Function and Importance of Antibodies Flashcards
Antibodies
Also called immunoglobulins- part of the adaptive immune system and produced by B cells.
Why are antibodies important?
- Immunity
- Research
- Medicine
Antibody structure
Y shaped, with 2 light chains and 2 heavy chains. These proteins come together through disulfide bonding to form a quaternary structure. The antigen binding site is located at the ends of the arms of the Y
Variable and constant regions
The constant regions are mostly invariant, especially within the same antibody class. The variable region, which contains the antigen binding site, is located at the end of the arms of the Y
Variable region
Composed of complementarity-determining regions (CDRs), which is the most variable portion of the antibody. This region is responsible for the specificity of the antibody, as it is complementary to the antigen it’s binding to. There are 3 CDRs per heavy chain and 3 per light chain. All 6 need to come together in the antibody structure, so the tertiary and quaternary structures are critical
Antigen-binding site specificity
Antigens with a high specificity have high affinity binding, while antigens with broad specificity have low affinity binding. As the immune response develops and somatic hypermutation occurs, antigen binding sites will have a higher affinity
Affinity
Strength of the antibody to antigen interaction. The affinity is independent of the number of antigen binding sites. Kd is the equilibrium dissociation constant and relates to the concentration of antibodies needed to promote binding to a specific antigen. A lower KD means you need less antibodies and have a better binding affinity
V region vs framework region
The variable region is composed of some CDRs, but the remainder is called the framework region. The framework forms a scaffold on which the CDRs can be arranged to form an antigen binding site. They are important structurally, but not functionally.
V-regions, C-regions, & Ig classes
There are 5 possible constant regions- A, G, D, M, or E on the heavy chain. There are also two possible constant regions on the light chain- kappa or lambda. The heavy chain constant regions correspond to the class of antibodies
Avidity
Total binding strength of an antibody, which takes into account the number of antigen binding sites
Immunoglobulin classes (5)
- Serum antibodies- IgG, IgD, IgE, and IgA (when present in the blood). These antibodies have the classic Y shape
- IgM- pentameric- 5 antibodies are joined with a J chain
- Secretory (mucosal) IgA- dimeric. Formed by a J chain but stabilized by a secretory component protein that it acquires as it makes its way to the mucosa
Fab fragments
Proteases, like papain or pepsin, can be used to cleave the hinge region of the antibody and generate antibody fragments. Cleavage generates 2 Fab fragments and one Fc. Fab stands for fragment antigen binding, and this is the arms of the Y. Contains the antigen binding site.
Fc fragments
Fragment crystallizable- bottom of the Y. Pepsin will degrade the Fc fragment
Antigens
Any foreign molecule- protein, lipid, or polysaccharide. If it’s different from molecules in the host, it will be recognized as foreign
Epitope
The piece of antigen that antibodies bind to
Continuous vs discontinuous epitopes
Continuous epitopes- if you were to denature an antigen and make it linear, the epitope would not be broken up. Discontinuous epitopes are the opposite- they only come together due to the way the antigen has folded. Denaturing the antigen would break up the epitope.
Agglutination
Antibodies can clump antigens together in a variety of different ways. Usually when the antigens are clumped together, they are neutralized and will be destroyed
Antibody hinge region
The hinge region is very flexible and helps the antibody to bind to epitopes
Primary antibody repertoire
Antibodies that have already been made by B cells, regardless of the specific infection or whether we’ve been exposed to a pathogen. Once infection occurs, these B cells are activated and antibodies are secreted. They have a broad and very diverse specificity, so they are prepared to come into contact with a variety of pathogens
Secondary antibody repertoire
Develops during an active infection. Antibodies are altered such that there is a great increase in their binding strength and the diversity of specificity. This process is known as affinity maturation
V(D)J recombination
Generates a primary antibody repertoire, occurring during development of B cells in the bone marrow and before we have come into contact with any pathogen. Each type of Ab chain (κ & λ L-chains, H-chains) is encoded by separate locus on separate chromosome- each locus has large number of V gene segments. Site-specific recombination occurs and produces different combinations of V, D, and J segments to make many different antigen binding sites of different specificities
L-chain V regions are encoded by
Encoded by DNA sequence from long V gene segment & short joining (J) gene segment
H-chain V regions are encoded by
Encoded by DNA seq from long V gene segment, short J gene segment and diversity (D) gene segment
V(D)J recombination – L-chain example
Recombination brings V3 (in this case) in close proximity with J3. Recombined DNA is transcribed to pre-RNA, and other J segments are spliced out during alternative splicing. Now, the antibody has its own unique variable region
V(D)J recombination – H-chain
Only difference between heavy and light V(D)J recombination is the extra D gene segment. D encodes amino acids in CDRs
Joining of gene segments in V(D)J recombination
The gene segments are joined by non-homologous end joining. A variable number of nucleotides may be lost or added. This increases the diversity of the variable regions
Junctional diversification
There is a random loss and gain of nucleotides at joining sites. It increases the diversification of V regions enormously
Why is V(D)J recombination considered site-specific?
There are conserved recombination signal sequences that flank each gene segment, and there are recognition sites for the joining process. This ensures that only appropriate gene segments recombine
V(D)J recombinase
The enzyme complex that mediates joining of the gene segments. Encoded by the recombination activating genes (rag) 1 & 2, which encode RAG1 & RAG2
V(D)J recombination process
- The V/D/J segments are flanked by sequences that are recognized by VDJ recombinase (the RAG proteins)
- That is going to cause curvature in the DNA, which brings 2 gene segments into close proximity
- The VDJ recombinase cleaves the DNA
- The 2 gene segments are joined through non-homologous end joining
V(D)J recombination at the κ light chain locus
Any of the 40 V gene segments may join to any of the 5 J segments. Encodes 200 different κ-chain V regions
V(D)J recombination at the heavy chain locus
Any of the 40 V gene segments may join to any of the 25 D & 6 J gene segments. Encodes 6000 different heavy chain V regions
How many different antigen binding sites can be produced by V(D)J recombination?
Humans can produce 320 different VL regions (200 κ and 120 λ) and 6000 different VH regions. Could be combined to make 1.9 X 106 different Ag-binding sites
Somatic hypermutation
Following stimulation, like infection or T cells, antibodies undergo mutations. This occurs long after the coding regions have been assembled. There is 1 mutation per V region coding sequence per generation. There is a 106 X greater than spontaneous mutation rate in other genes- not all mutations will result in an increased affinity for antigen
How does somatic hypermutation occur?
Very few mutations will result in an increased affinity for antigen. The antigen will stimulate preferentially those few B cells that do make antibodies with increased affinity, and clones of those altered B cells will preferentially survive and proliferate. Most other B cells die by apoptosis
Result of somatic hypermutation
Abs become of increasingly higher affinity as immune response progresses
Activation-induced deaminase (AID)
An enzyme that is expressed specifically in activated B cells and carries out somatic hypermutation. It deaminates C to U in the V region gene segments, producing U:G mismatches in the double helix. Repair of mismatches may leads to various mutations. Some mutations will be corrected to the original, while others result in a diverse range of mutations
Antibody class switch
As B cells are activated, antibodies typically undergo a class switch. Generally, the first antibodies released in an immune response will be IgM. With B cell activation, antibodies typically can switch from IgM to IgG. But they can switch to other isotypes as well, depending on the type of infection. IgA would be secreted for a mucosal infection
Why are antibodies so important to the immune response? (5)
- Specificity
- Neutralization
- Opsonization for phagocytosis
- Complement recruitment
- Lytic effects- C1q recruitment for MAC. Unique antibodies are bactericidal and fungicidal
Antibody opsonization
When the surface of the pathogen is coated with antibody. This improves phagocytosis, as macrophages and other phagocytes have lots of Fc receptors that recognize the Fc portions of the antibody
Complement functions (3)
Antibodies specifically recruit complement through the classical pathway. There are 3 possible outcomes- inflammatory cell recruitment, opsonization, and formation of the membrane attack complex, which will destroy the pathogen
Complement pathways (3)
- Classical pathway- binds antibody
- Lectin pathway- binds sugars on pathogen surfaces
- Alternative pathway- binds membranes
C3 convertase
An enzyme formed by the classical pathway. Once formed, C3 convertase cleaves C3, creating C3b and C3a. C3b is a potent opsonin, and C3a is a potent inflammatory mediator
C5 convertase
Formed by C3b binding to the C3 convertase. It produces C5a (inflammatory mediator) and C5b- it initiates polymerization reactions leading to MAC
C1q recruitment
Part of the classical pathway. Antibodies bind to antigens on the pathogen surface and then recruit complement component C1q, which recruits other components that lead to the 3 pathogen outcomes
Membrane attack complex (MAC)
Complements the binding activity of antibodies. C5b binds to C6 and C7, which then binds to the pathogen surface. It binds to C8, which inserts into the cell membrane. C9 molecules bind to the complex and polymerize, with 10-16 molecules binding to form a pore in the membrane. The MAC complements the binding activity of antibodies.
Complement-independent bactericidal antibodies
These antibodies can kill bacteria without using the MAC
How do antibodies have a bactericidal function?
They target antigens in lipid rafts. Many rafts aggregate together, creating a situation where there are large stretches of rafts and large stretches of non-raft regions. There is increased tension b/w LO and LD portions, which leads to membrane curvature. Membrane curvature leads to blebs
Bleb formation
Lipid rafts participate in bleb formation. Cholesterol and lipid rafts are critical for the effects of complement-independent antibodies
IgM functions (5)
- BCR (monomer)
- Early Ig responses
- Neutralization (+),
- C1q recruitment (+++) and formation of the MAC
- Opsonization (+)
IgG functions (4)
Subclasses: IgG1, IgG2, IgG3, IgG4.
1. Neutralization (++),
2. C1q recruitment (+++),
3. Opsonization (+++)
4. ADCC (++)
IgA functions (2)
- Neutralization (++)
- Mucosal immunity (+++)
IgD functions
BCR, no other known functions
IgE functions
Mast cell degranulation, associated with allergic responses
Secretory IgA transport
IgA comes from tissues underlying the mucosal tissues in a partial dimeric form. It is picked up by an Fc receptor on epithelial cells, and is then transcytosed to the mucosal surface. Following transcytosis, the Fc receptor remains bound to the IgA
IgM C1q recruitment
IgM binds in a flat orientation on the surface of a pathogen
What signals V(D)J recombination to stop occurring?
Once VDJ recombination has occurred, that will send signals in the B cell that turns off the RAG genes so that recombination will cease, and VDJ recombination will stop. Then, the B cell will make many antibodies, all with the same antigen binding sites- VDJ recombination results in antibodies with one specificity per B cell
