Chapter 4 – Antibody Structure and the Generation of B-Cell Diversity

I. Antibodies can recognize all types of biological macromolecules, but in practice, proteins and carbohydrates are the antigens most commonly encountered.

II. Collectively, antibodies are diverse in their antigen-binding specificities – the total number that can be made (antibody repertoire) might be as high as 1016; the number of B cells limits the actual repertoire to 109

III. Before encountering antigen, mature B cells only express membrane-bound immunoglobulin that serves as the cell receptor for antigen; when an antigen binds to this receptor, B cells are stimulated to proliferate and differentiate into plasma cells, which then secrete large amounts of antibodies with specificity for that membrane-bound immunoglobulin.

IV. The structural basis of antibody diversitya. Antigen binding/interaction with other immune system cells are carried out by different

part of the antibody moleculei. One part is highly variable (amino acid sequence differs considerably from

antibody to antibody)1. Contains sit of antigen binding and confers specificity

ii. One part is constant and interacts with other immune-system componentsb. Immunoglobulins are split into 5 classes (isotypes) – IgA, IgD, IgE, IgG and IgM

i. Distinguished based on structure differences in the constant part of the molecule; different effector functions

V. Antibodies are composed of polypeptides with variable and constant regionsa. Antibodies are glycoproteins built from a basic unit of four polypeptide chains (two

heavy [H] chains and two identical, smaller light [L] chains) – assembled to look like the letter Y.

i. Each arm is made of a complete light chain, paired with the amino terminal part of the heavy chain (chains are covalently linked by a disulfide bond)

b. Variability is the reason for great diversity of antigen-bindingspecificities among antibodies, because the paired variable (V) regions of a heavy and light chain form the antigen-binding site

i. Each antibody has two identical antigen binding sites(one at the end of each arm)

c. In IgG, a relatively unstructured portion in the middle of theheavy chain forms a flexible hinge region, at which it can becleaved to produce defined antibody fragments

i. Digestion with the plant protein papain produces 3 fragments –correspond to two arms and a stem

1. Arm fragments – FAB (FRAGMENT ANTIGEN BINDING)2. Stem fragment – Fc (FRAGMENT CRYSTALLIZABLE)

d. Differences in the H chain C-regions define the five isoptypes of immunoglobulini. Their heavy chains are denoted by ϒ (IgG), µ (IgM), δ (IgD), α (IgA), ε (IgE)

e. The L chain only has two isotype classes –κ and λi. Light chains of both isotypes are found associated with all H chain isotypes; each

antibody contains either the κ OR λ light chain isotype, NOT BOTH.VI. Immunoglobulin chains are folded into compact and stable protein domains

a. The V region at the amino-terminal end of each heavy or light chain is composed of a single variable domain (V domain) – VH in the heavy chain and VL in the light chain – these domains together form the antigen binding site

i. The other domains have little/no sequence diversity C domains make up the C regions

b. The structure of a single immunoglobulin domain can be compared to a bulging sandwich in which two β sheets are held together by strong hydrophobic interactions between their constituent amino acid side chains; structure stabilized by disulfide bonds between the two sheets.

VII. An antigen binding site is formed from the hypervariable regions of a heavy chain V domain and a light chain V domain

a. Comparison of V domains of heavy and light chains from different antibody molecules show the differences in amino acid sequences are concentrated within hypervariable regions, which are flanked by much less variable framework regions

i. Three HV regions are found in each V domainii. Framework regions correspond to the β strands and the remaining loops

b. The pairing of heavy and light chain in antibodies brings together the hypervariable loops from each V domain to create the composite hypervariable surface (forms antigen binding site at the tip of each FAB arm)

c. These hypervariable loops are also called complementarity-determining regions (CDR)

The structures of the human immunoglobulin classesNote differences in H chain C-regions, location of disulfide bonds linking the chains, and presence of a hinge region in IgG, IgA,

and IgD. All isotypes ccur as monomers in their membrane bound form; in their soluble, secreted form IgD, IgE and IgG are always monomers, IgA forms monomers and dimers, and IgM forms only pentamers.

VIII. Antigen-binding sites vary in shape and physical propertiesa. Antigenic determinant (epitope) is the part of antigen to which antibody binds

i. Are usually either carbohydrate or protein, because the surface molecules of pathogens are commonly glycoproteins, polysaccharides, glycolipids, and peptidoglycans

1. complex macromolecules such as these contain multiple epitopes, each of which can be bound to different antibody (multivalent)

b. Antigen binding site of antibodies vary according to size and shape of epitope they recognize

i. Epitopes can bind to pockets, grooves, extended surfaces, or knobs in antigen-binding sites

1. Linear epitopes-antibody binds to parts of the molecule that are adjacent in the linear sequence

2. Conformational (discontinuous) epitopes – formed by parts of a protein that are separated in the amino acid sequence, but are brought together in the folded protein.

ii. The binding of antigen to antibodies is based solely on non-covalent forces –electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions

1. Binding caused by van der Waals forces and hydrophobic interactions is complemented by the formation of electrostatic interactions and hydrogen bonds between particular chemical groups on the antigen and particular amino acid residues of the antibody

iii. Some antibodies have been found to catalyze reactions involving the antigen that they bind – catalytic antibodies

IX. Monoclonal antibodies are produced from a clone of anti-body producing cellsa. The traditional method for making antibodies of desired specificity is to immunize with

the appropriate antigen and the prepare antisera from their blood.b. More modern method – monoclonal antibodies

i. B cells are isolated from immunized animals and immortalized by fusion with a tumor cell to form hybridoma cell lines that will grow and produce antibodies indefinitely. Individual hybridoma cells are then separated, and cells making antibodies of the desired specificity are identified and selected for further propagation.

c. Monoclonal antibodies are used in therapy and diagnosis

i. First successful – mouse monoclonal antibody specific for CD3 antigen on human T cells; block T-cell response and prevent imminent T cell mediated rejection of transplanted kidneys

1. Limited because human immune system perceives mouse antibody as foreign; chimeric monoclonal antibodies reduce this problem (combine mouse V regions with human C regions)

2. Another approach to reducing the tendency for useful mouse monoclonal antibodies to provoke an immune response is to humanize them.

a. Sequences encoding CDR loops of mouse monoclonal antibody H and L chain are used to replace corresponding CDR sequences of a human immunoglobulin.

X. Generation of immunoglobulin diversity in B cells before encounter with antigen

a. For an immunoglobulin gene to be expressed, individual gene segments must be rearranged to assemble a functional gene – occurs only in developing B cells

i. Once complete, heavy and light chains can be produced and membrane bound immunoglobulin appears on B cell surface allowing the cell to recognize and response to antigen

b. In humans, immunoglobulin genes are found at thee chromosomal locationsi. Heavy chain locus on chromosome 14, the κ light chain locus on chromosome 2

and the λ light chain locus on chromosome 22.c. Gene segments encoding leader peptides and the C regions consist of exons and introns

i. In contrast, V regions encoded by two VL or three VH gene segments require rearrangement to produce an exon that can be transcribed

1. The two types of gene segments that encode the light chain V region are called variable (V) and joining (J) segments

2. the heavy chain locus includes an additional set of diversity (D) gene segments that lie between the V and J segments

d. Random recombination of gene segments produce diversity in the antigen-binding sites of immunoglobulins

i. During B cell development, arrays of V, D and J segments are cut and spliced by DNA recombination (somatic recombination)

1. Single gene segment is brought together to form a DNA sequence encoding the V region of an immunoglobulin chain

2. For light chains, a single recombination occurs between a VL and JL segment; for heavy chains, two recombinations are needed – first to join a D and JH segment and then to joined the combined DJ segment to a VH segment.

a. RANDOMLY SELECTED – numerous different combinations of V, D, and J segments are possible

3. Somatic recombination is carried out by enzymes that cut and rejoin the DNA; exploits mechanisms more universally used by cells for DNA recombination and repair

a. Recombination of V, D, and J segments is directed by recombination signal sequences (RSS) – which flank the 3’ side of the V segment, both sides of the D segment and the 5’ side of the J segment

i. Two types – 1. a heptamer sequence [7 base pairs: CACAGTG] and a nonamer [9 base pairs: ACAAAAACC] separated by a 12 base pair spacer; 2. The heptamer and the nonamer sequences separated by a 23 base pair spacer

ii. Recombination in the heavy chain CANNOT join VH directly to JH without the involvement of DH, because the VH and JH segments are flanked by the same type of RSS.

e. Recombination enzymes produce additional diversity in the antigen-binding sitei. V(D)J recombinase – set of enzymes needed to recombine the V, D, and J

segments1. Two of the component proteins are made only in lymphocytes –

specified by recombination-activating genes (RAG-1 and RAG-2)2. Other components are present in all nucleated cells and have activities

to repair double-stranded DNA, bend DNA, or modify ends of broken strands (DNA ligase IV, DNA-dependent protein kinase, the nuclease Artemis, and the Ku protein associated with DNA-PK)

3. RAG-1 and RAG-2 proteins interact with each other and with other proteins to form the RAG complex

a. One RAG complex binds to one type of RSS and another complex binds to the other type of RSS

b. Interaction between RAG complexes aligns the two RSSs and cleaves the DNA at the ends of the immunoglobulin gene segment in such as a way as to create a hairpin at the end of each segment and a clean break at the end of the two heptamer sequences

i. The DNA molecules are held in place by the RAG complexes while the broken ends are rejoined by DNA repair enzymes in a process called nonhomologous end-joining

(forms a coding joint and a signal joint)

c. Signal joint – joined ends of the removed DNA; Coding joint – joined ends of two gene segments

4. The enzymes that open hairpins and form the coding joint introduce additional diversity to the third hypervariable region (CDR3) of immunoglobulin heavy and light chains

a. The RAG complex generates palindromic “P” nucleotides, which allows the opened hairpins to be variably modified by exonucleases that remove germline encoded nucleotides and by the enzyme terminal deoxynucleotidyl transferase (TdT), which randomly adds nucleotides

i. Added nucleotides are called N nucleotides because they are non-templated (not encoded) in germline DNA.

b. Once single-strand tails of two gene segments are able to pair, the gaps are filled with complementary nucleotides to complete the coding joint.

c. Contribution of P nucleotides and N nucleotides to the resulting amino acid sequence diversity in CDR3 – junctional diversity

i. Important source of immunoglobulin variabilityii. The third hypervariable region of the light-chain V

domain is encoded by the junction between the V and J segments; the third hypervariable region of the heavy chain V domain is formed by the D segment and its junctions with the rearranged V and J segment

f. Developing and naïve B cells use alternative mRNA splicing to make IgM and IgDi. Naïve B cells – express both IgM and IgD on their surface ; have yet to

encounter antigen (IgM and IgD are the only isotypes that can be produced simultaneously by a B cell)

ii. Rearrangement of the V, D, and J segments of the heavy-chain locus that occurs during B-cell development brings a gene promoter and enhancer into closer juxtaposition, enabling transcription

1. Resulting mRNA transcript is spliced, processed and translated to give a heavy-chain protein

The generation of junctional diversity during gene rearrangement

Rearrangement of V, D, and J segments produces a functional heavy-chain gene

a. The exons encoding leader peptide and the V region are on the 5’ side (upstream) of the DNA encoding the nine different C regions

b. Closest to the rearranged V region is the µ-gene, followed by the δ-gene

2. In mature naïve B cells, transcription of the heavy chain starts upstream of the exons encoding the leader peptide and the V region, continues through the µ and δ C genes, and terminates downstream of the δ gene, before the ϒ3 C gene.

a. This long primary RNA transcript is then spliced and processed in two ways – one yields mRNA for the µ heavy chain and one yields mRNA for the δ heavy chain

i. In making the µ-chain mRNA, the entire δ gene RNA is removed along with the introns from the µ gene.

ii. In making the δ-chain mRNA, the entire µ gene RNA is removed as well as the δ-gene introns.

g. Each B cell produces immunoglobulin of a single antigen specificityi. Allelic exclusion - the process in a developing B cell of immunoglobulin-gene

rearrangement in which the process is tightly controlled so that only one heavy chain and one light chain are expressed

1. Even though every B cell has two copies of the heavy chain locus and two of each light chain locus, only ONE heavy-chain locus and ONE light chain locus are rearranged to produce functional genes

ii. Because an antigen-binding site is formed by the association of a heavy chain and a light chain, the combinatorial association makes an important contribution to the overall diversity of immunoglobulins

iii. Monospecific – encounter with a given pathogen engages a subset of B cells that will make antibodies that bind only to the pathogen

iv. Because the DNA sequence of expressed immunoglobulin genes varies from one clone of B cells to the next can be used to detect large clonal populations of cancer cells in patients with B-cell lymphoma or leukemia

h. Immunoglobulin is first made in a membrane-bound form that is present on the B cell surface

i. When a B cell first makes IgM and IgD, the heavy chains have a hydrophobic sequence near the carboxy terminus by which the immunoglobulins associate with cell membranes

ii. By themselves, these immunoglobulin molecules cannot be transported to the cell surface.

1. For this to happen, the must associate with two transmembrane proteins Igα and Igβ

a. Proteins are invariant in sequence, and travel to the B cell surface in a complex with the immunoglobulin molecule

b. At the surface, this complex forms the B cell receptor for antigen

XI. Diversification of antibodies after B cells encounter antigena. All the isotypes of immunoglobulin can be made in two forms:

i. one that is bound to the cell membrane and serves as the B cell receptor for antigen

ii. one, the antibody, that is secreted to bind to antigen and aid its destructionb. During differentiation to antibody-secreting plasma cells, B cells change from making

membrane-bound form to making the secreted form; plasma cells ONLY make secreted antibody

c. The difference between membrane-bound and secreted immunoglobulin lies at the carboxy terminus of the heavy chain:

i. Membrane-bound : has a hydrophobic anchor sequence that is inserted into the membrane

1. The hydrophobic anchor of the membrane-associated µ chain is encoded by two small, separate exons downstream.

2. The splicing to give membrane-bound µ chain: alternative splicing in the exon encoding the fourth C-region domain removes the sequence encoding the hydrophillic, whereas the exons encoding the hydrophobic carboxy terminus are retained and incorporated into the mRNA when the introns are spliced out.

ii. Secreted : has a hydrophilic sequence1. The hydrophilic caryboxy terminus of the secreted µ chain is encoded at

the 3’ end of the exon encoding the fourth C-region domain2. The splicing to give secreted µ chain: the sequence encoding the

hydrophilic carboxy terminus is retained and the sequences 3’ of that, including the exons encoding the hydrophobic membrane anchor, are discarded.

iii. Difference is determined by different patterns of RNA splicing and processing of the same primary RNA transcript; involves NO rearrangement of underlying genomic DNA.

d. Rearranged V-region sequences are further diversified by somatic hypermutationi. Diversity generated during gene rearrangement is concentrated in the third CDR

of the VH and VL regions. (somatic hypermutation)1. Randomly introduces single nucleotide substitutions (point mutations)

at a high rate throughout the rearranged V regions of the heavy and light chain genes

ii. Somatic mutation is dependent on the enzyme activation-induced cytidine deaminase (AID) – which is made ONLY by proliferating B cells

1. Converts cytosine in single-stranded DNA to uracil, a normal component of RNA but not DNA

2. Other enzymes, that are not specific to B cells but are components of general pathways of DNA repair and modification, can then act to convert the uracil to any one of the four bases of normal DNA

iii. Somatic hypermutation gives rise to B cells bearing mutant immunoglobulin molecules on their surface

1. Some mutant immunoglobulin molecules have substitutions in the antigen-binding site that increase its affinity for the antigen

iv. As the adaptive immune response proceeds, antibodies of progressively higher affinity for the infecting pathogens are produced – a phenomenon called affinity maturation

