodies are glycoproteins; with few exceptions, the sites of attachment for carbohydrates are re￾stricted to the constant region. We do not completely under￾stand the role played by glycosylation of antibodies, but it probably increases the solubility of the molecules. Inappro￾priate glycosylation, or its absence, affects the rate at which antibodies are cleared from the serum, and decreases the effi￾ciency of interaction between antibody and the complement system and between antibodies and Fc receptors. Chemical and Enzymatic Methods Revealed Basic Antibody Structure Our knowledge of basic antibody structure was derived from a variety of experimental observations. When the -globulin fraction of serum is separated into high- and low-molecular￾weight fractions, antibodies of around 150,000-MW, des￾ignated as immunoglobulin G (IgG) are found in the low￾molecular-weight fraction. In a key experiment, brief diges￾tion of IgG with the enzyme papain produced three frag￾ments, two of which were identical fragments and a third that was quite different (Figure 4-3). The two identical fragments Antibodies: Structure and Function CHAPTER 4 77 α β γ Globulins Albumin Absorbance Migration distance + − FIGURE 4-1 Experimental demonstration that most antibodies are in the -globulin fraction of serum proteins. After rabbits were im￾munized with ovalbumin (OVA), their antisera were pooled and elec￾trophoresed, which separated the serum proteins according to their electric charge and mass. The blue line shows the electrophoretic pattern of untreated antiserum. The black line shows the pattern of antiserum that was incubated with OVA to remove anti-OVA anti￾body and then electrophoresed. [Adapted from A. Tiselius and E. A. Kabat, 1939, J. Exp. Med. 69:119, with copyright permission of the Rockefeller University Press.] S S S S S S S S S S S S CHO CHO COO– Light chain κ or λ CH2 CH3 S S S S VH CH1 S S S S VL CL S S S S CH1 VH S S S S CL VL S S S S Heavy chain µ,γ,α,δ, or  Hinge NH3 + NH3 + NH NH 3 + 3 + COO– COO COO – – Biological activity Antigen binding CH2 CH3 446 214 FIGURE 4-2 Schematic diagram of structure of immunoglobulins derived from amino acid sequencing studies. Each heavy and light chain in an immunoglobulin molecule contains an amino-terminal variable (V) region (aqua and tan, respectively) that consists of 100– 110 amino acids and differs from one antibody to the next. The re￾mainder of each chain in the molecule—the constant (C) regions (purple and red)—exhibits limited variation that defines the two light-chain subtypes and the five heavy-chain subclasses. Some heavy chains (, , and ) also contain a proline-rich hinge region (black). The amino-terminal portions, corresponding to the V re￾gions, bind to antigen; effector functions are mediated by the other domains. The  and  heavy chains, which lack a hinge region, con￾tain an additional domain in the middle of the molecule. Go to www.whfreeman.com/immunology Animation Immunoglobulins 8536d_ch04_076-104 9/6/02 9:02 PM Page 77 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:

8536d_ch04076-104 9/6/02 9: 02 PM Page 78 mac85 Mac 85: 365 smm polsby et al./Immunology 5e: 78 PART I Generation of B-Cell and T-Cell Response Disulfide L chain bonds Fc fragmen Mercaptoethanol reduction HS HS SH Chains H chains FIGURE4-3 Prototype structure of IgG, showing chain structure treatments are also indicated. Light() chains are in gray and heavy and interchain disulfide bonds. The fragments produced by various(H) chains in blue (each with a MW of 45,000), had antigen-binding activity This question was answered by using antisera from goats that and were called Fab fragments ("fragment, antigen bind- had been immunized with either the Fab fragments or the Fc ing"). The other fragment(MW of 50,000)had no antigen- fragments of rabbit IgG. The antibody to the Fab fragment binding activity at all. Because it was found to crystallize could react with both the H and the L chains, whereas anti- during cold storage, it was called the Fc fragment("frag- body to the Fc fragment reacted only with the H chain. These ment,crystallizable). Digestion with pepsin, a different pro- observations led to the conclusion that the Fab fragment teolytic enzyme, also demonstrated that the antigen-binding consists of portions of a heavy and a light chain and that Fc properties of an antibody can be separated from the rest of contains only heavy-chain components. From these results the molecule. Pepsin digestion generated a single 100,000- and those mentioned above, the structure of IgG shown in MW fragment composed of two Fab-like fragments desig- Figure 4-3 was deduced. According to this model, the IgG nated the F(ab)2 fragment, which binds antigen. The Fc molecule consists of two identical H chains and two identical fragment was not recovered from pepsin