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Figures replaced or skipped for Ch 2
• Figure 2.3 - Enzyme digestion of Ab’s
• Flow cytometry - Fig 2.13
• Monoclonal Ab Fig 2.12 was replaced
Chapter 2
Antibody Structure and the Generationof B-Cell Diversity
• Molecular and structural basis of antibody diversity
• How B cells develop and function in the body
• How B cells are activated and participate in adaptive immunity
1. Antibodies are specific for individual epitopes2. Membrane bound form is present on a B-cell3. Ag binding to B cell stimulates it to secrete Ab
Location and production of ImmunoglobulinsHumoral Immunity - immunity dominated by antibodies that can be transferred to another person
Fab
Fc
Antibody structure
Heavy (5 classes), Light (2 classes), Constant (C) and Variable (V)Disulfide bonds link heavy chainsFab- fragment antigen bindingFc- fragment crystallizable
Immunoglobulin Isotypes or Classes
pentamers
Monomers or dimers
N-linked carbohydrateHinge regionDisulfide bondsMonomer as BCRMultimeric forms-Light chainIg domain
Figure 2-5Immunoglobulin (Ig) domain
~100 amino acid domain - very stableTwo types Ig domains- variable and constantAntigen Binding site - VH and VL
Heavy chain of IgG - four domains - VH, CH1, CH2, CH3
Figure 2-6
Anti-parallel -strands - contribute to the structural part
Loop region - in the V domain contribute to variability
Hypervariable Regions (CDR) of Antibodies
HV - hypervariable regions
CDR - complementarity determining regions
Framework region - strand region that has reduced variability
Amino Acid Sequence Variability in the V domain
110 amino acid light-chain V domain
Framework regions can have variability but variability is much higher in the HV regions
Physical Properties of Antigens-Antibody Binding
Epitope - part of the antigen bound by Ab
Antigen is usually carbohydrate or protein
• Lock and Key concept
• Variety of structures and sizes recognized by Ab’s
• Affinity vs Avidity - terms describing binding strength of an antibody for its epitope
Epitopes
• Epitope (antigenic determinant) is the part of the antigen bound by Ab
• Most antigens have multiple epitopes (multivalent)
• Usually carbohydrate or peptide.Fig. 2.9
Mechanisms of Epitope Recognition
• Linear and discontinuous epitopes
• Multivalent Antigens
• Polymeric Antibodies
• Affinity vs Avidity - terms describing binding strength of an antibody for its epitope
• Epitope binding mechanisms
Figure 2-9
Figure 2-26 part 1 of 2
First Ab to be made during during an immune response to an antigen
Monomers disulfide bonded together via the J-chain (joining chain)
Figure 2-30
Monomers disulfide bonded to the J-chain
Dimeric in mucosal lymphoid tissue - secreted into the gut to prevent pathogens binding to gut cells and can act as an antitoxin
Monomeric made by B-cells in lymph nodes/spleen and is not J-chain dependent
Antibody-antigen interaction• Non-covalent binding:
– Electrostatic– Hydrogen bonds– Van der Waals force– Hydrophobic forces
• Affinity: Strength of interaction between epitope and one antigen-binding site
• Avidity: Strength of the sum of interactions between antibody and antigen
Figure 2-8Poliovirus
VP1- blue, contains several epitopes (white) that can be recognized by human antibodies
PocketsAntibodies bind a Range of Structures
Extended surfacesGrooves Molecule DNA Lysozyme
Examples of Problematic Ab binding to various structures
Pocket:Penicillin - can bind to RBC surface proteins to create a foreign epitope. IgE binds to drug-RBC protein complex and initiates an inflammatory response
Groove:DNA - Systemic Lupus Erythematosus (Lupus) - autoimmune disease in which antibodies are made against DNA and other molecules leading to inflammatory reactions in joints, skin and kidney
Extended Surface:Lysozyme/Ovalbumin - allergies to hen egg components - more common in children under 5 - “desensitization” protocol can help
Haptens
Small molecules that are not immunogenic by themselves, but can bind immunoglobulins or TCRs.
Haptens can induce an immune response when linked to a larger protein.
Ag
1
2
3
4
Isolate serum
Ab-1, Ab-2Ab-3, Ab-4
Ag
1
2
3
4
Polyclonal antibodies
1 2
34
Isolate B-cellsSpleen
+
Myeloma cells
3 41 2
HybridomaCells
1 2 3 4
Ab-1
Polyclonal vs Monoclonal Antibodies
Ab-2 Ab-3 Ab-4
Monoclonal antibodies
Crossreactivity Occurs when an Antiserum is raised against antigen A but also reacts with antigen B
Antigen A and B share epitopes Antigen A and B have similar(but not identical) epitopes
Antibody Structure Summary
• Produced by B-cells
• Y shape, Four polypeptide chains, Ig domains
• Constant and Variable regions
• 5 classes - IgG, IgM, IgD, IgA, IgE
• CDR, Hypervariable regions
• Epitope recognition
Examples of Commercial uses for Ab’s
• Pregnancy test
• Rh disease therapy
• Antitoxin serum - Antivenin, rabies, etc
• Anti-cancer monoclonals - will be covered later in the course
Urine
Inputwindow
Pregnancywindow
Controlwindow
Reactionzone
Mouse Monoclonalanti-HCG
Ab-enzyme conjugate
Immobile Polyclonal
anti-HCG Ab+ dye
substrate
Immobile Goat Anti-mouse Ab
+ dye substrate
Pregnancy test
http://mcb.berkeley.edu/courses/mcb150/lecture5/Preg%20Test%20movie.swf
Rh diseaseRh disease (Erythroblastosis Fetalis) - Hemolytic disease of a newborn - occurs in Rh- woman carrying Rh+ fetus
RhoGAM - human plasma with anti-Rh+ (D antigen) antibodies. Works by binding any fetal RBC’s before the mother is able to produce an immune response and form anti-D IgG
Antitoxin serum
• Serum can be used in the prophylaxis and/or treatment of rabies, botulism, diphtheria, gas gangrene, snake and spider bites
• Antivenin Crotalidae Polyvalent (ACP) - horse serum based antivenom - gold standard for snake bites but can cause “serum sickness”
• New types of antitoxins– Fragmented antivenin (CroFab) where only the Fab fragment (sheep-
based) is used
– Humanized antibodies derived from mouse sources
Generation of Ig diversity in B cells
Unique organization
- Only B cells can express Ig protein
- Gene segments
-Light chain
, , , , - Heavy Chain
present on three chromosomes
The Gene Rearrangement Concept
• Germline configuration
• Gene segments need to be reassembled for expression
• Sequentially arrayed
• Occurs in the B-cells precursors in the bone marrow (soma)
• A source of diversity BEFORE exposure to antigen
Light Chain Variable - V, JConstant - C
Heavy ChainVariable - V, D, JConstant - C
V-variable, J-joining, D-diversity gene segments; L-leader sequences
Figure 2-14
Gene rearrangements during B-cell development
V-variable, J-joining, D-diversity gene segments; L-leader sequences
- 30 V & 4 pairs J & C (light chain) chs22
– 40 V & 5 J & 1 C (50% have 2x V) (light chain) chs2
H – 65 V & 27 D & 6 J chs14
CDR1 and CDR2 CDR3
V-region of light chain consists of one V and one J segment
C-region is encoded by one C segment
Variability comes from the V segment
D-diversity
CDR1 and CDR2 CDR3
Two recombination events needed to combine 3 segments to make the Variable region
Random Recombination of Gene segments is one factor contributing to
diversity
* Only one light chain loci gives rise to one functional polypeptide!
10,530 = 3,369,600 Ig molecules 120*)(200* + X
Mechanism of Recombination
• Recombination signal sequences (RSSs) direct recombination- V and J (L chain)- V D J (H chain)
• RSS types consist of:- nonamer (9 base pairs)
- heptamer (7 base pairs)- Spacer- Two types: [7-12-9], [7-23-9]
• RSS features:- recognition sites for recombination enzymes- recombination occurs in the correct order
(12/23 rule)
• V(D)J recombinase: all the protein components that mediate the recombination steps
• RAG complex: Recombination Activating Genes (RAG-1 and RAG-2) encode RAG proteins only made in lymphocytes
• Recombination only occurs through two different RSS bound by two RAG complexes (12/23 rule)
• DNA cleavage occurs to form a single stranded hairpin and a break at the heptamer sequences
• Enzymes that cut and repair the break introduce Junctional Diversity
Mechanism of Somatic Recombination
JunctionalDiversity
Recombination +
http://www.blink.biz/immunoanimations/#
Figure 2-19
Figure 2-18 part 2 of 3Junctional Diversity
• Nucleotides introduced at recombination break in the coding joint corresponding to CDR3 of light and heavy chains
- V and J of the light chain- (D and J) or (V and DJ) of the heavy chain
• P nucleotides generate short palindromic sequences
• N nucleotides are added randomly - these are not encoded
• Junctional Diversity contributes 3 x107 to overall diversity!
Generation of BCR (IgD and IgM)
• Rearrangement of VDJ of the heavy chain brings the gene’s promoter closer to C and C
• Both IgD and IgM are expressed simultaneously on the the surface of the B cell as BCR - ONLY isotypes to do this
• Alternative splicing of the primary transcript RNA generates IgD and IgM
• Naïve B cells are early stage B cells that have yet to see antigen and produce IgD and IgM
Figure 2-21Alternative Splicing of Primary Transcript to
generate IgM or IgD
Summary Biosynthesis of IgM in B cells
Figure 2-23• Long cytoplasmic tails interact with intracellular signaling proteins
• Disulfide-linked
• Ig and Ig - invariant
- Transmembrane proteins
- Dual-function
1) help the assembled Ig reach the cell surface from the ER
2) signal the B cell to divide and differentiate
Mature B cell
Principle of Single Antigen Specificity
• Each B cell contains two copies of the Ig locus (Maternal and Paternal copies)
• Only one is allowed to successfully rearrange - Allelic Exclusion
• All Igs on the surface of a single B cell have identical specificity and differ only in their constant region
• Result: B cell monospecificity means that a response to a pathogen can be very specific
Chromosome 22 2 14
* *
DNA hybridization of Ig genes can diagnose B-cell leukemias
Peripheral blood from healthy patient is made up of mostly neutrophils
Peripheral blood from a leukemia patient has an abnormally high proportion of B-cells.
Cancer cells are derived from one clonal line of B-cell which has V and C chains rearranged next to each other
Generation of B cell diversity in Ig’s before Antigen Encounter
1. Random combination of V and J (L chain) and V, D, J (H chain) regions
2. Junctional diversity caused by the addition of P and N nucleotides
3. Combinatorial association of Light and Heavy chains(each functional light chain is found associated with a different functional heavy chain and vice versa)
Concept of Combinatorial Association
Immature B cell Mature naive B cell(expressing BCR -
IgM and IgD)
1. Development before antigen
2. Development after antigen
plasma cell(expressing BCR and secretes Antibodies)
Developmental stages of B cells
Processes occurring after B cells encounter antigen
• Processing of BCR versus Antibody1. Plasma cells switch to secreted Ab2. Difference occurs in the c-terminus of the heavy chain3. Primary transcript RNA is alternatively processed to
yield transmembrane or secreted Ig’s
• Somatic Hypermutation1. Point mutations introduced to V regions2. 106 times higher mutation rate3. Usually targets the CDR
• Affinity maturation - mutant Ig molecules with higher affinity are more likely to bind antigen and their B cells are preferentially selected
• Isotype switching
RNA processing to generate BCR or Antibody
MC - membrane coding SC - secretion coding
Somatic Hypermutation(random introduction of point mutations)
Mutations occur throughout the V domain - especially CDROccurs on both gene copies - but only one expresses proteinAID - Activation induced Cytidine DeaminaseUNG - Uracil-DNA glycosylase
Process of Affinity Maturation
Hypermutation
IgG
IgM
IgM
Hypermutation leads to different B-cells
Mutant BCRs have various affinities
Higher affinity BCR’s are preferentially selected to mature
Isotype switching
1. IgM is the first Ab that is secreted in the IR
2. IgM is pentameric and each H chain can bind complement proteins
3. Isotypes with better effector functions are produced by activated B cells
4. Rearrangement of DNA using SWITCH regions - all C genes preceded by switch sequence (except - start from the gene and any other C gene (plus sequential)
5. Regulated by cytokines secreted by T cells
Switch regions flank each C gene (except delta)
Mu to any other isotypeSequential switchingAID is importantAID deficiency ---> Hyper IgM syndrome (antibodies not made after IgM and IgD )
Stages at which Isotype Switching occurs
antigen-independent
stem cell pre-B cell immature B cell(IgM +)
mature B cell(IgM , IgD +)
IgG
IgAIgE
IgMantigen-dependent
isotype switching
Does Isotype Switching occur in one B cell?
1. Activated B cell resides in the Germinal Center -some individuals will mature directly into
plasma cells
2. Some B cells in the germinal center divide and undergo hypermutation and/or isotype switching
3. After this stage they cannot divide and the higher affinity ones are selected
4. These cells can mature to plasma cells
5. End result: The B cell makes a different antibody isotype but with the same specificity
Figure 2-31 part 2 of 2Immunoglobulin classes
1. C regions determine the class of antibody and their effector function
2. Divided into Subclasses based on relative abundance in serum
3. Each class has multiple functions
1. IgM and IgG can bind complement2. IgG crosses placenta3. Receptors for constant regions (Fc Receptors)
- IgG (FcG receptors): mac, neutrophils, eosinophils, NK cells, others- IgE (FcE receptors): mast cells, basophils, others
IgM(plasma cells in lymph nodes,
spleen, and bone marrow and circulate in
blood/lymph)
Low affinity binding to antigen
via multiple binding sites
Exposure of constant region
Activate complement
Killdirectly
Phagocytose
Hypermutation and affinity maturation
Two binding sites sufficient for
strong binding
Isotype switching to IgG
Initial Immune Response mediated by IgM
IgG(lymph nodes,
spleen, and bone marrow)
Circulates in blood and lymph (most abundant Ab in internal
fluids)
Extravasation,Higher affinity
binding to antigen
Multiple effector
functions
Recruit phagocytes
Neutralize antigens
Activate complement
Monomeric IgAPlasma cells in lymph nodes, spleen, bone
marrow
Secreted into Blood
Dimeric IgAlymphoid tissue associated with
mucosal surfaces
Secreted into Gut lumen & body
secretions
Effector functions1. Mainly neutralization2. Minor opsonization
and activation of complement
IgA most made of any Ab
IgEPlasma cells in lymph nodes or germinal centers
Bind strongly to Mast cells via Fc
receptor
Cross-linking of receptor bound
Ab releases histamine and
other activatorsInflammation- Expulsion of large pathogens- Allergies
IgD
• Very low concentration in serum
• Primarily found with IgM on naïve mature B cells
• Function is not clear
Figure 2-32
Summary: Generation of B-cell diversity
• Diversity before Antigen exposure (Antigen Independent)- Random Recombination- Junctional Diversity- Combinatorial association
• Diversity after Antigen exposure (Antigen Dependent)- Switch to secreted Ab- Somatic Hypermutation- Affinity Maturation- Isotype Switching
• Immunoglobulin Classes- Properties- Effector functions