Antibody Structure and B-Cell Diversity (Human Immunology)

Antibody Structure and the Generation of B-Cell Diversity Based on Chapter 4 of the Parham textbook, 5th edition MMI 188 Human Immunology José V. Torres, PhD Professor and Vice Chair for Under...

Antibody Structure and the Generation of B-Cell Diversity Based on Chapter 4 of the Parham textbook, 5th edition MMI 188 Human Immunology José V. Torres, PhD Professor and Vice Chair for Undergraduate Education Medical Microbiology and Immunology School of Medicine University of California Davis, CA 95616 [email protected] 1 Antibody Structure and the Generation of B-Cell Diversity Learning Objectives for this Lecture: Ø Understand the functions of antibodies Ø Learn how different antibodies have different structures that are related to their function Ø Learn the major structural features of the B cell receptor and the antibody molecule Ø Learn the main factors involved in the generation of antibody diversity Ø Become familiar with the genetic basis of diversity Ø Learn how monoclonal antibodies are made and used 2 Function of antibodies: To bind to infectious agents Ø Different mechanisms: Ø Neutralization Ø Opsonization Ø Immunoglobulins = antigen-binding molecules made by B cells Ø Antibody = the secreted form of an immunoglobulin Ø B cell receptor = the membrane-bound immunoglobulin on the B cell surface Ø When B cells encounter antigen, they differentiate to plasma cells Ø Plasma cells secrete antibodies but do not have B cell receptors on their surface 3  Antibodies Ø produced by plasma cells Ø clear extracellular pathogens and their toxins Ø best source of protective immunity Ø antibody repertoire: 109 (1 billion) different specificities in a particular person Ø specificity of the secreted antibody is the same as that of the B cell receptor Figure 4.1 Plasma cells secrete antibody of the same antigen specificity as that of the antigen receptor of their B-cell precursor. A mature B cell expresses membrane- bound immunoglobulin (Ig) of a single antigen specificity. When a foreign antigen first binds to this immunoglobulin, the B cell is stimulated to proliferate. Its progeny differentiate into plasma cells that secrete antibody of the same specificity as that of the membrane-bound immunoglobulin. 4  Structure of the IgG Molecule Ø Two identical heavy chains (green) Ø Two identical light chains (yellow) Ø Repeating globular domains (individual rectangles) Ø N-terminal domains are Variable (red) Ø Variable region Ø C-terminal domains are Constant (blue) Ø Constant region Ø Connected by covalent and non-covalent bonds Ø Carbohydrates attached to heavy chains Ø Flexible hinge Ø Divalent: 2 antigen-binding sites Figure 4.2 The immunoglobulin G (IgG) molecule. As shown in the top panel, each IgG molecule consists of two identical heavy chains (green) and two identical light chains (yellow). Carbohydrate (turquoise) is attached to the heavy chains. The bottom panel shows the location of the variable (V) and constant (C) regions in the IgG molecule. The amino-terminal regions (red) of the heavy and light chains are variable in sequence from one IgG molecule to another; the remaining regions are constant in sequence (blue). The carbohydrate is omitted from this panel and from most subsequent figures for simplicity. In IgG, a flexible hinge region is located between the two arms and the stem of the Y. Covalent bonds form when two atoms share their electrons. There are four main types of noncovalent bonds in biological systems: hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds. 5  The Y-shaped immunoglobulin molecule can be dissected using a protease. Protease cleavage (papain digestion) and Reduction of disulfide bonds: – two Fab fragments (Fragment antigen binding) – one Fc subunit (Fragment crystallizable or constant) Figure 4.3 The Y-shaped immunoglobulin molecule can be dissected using a protease. By using a protease to cleave the hinge of each heavy chain (as shown by the scissors) and a reducing agent to break the disulfide bonds that connect the two hinges, the IgG molecule is dissected into three pieces: two Fab fragments and one Fc fragment. 6  IgG is very flexible! Ø The flexible hinge of the IgG molecule allows it to bind with both arms to many different arrangements of antigens on pathogens' surfaces. Ø Three IgG molecules are shown binding with both arms to antigens located at different distances apart on the surface of a bacterium. Figure 4.4 The flexible hinge of the IgG molecule allows it to bind with both arms to many different arrangements of antigens on the surfaces of pathogens. Three IgG molecules are shown binding with both arms to antigens that are located at different distances apart on the surface of a bacterium. 7 Differences in Human Immunoglobulin Classes (Isotypes) Secreted Forms: IgG monomer Ø heavy chain C region length IgM pentamer IgD monomer Ø location of disulfide bonds IgA monomer and dimer Ø carbohydrate groups IgE monomer Ø hinge region in IgG, IgD and IgA Two kinds of Light chains: kappa κ Ø no hinge region in IgM and IgE lambda λ Ø IgM and IgE are longer than all other monomeric forms (4 C domains in heavy chain) Figure 4.5 The structures of the human immunoglobulin classes. In particular, note the differences in length of the heavy-chain C regions, the locations of the disulfide bonds linking the chains, and the presence of a hinge region in IgG, IgA, and IgD, but not in IgM and IgE. The heavy-chain isotype in each antibody is indicated by the Greek letter. The isotypes also differ in the distribution of N-linked carbohydrate groups (turquoise). All these immunoglobulins occur as monomers in their membrane-bound form. In their soluble, secreted form, IgD, IgE, and IgG are monomers. IgA forms monomers and dimers, and IgM forms pentamers. 