digestion because it L chains, which are linked by disulfide bridges. The enzyme had been digested into multiple fragments papain cleaves just above the interchain disulfide bonds link A key observation in deducing the multichain structure of ing the heavy chains, whereas the enzyme pepsin cleaves just Igg was made when the molecule was subjected to mercap- below these bonds, so that the two proteolytic enzymes gen toethanol reduction and alkylation, a chemical treatment erate different digestion products. Mercaptoethanol reduc- that irreversibly cleaves disulfide bonds. If the sample is chro- tion and alkylation allow separation of the individual heav matographed on a column that separates molecules by size and light chains ng cleavage of disulfide bonds, it is clear that the intact 150,000-MW IgG molecule is, in fact, composed of subunits Each lgg molecule contains two 50,00o-dw polypeptide Obstacles to Antibody Sequencing chains, designated as light(L) chains(see Figure 4-3) Initial attempts to determine the amino acid sequence of the Antibodies themselves were used to determine how the heavy and light chains of antibody were hindered because in- enzyme digestion products--Fab, F(ab)2, and Fc-were re- sufficient amounts of homogeneous protein were available lated to the heavy-chain and light-chain reduction products. Although the basic structure and chemical properties of differ

(each with a MW of 45,000), had antigen-binding activity and were called Fab fragments (“fragment, antigen bind￾ing”). The other fragment (MW of 50,000) had no antigen￾binding activity at all. Because it was found to crystallize during cold storage, it was called the Fc fragment (“frag￾ment, crystallizable”). Digestion with pepsin, a different pro￾teolytic enzyme, also demonstrated that the antigen-binding properties of an antibody can be separated from the rest of the molecule. Pepsin digestion generated a single 100,000- MW fragment composed of two Fab-like fragments desig￾nated the F(ab)2 fragment, which binds antigen. The Fc fragment was not recovered from pepsin digestion because it had been digested into multiple fragments. A key observation in deducing the multichain structure of IgG was made when the molecule was subjected to mercap￾toethanol reduction and alkylation, a chemical treatment that irreversibly cleaves disulfide bonds. If the sample is chro￾matographed on a column that separates molecules by size following cleavage of disulfide bonds, it is clear that the intact 150,000-MW IgG molecule is, in fact, composed of subunits. Each IgG molecule contains two 50,000-MW polypeptide chains, designated as heavy (H) chains, and two 25,000-MW chains, designated as light (L) chains (see Figure 4-3). Antibodies themselves were used to determine how the enzyme digestion products—Fab, F(ab)2, and Fc—were re￾lated to the heavy-chain and light-chain reduction products. This question was answered by using antisera from goats that had been immunized with either the Fab fragments or the Fc fragments of rabbit IgG. The antibody to the Fab fragment could react with both the H and the L chains, whereas anti￾body to the Fc fragment reacted only with the H chain. These observations led to the conclusion that the Fab fragment consists of portions of a heavy and a light chain and that Fc contains only heavy-chain components. From these results, and those mentioned above, the structure of IgG shown in Figure 4-3 was deduced. According to this model, the IgG molecule consists of two identical H chains and two identical L chains, which are linked by disulfide bridges. The enzyme papain cleaves just above the interchain disulfide bonds link￾ing the heavy chains, whereas the enzyme pepsin cleaves just below these bonds, so that the two proteolytic enzymes gen￾erate different digestion products. Mercaptoethanol reduc￾tion and alkylation allow separation of the individual heavy and light chains. Obstacles to Antibody Sequencing Initial attempts to determine the amino acid sequence of the heavy and light chains of antibody were hindered because in￾sufficient amounts of homogeneous protein were available. Although the basic structure and chemical properties of differ- 78 PART II Generation of B-Cell and T-Cell Responses S Disulfide bonds L chain L chains HS SH Pepsin digestion F(ab')2 + + Fc fragments Fc Fab Fab Mercaptoethanol reduction H chain H chains S S S S S S S S S S S S S S S S S S S SH SH S S S S Papain digestion HS SH + SH SH + + FIGURE 4-3 Prototype structure of IgG, showing chain structure and interchain disulfide bonds. The fragments produced by various treatments are also indicated. Light (L) chains are in gray and heavy (H) chains in blue. 8536d_ch04_076-104 9/6/02 9:02 PM Page 78 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:

8536d_ch04_076-1049/6/02 9: 02 PM Page 79 macas Mac 85: 365_smm pldsby et al./ Immunology Se Antibodies: Structure and Function chapter 4 ent antibodies are similar, their antigen-binding specificities, was called the variable(v) region. The carboxyl-terminal and therefore their exact amino acid sequences, are very differ- half of the molecule, called the constant(C)region, had two ent. The population of antibodies in the serum Y-globulin basic amino acid sequences. This led to the recognition that fraction consists of a heterogeneous spectrum of antibodies. there were two light chain types, kappa(k)and lambda(A) Even if immunization is done with a hapten-carrier conjugate, In humans, 60% of the light chains are kappa and 40%are have different binding affinities. This heterogeneity of serum tains only one light chain type, either K or A, never both n the antibodies formed just to the hapten alone are heteroge- lambda, whereas in mice, 95% of the light chains are kappa neous: they recognize different epitopes of the hapten and and only 5% are lambda. A single antibody molecule antibodies made them unsuitable for sequencing studies. The amino acid sequences of x light chains show minor dif- ferences that are used to classify x light chains into subtypes In Pure Immunoglobulin Obtained from mice, there are three subtypes (Al, A2, and A3); in humans, Multiple Myeloma Patients Made there are four subtypes. Amino acid substitutions at only a few Sequencing Possible positions are responsible for the subtype differences. Sequencing analysis finally became feasible with the discov- Heavy-Chain Sequencing Revealed Five Basic ery of multiple myeloma, a cancer of antibody-producing Varieties of Heavy Chains plasma cells. The plasma cells in a normal individual are end stage cells that secrete a single molecular species of antibody For heavy-chain sequencing studies, myeloma proteins were for a limited period of time and then die. In contrast, a clone reduced with mercaptoethanol and alkylated, and the heavy of plasma cells in an individual with multiple myeloma has chains were separated by gel filtration in a denaturing sol escaped normal controls on their life span and proliferation vent. When the amino acid sequences of several myeloma and are not end-stage cells; rather, they divide over and over protein heavy chains were compared, a pattern similar to that in an unregulated way without requiring any activation by of the light chains emerged. The amino-terminal part of the ntigen to induce proliferation. Although such a cancerous chain, consisting of 100-110 amino acids, showed great se- plasma cell, called a myeloma cell, has been transformed, its quence variation among myeloma heavy chains and was protein-synthesizing machinery and secretory functions are therefore called the variable(V)region. The remaining part not altered; thus, the cell continues to secrete molecularly ho- of the protein revealed five basic sequence patterns, corre- mogeneous antibody. This antibody is indistinguishable sponding to five different heavy-chain constant (C)regions from normal antibody molecules but is called myeloma pro- (u, 8,Y,e and a). Each of these five different heavy chains is tein to denote its source. In a patient afflicted with multiple called an isotype. The length of the constant regions is ap- myeloma, myeloma protein can account for 95% of the proximately 330 amino acids for 8, y, and a, and 440 amino serum immunoglobulins. In most patients, the myeloma acids for H and E. The heavy chains of a given antibody mol cells also secrete excessive amounts of light chains. These ex- ecule determine the class of that antibody: IgM(u), IgG(Y) cess light chains were first discovered in the urine of IgA(a), IgD(8), or IgE(e). Each class can have either K or A myeloma patients and were named Bence-Jones proteins, light chains. A single antibody molecule has two identical for their discoverer heavy chains and two identical light chains, H2L2, or a multi Multiple myeloma also occurs in other animals. In mice it ple(H2L2) of this basic four-chain structure(Table 4 1) can arise spontaneously, as it does in humans, or conditions fa- Minor differences in the amino acid sequences of the a boring myeloma induction can be created by injecting mineral and y heavy chains led to further classification of the heavy oil into the peritoneal cavity. The clones of malignant plasma chains into subisotypes that determine the subclass of anti- cells that develop are called plasmacytomas, and many of these body molecules they constitute. In humans, there are two are designated MOPCs, denoting the mineral-oil induction of subisotypes of a heavy chains-al and a2-(and thus two plasmacytoma cells. A large number of mouse MOPC lines se- subclasses, IgAl and IgA2)-and four subisotypes of y heavy creting different immunoglobulin classes are presently carried chains: y1, y2, y3, and y4(therefore four subclasses, IgGI by the American Type-Culture Collection, a nonprofit repose- IgG2, IgG3, and IgG4). In mice, there are four subisotypes, tory of cell lines commonly used in research Yl, y2a, y2b, and y3, and the corresponding subclasses Light-Chain Sequencing Revealed That Immunoglobulins Have Constant and Immunoglobulin Fine Structure Variable Regions The structure of the immunoglobulin molecule is det When the amino acid sequences of several Bence-Jones pro- mined by the primary, secondary, tertiary, and quaternary teins (light chains) from different individuals were com- organization of the protein. The primary structure, the ared,a striking pattern emerged. The amino-terminal half amino acid sequence, accounts for the variable and constant of the chain, consisting of 100-110 amino acids, was found regions of the heavy and light chains. The secondary struc- to vary among different Bence-Jones proteins. This region ture is formed by folding of the extended polypeptide chain Gotowww.whfreeman.com/immunology5 Molecular Visualization

ent antibodies are similar, their antigen-binding specificities, and therefore their exact amino acid sequences, are very differ￾ent. The population of antibodies in the serum -globulin fraction consists of a heterogeneous spectrum of antibodies. Even if immunization is done with a hapten-carrier conjugate, the antibodies formed just to the hapten alone are heteroge￾neous: they recognize different epitopes of the hapten and have different binding affinities. This heterogeneity of serum antibodies made them unsuitable for sequencing studies. Pure Immunoglobulin Obtained from Multiple Myeloma Patients Made Sequencing Possible Sequencing analysis finally became feasible with the discov￾ery of multiple myeloma, a cancer of antibody-producing plasma cells. The plasma cells in a normal individual are end￾stage cells that secrete a single molecular species of antibody for a limited period of time and then die. In contrast, a clone of plasma cells in an individual with multiple myeloma has escaped normal controls on their life span and proliferation and are not end-stage cells; rather, they divide over and over in an unregulated way without requiring any activation by antigen to induce proliferation. Although such a cancerous plasma cell, called a myeloma cell, has been transformed, its protein-synthesizing machinery and secretory functions are not altered; thus, the cell continues to secrete molecularly ho￾mogeneous antibody. This antibody is indistinguishable from normal antibody molecules but is called myeloma pro￾tein to denote its source. In a patient afflicted with multiple myeloma, myeloma protein can account for 95% of the serum immunoglobulins. In most patients, the myeloma cells also secrete excessive amounts of light chains. These ex￾cess light chains were first discovered in the urine of myeloma patients and were named Bence-Jones proteins, for their discoverer. Multiple myeloma also occurs in other animals. In mice it can arise spontaneously, as it does in humans, or conditions fa￾voring myeloma induction can be created by injecting mineral oil into the peritoneal cavity. The clones of malignant plasma cells that develop are called plasmacytomas, and many of these are designated MOPCs, denoting the mineral-oil induction of plasmacytoma cells. A large number of mouse MOPC lines se￾creting different immunoglobulin classes are presently carried by the American Type-Culture Collection, a nonprofit reposi￾tory of cell lines commonly used in research. Light-Chain Sequencing Revealed That Immunoglobulins Have Constant and Variable Regions When the amino acid sequences of several Bence-Jones pro￾teins (light chains) from different individuals were com￾pared, a striking pattern emerged. The amino-terminal half of the chain, consisting of 100–110 amino acids, was found to vary among different Bence-Jones proteins. This region was called the variable (V) region. The carboxyl-terminal half of the molecule, called the constant (C) region, had two basic amino acid sequences. This led to the recognition that there were two light chain types, kappa () and lambda (). In humans, 60% of the light chains are kappa and 40% are lambda, whereas in mice, 95% of the light chains are kappa and only 5% are lambda. A single antibody molecule con￾tains only one light chain type, either or , never both. The amino acid sequences of light chains show minor dif￾ferences that are used to classify light chains into subtypes. In mice, there are three subtypes ( 1, 2, and 3); in humans, there are four subtypes. Amino acid substitutions at only a few positions are responsible for the subtype differences. Heavy-Chain Sequencing Revealed Five Basic Varieties of Heavy Chains For heavy-chain sequencing studies, myeloma proteins were reduced