Chapter 4- Antibody Structure and the Generation of B-Cell Diversity Flashcards
1
Q
Antibodies
A
The secreted form of the immunoglobulin expressed by a B cell. Each mature B cell expresses immunoglobulin with a different specificity for antigen. Antibodies are produced by the plasma cells that differentiate from B cells after activation by antigen
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Q
Where are antibodies found?
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They circulate as a major component of the plasma and lymph, and they are always present at mucosal surfaces
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Q
Which antigens do antibodies recognize?
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They recognize all types of biological macromolecules, but proteins and carbohydrates are the most common antigens that they encounter
4
Q
How do antibodies fight infection?
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Binding of antibody to a bacterium or a virus particle can disable the pathogen and render it susceptible to destruction by other components of the immune system. Antibodies are the best source of protective immunity, so most successful vaccines stimulate the production of high-quality antibodies
5
Q
Specific
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When molecules/receptors interact selectively with only one type or a few types of molecule or cell
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Q
Antibody repertoire
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The total number of different specific antibodies that can be made by an individual, estimated at around 10^9
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Q
Immunoglobulins
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The general name for antibodies and B-cell antigen receptors. During its development, each B cell is committed to producing immunoglobulin of a unique antigen specificity
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Q
Plasma cells
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A terminally differentiated B lymphocyte that is dedicated to the synthesis and secretion of antibodies
9
Q
Clonal selection
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The central principle of adaptive immunity. In the bone marrow, B and T cells are created. B cells divide to form millions of descendants, but each daughter cell is actually different from the parent cell and its siblings. Each daughter cell underwent shuffling in its DNA to produce a unique receptor that recognizes a unique pathogen. T cells develop in the thymus. Afterwards, the B and T cells may be stored in the lymph nodes. Dendritic cells capture pathogens and present them to lymphocytes in the lymph node. The lymphocyte with the unique receptor that matches the pathogen is activated and will then produce millions of identical clones. Some will act as effector cells and others will act as memory cells
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Q
Agammaglobulinemia
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The inability to make antibodies. It is reflected by abnormally low amounts or absence of antibodies in the blood. It makes patients highly susceptible to certain infections
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Function of an antibody
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To recognize and bind to its corresponding antigen and then deliver the bound antigen to other components of the immune system, which will destroy/clear the antigen
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Antigen-binding site
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The highly variable part of the antibody (variability in the amino acid sequence) which binds to a specific antigen. This region determines the specificity of the antibody
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Antibody isotypes
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The 5 classes of antibody- IgG, IgM, IgD, IgA, and IgE. They are distinguished by structural differences in the constant part of the molecule
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Q
Constant region
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The region of the antibody that has little variation in its amino acid sequence. It interacts with other immune-system components. The different immunoglobulin isotypes are distinguished by structural differences in the constant part of the molecule. The differences give each isotype a distinctive effector function, so they interact with different subsets of immune system proteins
15
Q
Basic antibody structure
A
Composed for 4 polypeptide chains- 2 identical heavy (H) chains and two identical light (L) chains. The chains form a Y shaped structure. Each arm of the Y is made up of one light chain that is paired with the amino-terminal part of a heavy chain and covalently linked to it by a disulfide bond. The stem of the Y consists of the paired carboxy-terminal parts of the two heavy chains, also linked by disulfide bonds
16
Q
Heavy (H) chains
A
Weigh 50 kDa and are the larger of the two types of polypeptide. Consists of one variable domain and a number of constant domains. The variable region of the H chain is called VH
17
Q
Light (L) chains
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Weigh 25 kDa and are the smaller of the two types of polypeptide. Consists of one variable and one constant domain, and is disulfide-bonded to a heavy chain in the immunoglobulin molecule. The variable region of the L chain is called VL
18
Q
Fab
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An IgG fragment that consists of the light chain and the amino-terminal half of the heavy chain held together by a disulfide bond between the chains. Called Fab because it is the fragment with antigen binding specificity
19
Q
Fc
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An IgG fragment that consists of the carboxy-terminal halves of the two heavy chains disulfide-bonded to each other by the residual hinge region. Fc is an abbreviation for fragment crystallizable because it readily crystallizes, mediates the effector functions of the antibody molecule by binding to serum proteins and cell-surface receptors
20
Q
IgG
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The class of immunoglobulin having gamma heavy chains. IgG is the most abundant class of immunoglobulin in plasma and has conformational flexibility
21
Q
IgM
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The class of immunoglobulin having mu heavy chains. IgM is the first immunoglobulin to appear on the surface of B cells and the first antibody secreted during an immune response. It’s secreted in pentameric form, which is joined by a J (joining) chain
22
Q
IgD
A
The class of immunoglobulin having delta heavy chains. It appears as surface immunoglobulin in mature naive B cells but its function is poorly understood. It is ubiquitious in species with an adaptive immune system. It is present in very low amounts in serum and has a very short half-life. However, IgD aids in signaling the B cell to become activated. Transcription of IgD is coordinated with that of IgM
23
Q
IgA
A
The class of immunoglobulin having alpha heavy chains. Dimeric IgA antibodies are the main antibodies present in mucosal secretions. Monomeric IgA is present in the blood. Protects mucosal surfaces- it is secreted in milk, saliva, tears, and sweat. A J chain holds together dimeric IgA
24
Q
IgE
A
The class of immunoglobulin having epsilon heavy chains. Activates mast cells and basophils. IgE is involved in reactions against internal parasites, particularly helminth worms, and in allergic reactions
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What differentiates the 5 isotypes of immunoglobulin?
Differences in the heavy chain C regions. The heavy chain of each isotype is denoted by one of 5 Greek letters. Additionally, IgG, IgA, and IgD have a hinge region while IgM and IgE do not. The isotypes also differ in the distribution of N-linked carbohydrate groups
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Light chain isotypes
There are only 2- kappa and lambda. No functional difference has been found between antibodies with kappa and lambda light chains. Each antibody contains each kappa or lambda light chains, never both. In humans, 2/3 of antibody molecules have kappa light chains
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Immunoglobulin domain
A compact and stable structure that is formed when antibody motifs (around 100 amino acids) fold. The structure is a sandwich of two beta sheets held together by a disulfide bond. Strong hydrophobic interactions between constituent amino acid side chains make up the "filling". Immunoglobulin heavy and light chains are made up of a linear series of immunoglobulin domains. The immunoglobulin domain helps the antibody to withstand harsh conditions in extracellular environments
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Immunoglobulin-like domains
Protein domain that resembles the immunoglobulin domain in structure and is present in numerous other proteins (MHC class 1 and 2, CD4, and CD8)
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Immunoglobulin superfamily
The name given to all the proteins that contain one or more immunoglobulin or immunoglobulin-like domains
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Hypervariable regions (HVs)
Also called complementarity-determining regions (CDRs). They are the most variable regions of the variable domains. Consists of a short region of high diversity in an amino acid sequence within the variable region of immunoglobulin and T-cell receptor chains. There are 3 CDRs (CDR1, CDR2, and CDR3) in each variable region (6 total), which collectively contribute to the antigen-binding site and determine the antigenic specificity
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Framework regions (FRs)
The relatively invariant amino acid sequences within the variable domains of immunoglobulins and T-cell receptors, which provide a structural scaffold, flanking the hypervariable regions
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Structure of antibody hypervariable regions
Differences in antibodies of the same isotype are concentrated within short hypervariable regions. The hypervariable regions are flanked by conserved framework regions. Every VH and VL domain contains 3 hypervariable regions
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Antigenic determinant
Also called an epitope. It is the part of an antigen to which a particular antibody binds. Individual epitopes are made of a cluster of amino acids or a small polysaccharide chain. These structures are usually either a carbohydrate (bacterial polysaccharides) or a protein (viral coat proteins). Complex macromolecules on pathogen surface have several different epitopes each of which can bind to a different antibody.
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Multivalent
An antigen that has two or more different epitopes or more than one copy of the same epitope
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Which sites can epitopes bind to?
Epitopes can bind to pockets, grooves, extended surfaces, or knobs in antigen-binding sites
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Linear epitopes
An antigenic structure in a protein that consists of a linear sequence of amino acids within the protein's primary structure. These epitopes are formed by accessible loops of a protein antigen
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Discontinuous epitopes
Formed by two or more parts of the protein antigen that are separated in the amino acid sequence but come together in the folded protein. Antibodies that bind discontinuous epitopes bind only to native folded proteins
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Which forces are used when antigens bind to antibodies?
Noncovalent forces- electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions
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Antisera
The fluid component of clotted blood that contains antibodies against a specific antigen. It is taken from an immune individual and contains a wide range of antibodies to bind the antigen
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Immunogen
The immunizing antigen in a vaccine, any substance that can provoke an immune response
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Polyclonal antiserum
Blood serum containing antibodies of many different specificities
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Hybridomas
In a method for making specific antibodies without a purified antigen, mice are immunized with any antigen source. B cells are collected from the spleen or lymph nodes and fused with cells of an immortalized mouse B-cell tumor (a myeloma) which lacks functional immunoglobulin genes. The hybrid cells are called hybridomas. They are each formed from one antibody-producing B cell and one myeloma tumor cell. Hybridomas are immortal like the parental myeloma cell
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Monoclonal antibody
An antibody produced by a single clone of B lymphocytes (like a hybridoma)- all the antibody molecules are identical in their structure and in their antigen specificity
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Flow cytometry
A technique in which individual cells can be counted or identified by their cell-surface molecules after fluorescent labeling. It can be used to analyze subpopulations of cells in peripheral blood and detect changes caused by disease
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Intravenous immunoglobulin (IVIG)
Preparation of serum gamma globulin from healthy blood donors, containing many different antibodies that is used as a treatment to replace antibodies and increase platelets in immunodeficiency and autoimmune diseases
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Chimeric monoclonal antibodies
Monoclonal antibody that combines mouse variable regions with human constant regions. Can be used in the treatment of cancer
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Humanized monoclonal antibodies
A mouse monoclonal IgG in which the conserved parts of the heavy and light chain V regions have been replaced by human versions. The only mouse parts of the antibody are the antigen-specific CDR loops. These therapeutic antibodies do not provoke a strong anti-IgG response in patients. Can be used to treat allergic asthma- prevents the recruitment of mast cells, eosinophils, and basophils in the allergic response
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Fully human monoclonal antibodies
Monoclonal antibody in which the light chains and heavy chains are completely of human origin. Can be made from mice, in which the immunoglobulin genes have been replaced by human versions
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Gene segments
A short DNA sequence that occurs in multiple alternative forms in immunoglobulin and Y receptor genes. The V-region gene is assembled from this sequence, each set of segments contains alternative versions of the same part of the V region. There are 3 different types- variable gene segment, joining gene segment, and diversity gene segment
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Germline form
Also called germline configuration. Inherited through the germline (egg and sperm). Immunoglobulin genes are inherited in this manner
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What must occur for an immunoglobulin gene to be expressed?
Individual gene segments have to be rearranged to assemble a functional gene. This process only occurs in immature B cells. The gene rearrangements occur as B cells develop from B cell precursors in the bone marrow. When the gene arrangements are complete, membrane-bound immunoglobulins appear at the B cell surface. B cells can recognize and respond to an antigen using these receptors
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Where are human immunoglobulin genes present in the genome?
At 3 locations- the heavy chain locus on chromosome 14, the kappa light chain locus on chromosome 2, and the lambda light chain locus on chromosome 22. Different gene segments encode the leader peptide (L), the V region, and the constant region of the heavy and light chains.
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Gene segments encoding C genes
Called C genes. The heavy-chain locus contains C genes for all of the different heavy-chain isotypes. These gene segments consist of exons and introns that are ready to be transcribed
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Gene segments encoding leader peptides
These gene segments consist of exons and introns that are ready to be transcribed
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Gene segments encoding the variable region
The V regions can't be transcribed because they are encoded in a fragmented form of two (VL) or three (VH) gene segments. Each segment is being represented by an array of alternative forms that differ in nucleotide sequence. To be transcribed, the segments are cut and splices to produce a V region exon with only one force of each gene segment- called V and J gene segments. The heavy chain locus has an array of D gene segments that lies between them
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Somatic recombination
The process by which arrays of immunoglobulin gene segments are cut and spliced. It generates a complete exon composed of a V gene segment and a J gene segment that encodes the variable region of an immunoglobulin (B cells) or a T cell receptor polypeptide chain. Humans inherit differences in the numbers of V and J gene segments
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Recombination signal sequences (RSSs)
A short stretch of DNA flanking each of the gene segments that are rearranged to generate V-region exons. They are the sites at which somatic recombination occurs
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12/23 rule
The fact that V(D)J recombination can only occur between gene segments with specific lengths of spacer in the recombination signal sequences. This means that a VH region can't be directly joined to a JH region without involving DH. Two types of spacer are 12 and 23 base pairs in length
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V(D)J recombinase
The set of enzymes needed to recombine V, D, and J segments in the rearrangement of immunoglobulin and T receptor genes. It includes the RAG-1 and RAG-2 proteins. The recombinase is specific to B and T cells and is expressed during the development/generation of antigen receptors.
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Recombination-activating genes
Includes RAG1 and RAG2. They are essential for the rearrangement of immunoglobulin and T-cell receptor genes in B cells and T cells. They encode the RAG-1 and RAG-2 proteins, which form a protein complex that catalyzes the recombination process
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RAG complex
Formed by two RAG-1 and two RAG-2 proteins. It acts as a scaffold that brings together the pair of RSSs that will be cut and spliced to achieve recombination.
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Palindromic (P) nucleotides
Nucleotides added into the junctions between immunoglobulin and T-cell receptor gene segments during the somatic recombinations that generate a rearranged variable-region sequence. They are an inverse repeat (palindrome) of the nucleotide sequence at the end of the adjacent gene segments
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Terminal deoxynucleotidyl transferase (TdT)
The enzyme that inserts non-templated nucleotides (N nucleotides) into the junctions between gene segments during the rearrangement of T cell receptor and immunoglobulin genes. Adds diversity during VDJ recombination
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N nucleotides
Non-templated nucleotides added at the junctions between gene segments of T cell receptor and immunoglobulin heavy-chain variable-region sequences during somatic recombination. N nucleotides aren't encoded by gene segments- they are inserted at random by TdT. Contribute to immunoglobulin and TCR diversity
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Junctional diversity
Variation present in immunoglobulin and T cell receptor polypeptides that is created during the process of gene rearrangement by the addition or removal of nucleotides at the junctions between gene segments
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Naive B cells
A mature B cell that has left the bone marrow to circulate in the blood but has not encountered a specific antigen. They express both IgM and IgD on their surfaces, which are the only immunoglobulin isotypes that are ever produced simultaneously by a B cell. Antigen binding induces proliferation and differentiation of B cells
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Allelic exclusion
After V(D)J rearrangement each mature B cell finally expresses only one immunoglobulin heavy chain (from the two possible heavy chain loci, maternal and paternal) and one light chain (from the 4 possible light chain loci). This ensures that each B cell produces immunoglobulin of a single specificity
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Rearrangement of the V, D, and J heavy chain segments during B cell development
Rearrangement brings a gene promoter and an enhancer into closer juxtaposition, which allows the rearranged gene to be transcribed.
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Ig-alpha and Ig-beta
Polypeptide chains associated with the B-cell receptor that transduce signals to the interior of the B cell when the B-cell receptor binds the antigen. They are encoded by genes that do not rearrange and don't vary in sequence between different clones of B cells
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The B-cell receptor complex
The membrane-bound antigen receptor on B cells. Each B cell makes a single type of immunoglobulin. The cell-surface form of this immunoglobulin serves as a B cell receptor for the specific antigen
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What triggers a naive B cell's proliferation and differentiation?
Binding of an antigen to the surface immunoglobulin of a B cell
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2 forms of immunoglobulin
1. On the outside of cell, attached to the plasma membrane- acts as the B cell's receptor for antigen. Only made in small amounts
2. Antibody- a secreted and soluble effector molecule. Made in larger quantities
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Difference between membrane bound and secreted immunoglobulin
The main difference is at the carboxy-terminal part of the heavy chain. The B-cell receptor has a hydrophobic sequence, which anchors it in the membrane, while the antibody has a hydrophilic sequence. The alternate structures are determined by differential RNA splicing and processing of the same primary RNA transcript. It involves no gene rearrangements
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Somatic hypermutation
Maturation that occurs at high frequency in the rearranged variable-region DNA segments of immunoglobulin genes in activated B cells, resulting in the production of variant antibodies. Some of these antibodies have a higher affinity for the antigen. Somatic hypermutation introduces single-nucleotide substitutions almost randomly throughout the rearranged V regions of heavy and light chain genes. The immunoglobulin constant regions aren't affected, and neither are other B-cell genes.
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Activation-induced cytidine deaminase (AID)
The enzyme that deaminates DNA at cytosine residues and converts them to uracil. It is only made by proliferating B cells, mainly in germinal centers. AID converts cytidine in ssDNA to uridine. The activity of this enzyme and the consequent repair of the damaged DNA are the basis of somatic hypermutation and isotype switching in activated B cells
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Affinity maturation
Affinity maturation is an evolutionary process. Variant immunoglobulins are generated in a random manner and are selected for improved binding to a pathogen. This process only takes a few days opposed to the thousands or millions of years required for Darwinian evolution. It is the result of somatic hypermutation and the rearranged immunoglobulin V-gene region
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Isotype switching
Also called class switching. It is the process by which a B cell changes the class of immunoglobulin it makes. The antigenic specificity is preserved during this process. IgM is the first antibody produced when naive B cells encounter an antigen. Isotype switching is required to produce antibodies with other effector functions
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Switch sequences
Also called switch regions. They are short DNA sequences preceding each heavy chain constant-region gene. Somatic recombination occurs here when B cells switch from production of one immunoglobulin isotype to another
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Hyper-IgM syndrome
An autosomal recessive condition where patients lack a functional AID gene. The B cells can't undergo somatic hypermutation or isotype switching. These individuals can only make low-affinity IgM, not any other antibodies. IgM is present in plasma at greater amounts than in normal individuals, while other isotypes are only present at low levels. Patients exhibit recurrent upper and lower respiratory tract bacterial infections. Immunoglobulin replacement therapy is effective, but is not a cure. Antibodies only last 21-30 days
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Subclasses of IgG
IgG1, IgG2, IgG3, and IgG4. They are numbered according to their relative abundance in plasma, with IgG1 being most abundant. They differ in the constant region of the heavy chain, with many of the differences being located in the hinge
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Subclasses of IgA
IgA1 and IgA2
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Neutralizing antibodies
High-affinity IgA and IgG antibodies that bind to pathogens and prevent their growth or entry into cells
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Opsonins
General name for antibodies and complement proteins that coat pathogens, facilitating phagocytosis
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Affinity
Measure of the strength with which one molecule binds to another at a single binding site. Involves a single antigenic determinant binding to a single binding site on an antibody. The higher the affinity, the more stable the interaction
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Avidity
The overall strength of binding of an antibody with multiple binding sites to an antigen (also with multiple sites- a multivalent antibody). You can have an antibody with low affinity but high avidity
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Fc receptors
A cell-surface receptor for the Fc portion of an immunoglobulin isotype. There are different Fc receptors for the different isotypes and subtypes
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Monomeric IgA
Made by plasma cells in the lymph nodes, spleen, and bone marrow, and is secreted into the bloodstream
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Dimeric IgA
Made mainly in the lymphoid tissues underlying mucosal surfaces and is the antibody secreted into the lumen of the gut. The GI tracts provides an extensive surface of contact between the human body and the environment, so more IgA is made than any other isotype. It functions to keep populations of resident microorganisms in check. Also the main antibody in other secretions- milk, saliva, sweat, and tears. Structure- 2 monomers joined by a J chain identical to that in pentameric IgM.
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IgM
When bound to antigen, sites in the IgM constant region become exposed and initiate reactions with complement that can kill microorganisms directly or facilitate phagocytosis. Switching isotype allows for different effector functions while preserving antigen specificity. IgM has low affinity and is good at complement activation
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IgE
This class is highly specialized at recruiting the effector functions of mast cells in the epithelium, activated eosinophils at mucosal surfaces, and basophils in the blood. These 3 cell types carry a high affinity receptor that binds IgE in the absence of antigen. When antigen binds to cell-surface IgE, it triggers strong physical and inflammatory reactions that expel and kill infecting parasites
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IgG
The most abundant antibody in the internal body fluids. Mainly made in the lymph nodes, spleen, and bone marrow, and circulates in lymph and blood. IgG is smaller and more flexible than IgM, which gives it easier access to antigens in the extracellular spaces of damaged and infected tissues. It has higher affinity and is good at neutralization and opsonization
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IgD
Basophils have a high affinity receptor that binds IgD in the absence of antigen and is different from the IgE receptor. When antigen is present and binds to cell surface IgD, it triggers basophils to orchestrate a local immune response that eliminates the bacteria
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IgG1
The most abundant and versatile of the 4 subclasses, "good all-round" subclass. Intermediate in its flexibility, susceptibility to proteolysis, and capacity to activate complement. Constitutes most of the antibody made against protein antigens
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IgG2
The hinge is of similar length to that of IgG1 but contains additional disulfide bonds that reduce its flexibility, susceptibility to proteolysis, and capacity to activate complement. Preferentially made against the highly repetitive carbohydrate antigens of microbial surfaces. This puts fewer demands on an antibody's flexibility. IgG2 is most effective against encapsulated bacteria
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IgG3
The best subclass at activating complement. Has a much longer hinge region than other subclasses. This long hinge gives IgG3 greater flexibility in binding to antigens and also makes the Fc more accessible for binding to C1. These qualities contribute to its superior activation of complement. Disadvantage of the long hinge- IgG3 is especially susceptible to cleavage by proteases and therefore has a shorter half-life in circulation than the other subclasses
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IgG4
Has an Fc region that can't activate complement because it binds poorly to C1q. IgG4 has a unique capacity to exchange modules composed of one heavy chain and its associated light chain. The reaction produces hybrid IgG4 molecules with two different heavy chains, two different light chains, and two antigen-binding sites of different specificity. This makes IgG4 functionally monovalent- it can only impede pathogens by neutralization. IgG4 is anti-inflammatory and is frequently elevated in allergic individuals- it can reduce the severity of an allergic reaction by binding to an allergen, blocking IgE from binding
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Adaptive immunity
Involves clearance of extracellular pathogens and their toxins by means of antibodies. Produced by the effector B lymphocytes or plasma cells and are the secreted form of the B-cell receptor for antigen
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Antibodies
The secreted form of the B cell receptor for antigen, which are secreted by B cells and plasma cells. They are soluble and secreted in larger quantities as the immune response progresses. Antibodies circulate in the blood and lymph, and are present at mucosal surfaces. They recognize all types of macromolecules, but proteins and carbohydrates are the most commonly encountered.
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What happens when an antibody binds to a pathogen?
Binding of antibody to a pathogen can disable the pathogen and make it more susceptible to destruction by other elements of the immune system (innate elements). Antibodies are the best source of protective immunity
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Immunoglobulins
The general term for cell surface B-cell antigen receptors and secreted antibodies. A mature B cell expresses membrane-bound immunoglobulin of a single antigen specificity. When a foreign antigen binds to this Ig, the B cell is then stimulated to proliferate. Its progeny differentiate into plasma cells that secrete antibodies of the same specificity as the membrane-bound Ig
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Papain
A protease that can cleave the hinge region of the antibody to produce 2 types of fragments (3 fragments in total)- 2 Fab fragments and one Fc fragment
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Isotypes differ in (4)
1. Length of Heavy chain C regions
2. Location of disulfide bonds linking the chains
3. Presence of hinge regions in IgG, IgD and IgA
4. Differences in N-linked carbohydrate groups (stability, aggregation, secretion, activities)
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Production of polyclonal antibodies (6)
1. Antigen design, synthesis, and conjugation
2. Immunization of 2-4 rabbits with the antigen
3. Additional boosts and screenings (ELISA, dot blot)
4. Serum (antibodies in the blood) is separated from the rabbit blood and collected
5. Affinity purification (protein A/G or antigen)
6. Antibody validation (ELISA, dot blot)
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VDJ recombination
Forms the variable (V) region of the antibody by randomly carrying out somatic recombination. The 3 gene segments that are recombined are the variable (V) gene, the joining (J) gene, and the diversity (D) gene. CDR regions are determined by the V gene selected and the junctions between V and J genes
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Light chain V region (VL)
Made up of a V gene and J gene
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Heavy chain V region (VH)
Made up of a D gene, J gene, and V gene
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Loci
The area of the chromosome with the cluster of genes that encode heavy and light chains. On the heavy chain, this is chromosome 14. On the light chain, this is chromosome 2 (kappa) or chromosome 22 (32)
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Germline configuration
When the orientation of genes is inherited ("complete set")
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Somatic
Genes that are expressed in all cells
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DNA repair enzymes (4)
1. Ku70:Ku80
2. DNA-dependent protein kinase (DNA-PK)
3. Artemis
4. DNA ligase
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General steps of VDJ recombination (4)
1. Synapsis
2. Cleavage
3. Hairpin opening and end-processing
4. Processing and joining
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Synapsis
The first step in VDJ recombination. Two selected coding segments and their adjacent RSS are brought together by a chromosomal looping event
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Cleavage
The second step of VDJ recombination. The endonuclease activity of RAG creates double stranded breaks at RSS-coding sequence junctions; closed DNA hairpin ends are formed
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Hairpin opening and end processing
The third step of VDJ recombination. Ku70:Ku80 binds to DNA ends. Then, DNA-PK and Artemis are recruited; endonuclease activity opens hairpins. Hairpins are opened by Artemis proteins; ends are modified by addition or removal of bases via TdT enzyme
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Processing and joining
The fourth and final step of VDJ recombination. TdT randomly adds and removes nucleotides to open DNA ends. Then, DNA ligase joins the two coding regions together. Signal ends are ligated together to form a signal joint
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Combinational diversity
Random pairing of V, D, and J genes
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P-nucleotides
Added after Artemis:DNA-PK complex opens the hairpins loops. Contribute to junctional diversity
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Isotype switching mechanism (4)
1. AID binds to repetitive DNA sequence flanking each C gene- switch regions
2. AID activity leads to nicks in both DNA strands
3. Nicks result in recombination between switch regions. Cμ, Cδ, and any other intervening C genes are excised as a circular DNA molecule
4. VDJ segments are brought in line with the next C gene. Antigen specificity does NOT change, but the Effector function DOES change