8 The Antibodies Electron micrograph of pentameric IgM molecules, the first antibody made in an immune response. 9 Major Classes (Isotypes) of Antibodies ØIgM ØPentamer ØThe first antibody made after infection ØBinds complement proteins ØCannot bind Fc receptors on phagocytes (no opsonization) ØIgG ØAfter acute infection, IgG becomes the dominant antibody in circulation ØNeutralizes toxins ØOpsonizes pathogens ØBinds complement proteins ØBinds Fc receptors on phagocytes (after opsonization) ØIgA ØProtects mucosal surfaces as a dimer ØIgE ØMediates mast cell and eosinophil degranulation (allergy, parasites) ØIgD ØCo-expressed with IgM on the surface of naïve B cells (B cell receptor) 10  Protein Domains in Immunoglobulins For IgG: Ø 3 globular portions of similar size Ø each portion has 4 immunoglobulin domains Ø One VL and one CL domain in each light chain Ø One VH and three CH domains in each heavy chain Figure 4.6 IgG is built from 12 similarly shaped immunoglobulin domains. The diagram in panel a is based on three-dimensional crystallographic structures. The polypeptide backbones of the heavy chains (one chain is yellow and the other purple) and the light chains (both red) are shown as ribbons. Each Fab and Fc fragment is made up of four immunoglobulin domains that produce a similarly shaped globular structure. Panel b shows how the different types of domain are organized and connected within the three-dimensional structure. 11  3D structure of Immunoglobulin Constant and Variable domains Ø β strands of β sheet Ø antiparallel sheets connected by loops Figure 4.7 The three-dimensional structure of immunoglobulin C and V domains. The structure of one light chain with its constituent C domain (left panel) and V domain (right panel) is shown here. The inset shows the location of the light chain in a Fab of IgG. The folding of the polypeptide is depicted as a ribbon diagram in which the thick colored arrows correspond to the parts of the chain that form the β strands of the β sheet. The arrows point from the amino terminus to the carboxy terminus. Adjacent strands in each β sheet run in opposite directions (antiparallel), as shown by the arrows, and are connected by loops. The C domain has four β strands in the upper β sheet (yellow) and three in the lower (green). The V domain has five strands in the upper β sheet (blue) and four in the lower (red). 12  Hypervariable Regions of Variable Domains Hypervariable loops determine antigenic specificity of antigen binding site. Variable domains of heavy and light chains (VH and VL) contain three hypervariable loops that determine antigen specificity. Ø Hypervariable loops (HV1, HV2, HV3) are also called complementarity-determining regions (CDR1, CDR2, CDR3) Ø Regions flanking hypervariable loops are called framework regions (FR1-4) Figure 4.8 The hypervariable regions of antibody V domains lie in discrete loops at one end of the domain structure. Top panel: the variability plot for the 110 positions within the amino acid sequence of a light-chain V domain. It is obtained from comparison of many light-chain sequences. Variability is the ratio of the number of different amino acids found at a position to the frequency of the most common amino acid at that position. The maximum value possible for the variability is 400, the square of 20, the number of different amino acids found in antibodies. The minimum value is 1. Three hypervariable regions (HV1, HV2, and HV3) can be discerned (red) flanked by four framework regions (FR1, FR2, FR3, and FR4) (yellow). Center panel: the correspondence of the hypervariable regions to three loops at the end of the V domain farthest from the C region. The location of hypervariable regions in the heavy-chain V domain is similar (not shown). The hypervariable loops contribute much of the antigen specificity of the antigen-binding site. One antigen-binding site is located at the tip of each arm of the antibody molecule. Hypervariable regions are also known as complementarity-determining regions: CDR1, CDR2, and CDR3. Bottom panel: the location of the light-chain V region in the Fab part of the IgG molecule. 13  Binding to Multivalent Antigens Two kinds of multivalent antigen A pathogen can be recognized at: Ø multiple epitopes by different antibodies and/or Ø repeated epitopes by the same antibody Figure 4.9 Two kinds of multivalent antigen. Many soluble protein antigens have several different epitopes, but each is represented only once on the surface of the protein. This situation is depicted in the upper panel, where four IgG molecules with different specificities all bind to the protein antigen using a single Fab arm. On pathogen surfaces there are numerous copies of the same epitope. This situation, depicted in the lower panel, allows many IgG molecules with identical antigenic specificity to bind to the multivalent antigen with both Fab arms. 14  Antibodies bind epitopes of different shapes Ø Hypervariable loops of antibody V domains vary in shape to fit different epitopes Ø lock and key mechanism Figure 4.10 Epitopes can bind to pockets, grooves, extended surfaces, or knobs in antigen-binding sites. Shown are schematic representations of antibodies (blue) binding to four different types of epitope (red). First panel: a small compact epitope binding to a pocket in the antigen-binding site. Second panel: an epitope consisting of a relatively unfolded part of a polypeptide chain binding to a shallow groove. Third panel: an epitope with an extended surface binding to a similarly sized but complementary surface in the antibody. Fourth panel: the epitope is a pocket in the antigen into which the antigen-binding site of the antibody intrudes. Only one Fab arm of each antibody is shown. 