with mercaptoethanol and alkylated, and the heavy chains were separated by gel filtration in a denaturing sol￾vent. When the amino acid sequences of several myeloma protein heavy chains were compared, a pattern similar to that of the light chains emerged. The amino-terminal part of the chain, consisting of 100–110 amino acids, showed great se￾quence variation among myeloma heavy chains and was therefore called the variable (V) region. The remaining part of the protein revealed five basic sequence patterns, corre￾sponding to five different heavy-chain constant (C) regions (, , ,  and ). Each of these five different heavy chains is called an isotype. The length of the constant regions is ap￾proximately 330 amino acids for , , and , and 440 amino acids for  and . The heavy chains of a given antibody mol￾ecule determine the class of that antibody: IgM(), IgG(), IgA(), IgD(), or IgE(). Each class can have either or light chains. A single antibody molecule has two identical heavy chains and two identical light chains, H2L2, or a multi￾ple (H2L2)n of this basic four-chain structure (Table 4-1). Minor differences in the amino acid sequences of the and heavy chains led to further classification of the heavy chains into subisotypes that determine the subclass of anti￾body molecules they constitute. In humans, there are two subisotypes of heavy chains—1 and 2—(and thus two subclasses, IgA1 and IgA2)—and four subisotypes of heavy chains: 1, 2, 3, and 4 (therefore four subclasses, IgG1, IgG2, IgG3, and IgG4). In mice, there are four subisotypes, 1, 2a, 2b, and 3, and the corresponding subclasses. Immunoglobulin Fine Structure The structure of the immunoglobulin molecule is deter￾mined by the primary, secondary, tertiary, and quaternary organization of the protein. The primary structure, the amino acid sequence, accounts for the variable and constant regions of the heavy and light chains. The secondary struc￾ture is formed by folding of the extended polypeptide chain Antibodies: Structure and Function CHAPTER 4 79 Go to www.whfreeman.com/immunology Molecular Visualization An Introduction to Immunoglobulin Structure 8536d_ch04_076-104 9/6/02 9:02 PM Page 79 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:

8536d_ch04-076-1049/5/02 6: 19 AM Page 80 mac76mac76: 385 Goldapy et al/Immunology5e 80 PART I Generation of B-Cell and T-Cell Response TABLE 4.1 Chain composition of the five several homologous units of about 110 amino acid residues mmunoglobulin classes in humans Within each unit termed a domain an intrachain disulfide bond forms a loop of about 60 amino acids. Light chains con Heavy Light Molecular tain one variable domain(Vi), and one constant domain Class chain Subclasses chain formula (CL); heavy chains contain one variable domain(VH), and ei- ther three or four constant domains(CHl, CH2, CH3, and y1,y2.y3,y4Korλ CH4), depending on the antibody class( figure 4-6) X-ray crystallographic analysis revealed that im- munoglobulin domains are folded into a characteristic com- pact structure called the immunoglobulin fold. This structure consists of a"sandwich"of two B pleated sheets, K ora(a2k2 each containing antiparallel B strands of amino acids, which are connected by loops of various lengths( Figure 4-7). The B n=1, 2, 3, or 4 strands within a sheet are stabilized by hydrogen bonds that KorA∈2K2 connect the-NH groups in one strand with I groups of an adjacent strand(see Figure 4-4). The B strands are characterized by alternating hydrophobic and hydrophilic amino acids whose side chains are arranged perpendicular to the plane of the sheet; the hydrophobic amino acids are ori- ented toward the interior of the sandwich, and the hy drophilic amino acids face outward. back and forth upon itself into an antiparallel p pleated sheet The two B sheets within an immunoglobulin fold are sta- Figure 4-4). The chains are then folded into a tertiary struc- bilized by the hydrophobic interactions between them and by ture of compact globular domains, which are connected to the conserved disulfide bond. An analogy has been made to neighboring domains by continuations of the polypeptide two pieces of bread, the butter between them, and a tooth chain that lie outside the p pleated sheets. Finally, the glob- pick holding the slices together. The bread slices represent the lar domains of adjacent heavy and light polypeptide chains two B pleated sheets; the butter represents the hydrophobic interact in the quaternary structure(Figure 4-5), forming interactions between them; and the toothpick represents the functional domains that enable the molecule to specifically intrachain disulfide bond. Although variable and constant bind antigen and, at the same time, perform a number of bi- domains have a similar structure, there are subtle differences ological effector functions between them. The