1. Process of evolution in which variant immunoglobulins generated in a random manner are subjected to selection for improved binding to a pathogen

e. Isotype switching produces immunoglobulins with different C regions but identical antigen specificities

i. IgM is the first antibody made in primary immune response1. Membrane-bound IgM of the B cell receptor is monomeric, whereas

secreted IgM consists of a circular pentamer of the Y shaped immunoglobulin monomers

2. IgM binds strongly to the surface of pathogens with multiple repetitive epitopes, but is limited in effector mechanisms that it uses to clear antigen from the body

ii. Isotype switching (class switching) – a further DNA recombination event enables the rearranged V region coding sequence to be used with other heavy chain C-genes.

1. Is dependent on AID and similarly only occurs in B cells proliferating in response to antigen

2. Accomplished by recombination within the cluster of C genes that previously expressed C gene and brings a different one into juxtaposition with the assembled V-region sequence

a. Thus, antigen specificity remains unchanged; isotype changes

3. Flanking the 5’ side of each C gene, are highly repetitive sequences that mediate recombination – switch regions (except on the δ gene)

Isotype switching involves recombination between specific switch regionsRepetitive DNA sequences are found to the 5’ side of each heavy chain C-gene (except δ gene). Immunoglobulin isotype

switching occurs by recombination between the 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 DNA. The nicks facilitate recombination between the switch regions, which leads to excision of intervening DNA as a non-functional circle of DNA and brings the rearranged VDJ segments 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 ϒ1 isotype is shown here. Further switching to other isotypes can take place subsequently.

iii. In patients that lack a functional AID gene cannot undergo somatic hypermutation or isotype switching; only produce low-affinity IgM (Hyper IgM Immunodeficiency)

1. Main consequence is susceptibility to infection by pyogenic bacteria, particularly in sinuses, ears and lungs

f. Antibodies with different C regions have different effector functionsi. The certain classes of immunoglobulin are further divided into subclasses, which

differ in both nomenclature and properties between species.1. IgA IgA1 and IgA2 ; IgG IgG1, IgG2, IgG3, and IgG4

(numbered according to relative number in plasma)2. Heavy chains of IgA subclasses are designated α1 and α2 and the heavy

regions of IgG subclasses are ϒ1, ϒ2, ϒ3, and ϒ4ii. Neutralizing Antibodies – directly inactivate pathogens/toxins and prevent it

from interacting with human cells

1. Bind to a site on pathogen that is normally used to infect cells ; opsonization (coating pathogens with immune system protein)

a. Opsonized pathogen are more efficiently ingested by phagocytes, which have receptors for the Fc region of some antibodies and for certain complement proteins

iii. Overall strength of binding at multiple sites – avidityiv. Strength of binding to a single site – affinityv. By isotype switching, different effector functions can be brought into play

while preserving antigen specificity: synthesis of IgM gives way to synthesis of IgG

XII. IgM is made primarily by plasma cells resident in lymph nodes, spleen and bone marrow; circulates in blood and lymph

XIII. IgG is the most abundant antibody in internal fluidsa. Made principally in lymph nodes, spleen and bone marrow ; circulates in lymph and

blood. b. Structure is smaller and more flexible than IgM, giving it easier access to antigens in the

extracellular spaces of damaged and infected tissuesc. Can be transferred across the placenta to provide the fetus with protective antibodies

from the mother, in advance of possible infectionXIV. Monomeric IgA is made by plasma cells in lymph nodes, spleen and bone marrow and is

secreted into the blood streama. Can also be made as a dimer

i. Dimeric IgA is principally found in lymphoid tissues underlying mucosal surfaces and is the antibody secreted into the lumen of the gut; is also the main antibody in other secretions including milk, saliva, sweat and tears

ii. Some effector function is directed against resident microogansisms that colonize mucosal surfaces, keeping their population in check

XV. IgE is highly specialized toward recruiting the effector functions of mast cells in epithelium, activated eosinophils present at mucosal surfaces, and basophils in blood.