15  Linear and discontinuous (conformational) epitopes A discontinuous epitope is formed from amino acids from different parts of the polypeptide chain that are brought together when the chain folds. Figure 4.11 Linear and discontinuous epitopes. A linear epitope of a protein antigen is formed from contiguous amino acids. A discontinuous epitope is formed from amino acids from different parts of the polypeptide that are brought together when the chain folds. 16 The Production of Monoclonal Antibodies Monoclonal antibodies are produced from one single clone of a transformed plasma cell A hybridoma results from the fusion of a single antibody-producing cell and a myeloma cell (cancer cell line) Hybridoma cell clones are selected for antigen specificity and can be cultured indefinitely Humanized monoclonal antibodies can be produced by genetic engineering techniques Monoclonal antibodies produced by a hybridoma come from the same cell clone, so they are identical copies of antibodies specific for the same epitope ØIn contrast, polyclonal antisera produced during an infection contains many specificities and isotypes Figure 4.12 Production of a mouse monoclonal antibody. Lymphocytes from a mouse immunized with the antigen of choice are fused with myeloma cells using polyethylene glycol. The fused cells are then grown in the presence of a drug that kills the myeloma cells but permits the growth of hybrid cells. Unfused lymphocytes also die. Cultures of hybrid cells (hybridomas) are tested to determine whether they make the desired antibody. Cultures that contain a hybridoma making the desired antibody are then cloned to produce a homogeneous culture of cells making the monoclonal antibody. Myelomas are tumors of plasma cells; those used to make hybridomas were selected not to express antibody heavy and light chains. Thus, hybridomas only express the antibody made by the B-cell partner. 17  Use of Monoclonal Antibodies in Research and Clinical Settings Ø Flow Cytometry is used to determine the number of each different cell type in a mixture Ø 18 cell-surface markers can be analyzed simultaneously! Ø Fluorescence-Activated Cell Sorting (FACS) is used to separate different types of cells Number of Receptors: 100K BCR/B cell 30K TCR/T cell Figure 4.13 Flow cytometry enables cells to be distinguished by their cell-surface molecules. The left-hand panels illustrate the principles of flow cytometry. Human cells are labeled with mouse monoclonal antibodies specific for human cell-surface proteins, antibodies of different specificity being tagged with fluorescent dyes of different colors. The labeled cells pass through a nozzle, forming a stream of droplets each containing one cell. The stream of cells passes through a laser beam, which causes the fluorescent dyes to emit light of different wavelengths. The emitted signals are analyzed by a detector: cells with particular characteristics are counted, and the abundance of each type of labeled cell-surface molecule is measured. The right-hand panels show how the data obtained can be represented, as exemplified by the presence of IgM and T-cell receptor (TCR) on lymphocytes from the human spleen. The expression of IgM and TCR distinguishes between B cells and T cells, respectively. When the presence of one type of molecule is analyzed, the data are usually displayed as a histogram, as shown in the upper right panel for TCR. Two populations of cells are distinguished in the histogram. The larger peak to the left consists of lymphocytes (mostly B cells) that do not bind the anti-TCR monoclonal antibody; it comprises 58% of the total cell number. The smaller peak to the right corresponds to the T cells that bind the anti-TCR antibody and comprises 32% of the total. Two-dimensional plots (lower right panel) are used to compare the expression 18 of two cell-surface molecules. The horizontal axis represents the amount of fluorescent anti-TCR antibody bound by a cell; the vertical axis represents the amount of fluorescent anti-IgM antibody bound. Each dot represents the values obtained for a single cell. Many thousands of cells are usually analyzed, and the dots can blend into each other in those parts of the plot that are heavily populated. The dot plot is divided into four quadrants that roughly correspond to the four cell populations distinguished by analysis with two antibodies. In the top left quadrant are B cells; these bind anti-IgM antibody but not anti-TCR antibody and comprise 60% of the cells. In the bottom right quadrant are the T cells; these bind anti-TCR antibody but not anti-IgM antibody and comprise 31% of the cells. In the bottom left quadrant are cells that bind neither anti-IgM nor anti-TCR antibody; these comprise 8% of the cells and probably include natural killer (NK) cells and some contaminating non-lymphoid leukocytes. Theoretically, cells in the upper right quadrant would correspond to lymphocytes that bind both anti-IgM and anti-TCR. As no lymphocytes express both IgM and TCR at their surface, these double reactions (1% of the total) arise from imprecision in using quadrants to separate the cell population and from experimental artifact, for example the nonspecific sticking of antibody molecules to cells that do not express their specific antigen. Flow cytometry is used both to analyze and to purify cell populations. Although this example shows the simultaneous analysis of two cell-surface molecules, the technology has advanced to where up to 18 cell- surface markers can be analyzed simultaneously. 18  Monoclonal antibodies as treatments for disease. Some examples: Figure 4.14 Monoclonal antibodies as treatments for disease. Mouse antibodies were the first monoclonal antibodies used therapeutically. Their drawback was that patients responded to the foreign mouse protein by making antibodies against it, which reduced the clinical effect of the mouse antibodies. This problem was lessened by making chimeric antibodies, in which the mouse antibody (blue) constant regions are replaced with human (yellow) constant regions. Extending this approach are humanized antibodies, in which only the CDR loops are of mouse origin. Fully human monoclonal antibodies are made by immunizing mice in which the mouse immunoglobulin genes have been replaced by human immunoglobulin genes. All four types of antibody are being used therapeutically. CTLA-4, cytotoxic T- lymphocyte-associated protein 4; HER2, human epidermal growth factor receptor 2; IL, interleukin; PSMA, prostate-specific membrane antigen. 19 Over 100 monoclonal antibodies have been approved by the FDA for various medical purposes. This number includes mAbs used in treating conditions such as: 1.Cancer: Many mAbs target specific antigens on cancer cells to inhibit their growth or mark them for destruction by the immune system. 2.Autoimmune Diseases: Some mAbs modulate the immune response to treat conditions like rheumatoid arthritis or multiple sclerosis. 3.Infectious Diseases: Certain mAbs have been developed to treat viral infections, including COVID-19. 4.Other Conditions: There are also mAbs approved for other indications, such as cardiovascular diseases and metabolic disorders. Detailed Breakdown of Approved Monoclonal Antibodies: 1.Oncology: Most approved mAbs fall into this category due to the high demand for targeted cancer therapies. Examples include trastuzumab (Herceptin) for breast cancer and rituximab (Rituxan) for non-Hodgkin lymphoma. 2.Immunology: This category includes drugs like adalimumab (Humira), used for various inflammatory conditions. 3.Infectious Disease Treatments: The emergence of COVID-19 led to the rapid development and approval of several mAbs aimed at neutralizing the virus. 4.Cardiovascular Applications: Some mAbs are designed to lower cholesterol levels or manage cardiovascular risks. 5.Rare Diseases: A smaller subset of mAbs has been approved for rare genetic disorders or conditions with limited treatment options. 20  What factors account for antibody diversity? (How can we recognize so many antigens?) During B-cell maturation, before the B-cell encounters its antigen: 1. Genetic diversity 2. Combinatorial diversity 3. Junctional diversity 1. Genetic Diversity Inherited germline organization of the immunoglobulin heavy chain and light chain loci (gene segments) Ø Germline sequence (inherited from egg and sperm) is different from mature B cell (somatic) 3 loci located in 3 different chromosomes Gene Segments: Ø Variable (V) Ø Joining (J) Ø Diversity (D) Ø Constant (C) Figure 4.15 The germline organization of the human immunoglobulin heavy-chain and light-chain loci. Top row: the λ light-chain locus, which has about 30 functional Vλ gene segments and 4 pairs of a functional Jλ and a Cλ gene segment. Center row: the κ locus is organized in a similar way, with about 35 functional Vκ gene segments accompanied by a cluster of 5 Jκ gene segments but with a single Cκ gene segment. In approximately 50% of the human population, the entire cluster of Vκ gene segments is duplicated (not shown, for simplicity). Bottom row: the heavy-chain locus has about 40 functional VH gene segments, a cluster of about 23 D segments, and 6 JH gene segments. For simplicity, a single CH gene (CH1–9) is shown in this diagram to represent the 9 C genes. The diagram is not to scale: the total length of the heavy- chain locus is more than 2 megabases (2 million bases), whereas some of the D segments are only 6 bases long. L, leader sequence. 21  2. Combinatorial Diversity Rearrangement Random selection of one gene segment of each type: Ø Variable (V) Ø Joining (J) Ø Diversity (D) (only for heavy chain) Figure 4.16 V-region sequences are constructed from gene segments. Left panels: light-chain V-region genes are constructed from two segments. A variable (V) gene segment and a joining (J) gene segment in the genomic DNA are joined to form a complete light-chain V-region (VL) exon. After rearrangement, the light-chain gene consists of three exons, which encode the leader (L) peptide, the V region, and the C region and are separated by introns. Right panels: heavy-chain V regions are constructed from three gene segments. First the diversity (D) and J gene segments join, then the V gene segment joins to the combined DJ sequence, forming a complete heavy-chain V-region (VH) exon. For simplicity, only the first of the heavy- chain genes, Cμ, is shown here. Each immunoglobulin domain is encoded by a separate exon, and two additional membrane-coding exons (MC; light blue) specify the hydrophobic sequence that will anchor the heavy chain to the B-cell membrane. 22  Immunoglobulin Diversity (# of gene segments/locus) Determine isotype Figure 4.17 The numbers of functional gene segments available to construct the variable and constant regions of human immunoglobulin heavy chains and light chains. 23 Successful recombination leads to allelic exclusion Heavy chain loci on both chromosomes begin rearrangement first: Ø Heavy chains have two chances to rearrange (two copies of chromosome 14). Ø The process stops as soon as one chromosome reaches a productive (in-frame) recombination. Ø From then on, only the successfully rearranged locus is used to make immunoglobulins by that particular B cell. Ø The other allele is “excluded”. Light chain loci rearrange after heavy chains: Ø Light chains have four chances to rearrange. Ø kappa chain (both copies of chromosome 2), followed by lambda chain (both copies of chromosome 22). Ø Once a successful rearrangement is found, everything stops. Ø Immunoglobulins use kappa chains more frequently than lambda chains (66% kappa and 33% lambda). 24 Mechanism of recombination ensures the correct orientation of gene segments RSS (Recombination Signal Sequences) between gene segments. Ø RSS determine where DNA is cut: Ø Two types: Ø 23 nucleotide (nt) spacer with 7 and 9 nucleotides before and after spacer Ø 12 nucleotide (nt) spacer with 9 and 7 nucleotides before and after spacer 7 nucleotide heptamer (orange) 9 nucleotide nonamer (purple) Figure 4.18 Each V, D, or J gene segment is flanked by recombination signal sequences (RSSs). There are two types of RSS. One consists of a nonamer (9 bp, shown in purple) and a heptamer (7 bp, shown in orange) separated by a spacer of 12 bp (white). The other consists of the same 9- and 7-bp sequences separated by a 23- bp spacer (white). 25 Rearrangement of V gene segments is required to make immunoglobulin genes functional RAG (Recombination Activating Genes) complex that binds intergenic sequences Ø One RAG-1 subunit binds to the nonamer and heptamer of the V gene segment. Ø Another RAG-1 subunit binds to the nonamer and heptamer of the J gene segment. Ø The cleavage site is between the heptamer and the gene segment. Ø V and J segments are ligated and a circular loop containing unused segments is excised Figure 4.19 Rearrangement of V gene segments is required to make immunoglobulin genes functional. The V(D)J recombinase is a Y-shaped structure that consists of two RAG-1 and two RAG-2 subunits. Each RAG-1 subunit has binding sites for the nonamer (N) and heptamer (H) nucleotide motifs that flank the V and J gene segments. In the V gene segment the nonamer and heptamer are separated by a 12-bp spacer, and in the J gene segment they are separated by a 23-bp spacer. One RAG-1 subunit binds to the nonamer and heptamer of the V gene segment, while the other binds to the nonamer and heptamer of the J gene segment. These interactions create a scaffold that brings together the V and J gene segments that will be cut and then spliced together. 26 3. Junctional Diversity: Random addition of nucleotides during joining The process is illustrated for a D to J rearrangement. Ø The RSSs are brought together, and the RAG complex cleaves (arrows) between the heptamer sequences and the gene segments (top panel). Ø This leads to the excision of the DNA that separates the D and J segments. The ends of the two strands of the DNA double helix in the D and J segments are joined to form structures known as hairpins. Ø Further cleavage (arrows) on one DNA strand of the D and J segments opens the hairpins and generates short single-stranded sequences at the ends of the D and J segments. Ø Terminal deoxynucleotidyl Transferase (TdT) adds nucleotides randomly to the ends of the single strands. These nucleotides are not encoded in the germline and are known as N nucleotides. Figure 4.20 (Part 1) The generation of junctional diversity during gene rearrangement. The process is illustrated for a D to J rearrangement. The RSSs are brought together and the RAG complex cleaves (arrows) between the heptamer sequences and the gene segments (top panel). This leads to excision of the DNA that separates the D and J segments. The ends of the two strands of the DNA double helix in the D and J segments are joined to form structures known as ‘hairpins.’ Further cleavage (arrows) on one DNA strand of the D and J segments opens the hairpins and generates short single-stranded sequences at the ends of the D and J segments. The extra nucleotides are known as P nucleotides because they make a palindromic sequence in the final double-stranded DNA (as indicated on the diagram). Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the ends of the single strands. These nucleotides, which are not encoded in the germline, are known as N nucleotides. The single strands pair, and through the action of exonuclease, DNA polymerase, and DNA ligase, the double-stranded DNA molecule is repaired to give the coding joint. 27  Junctional Diversity Continued: Ø The single strands pair, and through the action of exonuclease, DNA polymerase, and DNA ligase, the double-stranded DNA molecule is repaired to give the coding joint. Ø The extra nucleotides are known as P nucleotides because they make a palindromic sequence in the final double-stranded DNA. Ø P-nucleotides (palindrome) come from imprecise cleavage Ø The nucleotides added by Terminal deoxynucleotidyl Transferase (TdT) randomly to the ends of the single strands, which are not encoded in the germline, are known as N nucleotides. Ø N-nucleotides (non-germline) are randomly added by Terminal deoxynucleotidyl Transferase (TdT) Figure 4.20 (Part 2) The generation of junctional diversity during gene rearrangement. The process is illustrated for a D to J rearrangement. The RSSs are brought together and the RAG complex cleaves (arrows) between the heptamer sequences and the gene segments (top panel). This leads to excision of the DNA that separates the D and J segments. The ends of the two strands of the DNA double helix in the D and J segments are joined to form structures known as ‘hairpins.’ Further cleavage (arrows) on one DNA strand of the D and J segments opens the hairpins and generates short single-stranded sequences at the ends of the D and J segments. The extra nucleotides are known as P nucleotides because they make a palindromic sequence in the final double-stranded DNA (as indicated on the diagram). Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the ends of the single strands. These nucleotides, which are not encoded in the germline, are known as N nucleotides. The single strands pair, and through the action of exonuclease, DNA polymerase, and DNA ligase, the double-stranded DNA molecule is repaired to give the coding joint. 28  What is the final gene organization? ØThe VDJ region (left) provides the specificity for antigen recognition ØAll the different gene segments for constant (C) regions are still present ØThe B cell clone can continue to differentiate and switch to a different antibody isotype: from IgM to IgG, for example. ØDifferent antibody isotypes made by the same B cell clone will have the same antigen specificity since they have the same VDJ region. ØB cells are monospecific. Figure 4.21 Rearrangement of V, D, and J segments produces a functional heavy- chain gene. The assembled VDJ sequence lies some distance from the cluster of C genes. Only functional C genes are shown here. The four different γ genes specify four different subtypes of the γ heavy chain, whereas the two α genes specify two subtypes of the α heavy chain. For simplicity, individual exons in the C genes are not shown. The diagram is not to scale. 29  Genetics of Antibody Diversity Events after a B cell encounters antigen: Ø Switch to secreted immunoglobulin Ø Somatic hypermutation Ø Isotype switching Coexpression of IgM and IgD as B cell receptor is regulated by alternative RNA processing: Ø a different pattern of splicing Figure 4.22 Coexpression of IgD and IgM is regulated by RNA processing. In mature B cells, transcription initiated at the VH promoter extends through both the Cμ and Cδ genes. For simplicity we have not shown all the individual C-gene exons but only those relevant to the production of IgM and IgD. The long primary transcript is then processed by cleavage, polyadenylation, and splicing. Cleavage and polyadenylation at the μ site (pAμm; the ‘m’ denotes that this site produces membrane-bound IgM) and splicing between Cμ exons yields an mRNA encoding the μ heavy chain (left panel). Cleavage and polyadenylation at the δ site (pAδm) and a different pattern of splicing that removes the Cμ exons yields mRNA encoding the δ heavy chain (right panel). AAA designates the poly(A) tail. MC, exons that encode the transmembrane region of the heavy chain. 30  Membrane-bound immunoglobulins are associated with two other proteins, Igα and Igβ. Ø Igα and Igβ are disulfide-linked. Ø They have long cytoplasmic tails that can interact with intracellular signaling proteins. Ø The complex of immunoglobulin with Igα and Igβ serves as the functional B-cell receptor. Figure 4.23 Membrane-bound immunoglobulins are associated with two other proteins, Igα and Igβ. Igα and Igβ form a disulfide-linked complex that interacts with the immunoglobulin. They have long cytoplasmic tails that interact with intracellular signaling proteins, and both Igα and Igβ have binding sites for the immunoglobulin C region on their extracellular portions. The complex of immunoglobulin with Igα and Igβ serves as the functional B-cell receptor. The immunoglobulin shown here is IgM, but all isotypes can serve as B-cell receptors. Igα and Igβ are also called CD79a and CD79b, respectively. 31  Switch from Membrane-Bound to Secreted Immunoglobulin After a naïve B cell encounters antigen, alternative RNA processing results in a switch from membrane-bound to secreted immunoglobulin: Two exons: Ø Membrane-Coding (MC) sequence (light blue) encoding the transmembrane region Ø Secretion-Coding (SC) sequence (orange) encoding the carboxy terminus of the secreted form Ø Two potential polyadenylation sites (shown as pAμs and pAμm) Figure 4.24 The surface and secreted forms of an immunoglobulin are derived from the same heavy-chain gene by alternative RNA processing. Each heavy-chain C gene has two exons (membrane-coding, MC; light blue) encoding the transmembrane region and cytoplasmic tail of the surface form of that isotype and a secretion-coding (SC) sequence (orange) encoding the carboxy terminus of the secreted form. The events that dictate whether a heavy-chain RNA will result in a secreted or transmembrane immunoglobulin occur during processing of the initial transcript and are shown here for IgM. Each heavy-chain C gene has two potential polyadenylation sites (shown as pAμs and pAμm). Left panel: the transcript is cleaved and polyadenylated at the second polyadenylation site (pAμm). Splicing between a site located between the fourth Cμ exon and the SC sequence and a second site at the 5ʹ end of the MC exons removes the SC sequence and joins the MC exons to the fourth Cμ exon. This generates the transmembrane form of the heavy chain. Right panel: the primary transcript is cleaved and polyadenylated at the first polyadenylation site (pAμs), eliminating the MC exons and giving rise to the secreted form of the heavy chain. AAA designates the poly(A) tail. 32 Somatic Hypermutation Antigen-binding sequences are further diversified after antigen exposure by: Ø Random point mutations in V region genes Ø > 106 times more frequent than the normal mutation rate Ø A further selection of best-fit results in affinity maturation of immunoglobulin Figure 4.25 Somatic hypermutation is targeted to the rearranged gene segments that encode immunoglobulin V regions. The frequency of mutations at positions in and around the rearranged VJ sequence—which encodes the V region—of an expressed light-chain gene is shown here. 33  Somatic Hypermutation enables selection of B cells making higher-affinity antibodies Red bars represent amino acid positions that differ from the original sequence due to mutation. Figure 4.26 Somatic hypermutation enables selection of B cells making higher- affinity antibodies. B cells were collected 1 and 2 weeks after immunization with the same epitope and used to make hybridomas secreting monoclonal antibodies. The amino acid sequences of the heavy and light chains of the epitope-specific monoclonal antibodies were determined. Each thick horizontal line represents one variant, and the red bars represent amino acid positions that differ from the prototypic sequence. One week after primary immunization, most of the B cells make IgM, which shows some new sequence variation in the V region. This variation is confined to the CDRs, which form the antigen-binding site. Two weeks after immunization, both IgG- and IgM-producing B cells are present, and their antibodies show increased variation and higher affinity that involves all six CDRs of the antigen- binding site. 34 Heavy chain isotype switching produces antibodies with different C regions but identical antigen specificity Ø Recombination between the rearranged VDJ segment and different CH gene segments Ø DNA loops out, and circular DNA containing the intervening sequences is deleted Ø Sequential switching to different isotypes and subclasses can occur during an immune response, but only in one direction 35  Joining Chain Figure 4.27 IgM is secreted as a pentamer of immunoglobulin monomers. The left two panels show schematic diagrams of the IgM monomer and pentamer. The IgM pentamer is held together by a polypeptide called the J chain, for joining chain (not to be confused with a J segment). The monomers are cross-linked by disulfide bonds to each other and to the J chain. The right panel is an electron micrograph of an IgM pentamer (×900,000), showing the arrangement of the monomers in a flat disc. The lack of a hinge region in the IgM monomer makes the molecule less flexible than, say, IgG, but this is compensated for by the pentamer having five times as many antigen- binding sites as IgG. Faced with a pathogen that has multiple identical epitopes on its surface, IgM can usually attach to it with several binding sites simultaneously. Electron micrograph courtesy of K.H. Roux and J.M. Schiff. 36 Isotype switching occurs at Switch regions (S) AID enzyme (activation-induced cytidine deaminase) Ø Immunoglobulin isotype switching occurs by recombination between these switch regions (S), with deletion of the intervening DNA. Ø AID targets the switch regions, causing nicks in both strands of DNA. Ø Excision of the intervening DNA as a nonfunctional circle of DNA juxtaposes the rearranged VDJ sequence with a different C gene. From IgM and IgD to IgG1 Figure 4.28 Isotype switching involves recombination between specific switch regions. Repetitive DNA sequences are found to the 5ʹ side of each of the heavy- chain C genes, with the exception of the δ gene. Immunoglobulin isotype switching occurs by recombination between these switch regions (S), with deletion of the intervening DNA. The switch regions are targeted by AID, which leads to nicks being made in both strands of the DNA. These nicks facilitate recombination between the switch regions, which leads to excision of the intervening DNA as a nonfunctional circle of DNA and brings the rearranged VDJ sequence into juxtaposition with a different C gene. The first switch a clone of B cells makes is from the μ isotype to another isotype. A switch from μ to the γ1 isotype is shown here. Further switching to other isotypes can take place subsequently. 37  Properties of human immunoglobulin isotypes Antibodies with different C regions have different properties and functions 0.00005 Figure 4.29 The physical properties of the human immunoglobulin isotypes. The molecular mass given for IgM is that of the pentamer (see Figure 4.27), the form present in serum. The molecular mass given for IgA is that of the monomer. Large amounts of IgA are also produced in the form of dimers, which are secreted at mucosal surfaces. 38  Properties and Functions of Ig Subclasses Remember the ones with +++ ++ for sensitization for killing by NK cells Figure 4.30 Each human immunoglobulin isotype has specialized functions correlated with distinctive properties. The major effector functions of each isotype (+++) are shaded in dark red; lesser functions (++) are shown in dark pink, and minor functions (+) in pale pink. Other properties are similarly marked. Opsonization refers to the ability of the antibody itself to facilitate phagocytosis. Antibodies that activate the complement system indirectly cause opsonization via complement. The properties of IgA1 and IgA2 are similar and are given here under IgA. *IgG2 acts as an opsonin in the presence of one genetic variant of its phagocyte Fc receptor, which is found in about 50% of people of European origin. 39  Mucosal IgA is a Dimer Ø J chain Ø Disulfide bonds Ø the monomers have disulfide bonds to the J chain but not to each other Figure 4.31 IgA molecules can form dimers. In mucosal lymphoid tissue, IgA is synthesized as a dimer in association with the same J chain that is present in pentameric IgM. In dimeric IgA, the monomers have disulfide bonds to the J chain but not to each other. The bottom panel shows an electron micrograph of dimeric IgA (×90,000). Electron micrograph courtesy of K.H. Roux and J.M. Schiff. 40  IgG is a highly flexible molecule. Figure 4.32 IgG is a highly flexible molecule. The most flexible part of the IgG molecule is the hinge, which allows the Fab arms to wave and rotate and thus accommodate the antibody to the orientation of epitopes on pathogen surfaces. Adding to further flexibility in binding antigen is the ‘elbow’ within the Fab that allows the variable domains to bend with respect to the constant domains. Similarly, the wagging of the Fc tail allows IgG molecules that have bound to antigen to accommodate to the binding of C1q and other effector molecules. Shown is a molecule of the IgG1 subclass, which represents IgG in figures throughout this book. 41  Different hinge structures distinguish the four subclasses of IgG Number of disulfide bonds 2 4 11 2 Ø A disulfide bond is a covalent bond between two sulfur atoms (–S–S–) formed by coupling two thiol (–SH) groups. Ø Cysteine, one of 20 amino acids, has a –SH group in its side chain and can easily be dimerized in aqueous solution by forming a disulfide bond. Ø Disulfide bonds between cysteine residues are essential for protein folding in many proteins. Ø Disulfide bonds in proteins are cleaved by heating or the addition of reducing reagents, which leads to protein denaturation. Ø A reducing agent is a chemical species that "donates" an electron to an electron recipient. Figure 4.33 Different hinge structures distinguish the four subclasses of IgG. The relative lengths of the hinge and the number of disulfide bonds in the hinge that cross-link the two heavy chains are shown. Not shown are other differences in the amino acid sequence, particularly glycine and proline residues, that influence hinge flexibility. 42  The four subclasses of IgG have different and complementary functions. Remember the ones with ++ and +++ Figure 4.34 The four subclasses of IgG have different and complementary functions. 43  IgG4 is present in the circulation in a functionally monovalent form. Ø IgG4 is synthesized in a form that has two heavy chains, two light chains, and two identical antigen-binding sites. Ø Molecules of IgG4 can interact in the circulation and exchange one heavy chain and its associated light chain. Ø Most IgG4 molecules in the circulation have two different binding sites for antigen. Thus, they only interact with a pathogen or a protein antigen through one binding site. This dual binding capability can: Ø Enhance the specificity and strength of the immune response by allowing the IgG4 molecule to bind to multiple epitopes on the antigen, increasing the likelihood of neutralizing or marking it for destruction. Ø Promote the formation of immune complexes, which are crucial for efficiently removing antigens from circulation. Ø This mechanism helps effectively remove foreign substances and pathogens from the body. Figure 4.35 IgG4 is present in the circulation in a functionally monovalent form. Like other IgG subclasses, IgG4 is synthesized in a form that has two heavy chains, two light chains, and two identical antigen-binding sites. Unlike other IgGs, however, molecules of IgG4 can interact in the circulation and exchange one heavy chain and its associated light chain. Because of this property, most IgG4 molecules in the circulation have two different binding sites for antigen. Thus they only interact with a pathogen or a protein antigen through one binding site. 44  Summary of gene segment rearrangement and the synthesis of cell-surface IgM in B cells Figure 4.36 Gene rearrangement and the synthesis of cell-surface IgM in B cells. Before immunoglobulin light-chain (center panel) and heavy-chain (right panel) genes can be expressed, rearrangements of gene segments are needed to produce exons encoding the V regions. Once this has been achieved, the genes are transcribed to give primary transcripts containing both exons and introns. The latter are spliced out to produce mRNAs that are translated to give κ or λ light chains and μ heavy chains that assemble inside the cell and are expressed as membrane-bound IgM at the cell surface. The main stages in the biosynthesis of the heavy and light chains are shown in the panel on the left. 45  Summary and Review: Figure 4.37 Changes in the immunoglobulin genes that occur during a B cell’s lifetime. 46