V domain is slightly longer than the C do main and contains an extra pair of B strands within the B- Immunoglobulins Possess Multiple Domains this pair of B strands (see Figure 4-7) The basic structure of the immunoglobulin fold con Careful analysis of the amino acid sequences of immunoglob- tributes to the quaternary structure of immunoglobulins ulin heavy and light chains showed that both chains contain by facilitating noncovalent interactions between domains N FIGURE Structural formula of a B pleated sheet containing two ular to the plane of the sheet. (Adapted from H. Lodish et al., 1995, antiparallel B strands. The structure is held together by hydrogen Molecular Cell Biology, 4th ed, Scientific American Books, New York, bonds between peptide bonds of neighboring stretches of polypep. reprinted by permission of W H Freeman and Company/ tide chains. The amino acid side groups(R)are arranged perpendic

back and forth upon itself into an antiparallel  pleated sheet (Figure 4-4). The chains are then folded into a tertiary struc￾ture of compact globular domains, which are connected to neighboring domains by continuations of the polypeptide chain that lie outside the  pleated sheets. Finally, the globu￾lar domains of adjacent heavy and light polypeptide chains interact in the quaternary structure (Figure 4-5), forming functional domains that enable the molecule to specifically bind antigen and, at the same time, perform a number of bi￾ological effector functions. Immunoglobulins Possess Multiple Domains Based on the Immunoglobulin Fold Careful analysis of the amino acid sequences of immunoglob￾ulin heavy and light chains showed that both chains contain several homologous units of about 110 amino acid residues. Within each unit, termed a domain, an intrachain disulfide bond forms a loop of about 60 amino acids. Light chains con￾tain one variable domain (VL), and one constant domain (CL); heavy chains contain one variable domain (VH), and ei￾ther three or four constant domains (CH1, CH2, CH3, and CH4), depending on the antibody class (Figure 4-6). X-ray crystallographic analysis revealed that im￾munoglobulin domains are folded into a characteristic com￾pact structure called the immunoglobulin fold. This structure consists of a “sandwich” of two  pleated sheets, each containing antiparallel  strands of amino acids, which are connected by loops of various lengths (Figure 4-7). The  strands within a sheet are stabilized by hydrogen bonds that connect the –NH groups in one strand with carbonyl groups of an adjacent strand (see Figure 4-4). The  strands are characterized by alternating hydrophobic and hydrophilic amino acids whose side chains are arranged perpendicular to the plane of the sheet; the hydrophobic amino acids are ori￾ented toward the interior of the sandwich, and the hy￾drophilic amino acids face outward. The two  sheets within an immunoglobulin fold are sta￾bilized by the hydrophobic interactions between them and by the conserved disulfide bond. An analogy has been made to two pieces of bread, the butter between them, and a tooth￾pick holding the slices together. The bread slices represent the two  pleated sheets; the butter represents the hydrophobic interactions between them; and the toothpick represents the intrachain disulfide bond. Although variable and constant domains have a similar structure, there are subtle differences between them. The V domain is slightly longer than the C do￾main and contains an extra pair of  strands within the - sheet structure, as well as the extra loop sequence connecting this pair of  strands (see Figure 4-7). The basic structure of the immunoglobulin fold con￾tributes to the quaternary structure of immunoglobulins by facilitating noncovalent interactions between domains 80 PART II Generation of B-Cell and T-Cell Responses TABLE 4-1 Chain composition of the five immunoglobulin classes in humans Heavy Light Molecular Class chain Subclasses chain formula IgG 1, 2, 3, 4 or 2 2 2 2 IgM  None or (2 2)n (2 2)n n  1 or 5 IgA 1, 2 or (2 2)n (2 2)n n  1, 2, 3, or 4 IgE  None or 2 2 2 2 IgD  None or 2 2 2 2 H H H O O O O N N N C C C C C C H H O N N C C O H N C O H N C O H N C C C H O N C C H O N C C C H O C N H O C N H O C N C C H O N C C C H O N C C C C C C C R R R R R R R R R R R R R R R R H O C N FIGURE 4-4 Structural formula of a  pleated sheet containing two antiparallel  strands. The structure is held together by hydrogen bonds between peptide bonds of neighboring stretches of polypep￾tide chains. The amino acid side groups (R) are arranged perpendic￾ular to the plane of the sheet. [Adapted from H. Lodish et al., 1995, Molecular Cell Biology, 4th ed., Scientific American Books, New York; reprinted by permission of W. H. Freeman and Company.] 8536d_ch04_076-104 9/5/02 6:19 AM Page 80 mac76 mac76:385 Goldsby et al./Immunology5e: