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ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

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ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY WHAT ARE ANTIBODIES? Antigen specific proteins produced by plasma cells Belong to immunoglobulin superfamily ... – PowerPoint PPT presentation

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Title: ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY


1
ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL
DIVERSITY
2
WHAT ARE ANTIBODIES?
  • Antigen specific proteins produced by plasma
    cells
  • Belong to immunoglobulin superfamily
  • Located in blood and extravascular tissues,
    secretions and excretions
  • Bind pathogenic microorganism and their toxins in
    extracellular compartments
  • Secreted form of immunoglobulins

3
WHAT ARE IMMUNOGLOBULINS?
  • Antigen specific proteins produced by B
    lymphocytes
  • Belong to immunoglobulin superfamily
  • Bound to surface of B lymphocytes
  • Function as binding (receptor) sites for specific
    antigens
  • Antigen receptor sites on mature B lymphocytes
  • IgM
  • IgD
  • Membrane-bound form of immunoglobulins

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WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY
  • Proteins with structural feature first defined in
    immunoglobulins
  • Characteristic structural feature
  • Sequence of Domains providing stable conformation
  • Domain
  • Polypeptide (100 to 110 amino acids) chain folded
    into sandwich (2 slices of bread) held together
    by disulfide bond
  • IG superfamily members
  • Antibodies, B cell receptors, T cell receptors,
    MHC molecules and others

6
STRUCTURE OF ANTIBODIES
  • Antibodies are glycoproteins composed of
  • Polypeptide chains and carbohydrate
  • Monomeric structure
  • Polypeptide chains
  • 2 identical heavy chains
  • 2 identical light chains
  • Polypeptide chains joined by disulfide bonds
  • Carbohydrate

7
STRUCTURE OF ANTIBODIES
  • Polypeptide chains have variable and constant
    regions
  • Variable
  • N (amino)-terminal of polypeptide chain
  • Antigen binding site
  • Constant
  • C (carboxyl)-terminal of polypeptide chain
  • Binding sites for cell surface receptors and
    complement
  • Structure represented by the letter Y
  • Y shaped molecule cleaved by protease papain
  • Fragment antigen binding (Fab)
  • Fragment crystallizable (Fc)

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CLASSES (ISOTYPES) OF IMMUNOGLOBULINS
  • Classes based on constant region of heavy chains
  • Immunoglobulin A (IgA)
  • Immunoglobulin D (IgD)
  • Immunoglobulin E (IgE)
  • Immunoglobulin G (IgG)
  • Immunoglobulin M (IgM)
  • Differentiation of heavy chains
  • Length of C region, location of disulfide bonds,
    hinge region, distribution of carbohydrate
  • Classes have different effector functions

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CLASSES (ISOTYPES) OF IMMUNOGLOBULINS
  • Additional classification based on light chains
  • Kappa
  • Lambda
  • Each IG has either kappa or lambda, not both
  • IgG kappa
  • IgG lambda
  • No functional differences between light chains

13
ITS GREEK TO ME
  • Heavy chains, light chains and other molecules of
    the immune system identified using letters of the
    Greek alphabet

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THREE DIMENSIONAL STRUCTURE OF ANTIBODIES
  • Antibodies function in setting of infectious
    process
  • Proteolytic enzymes, salt and pH differences
  • Antibodies remain stable based on
  • Sequence of domains
  • Single domain consists of
  • 100 110 amino acids folded into compact and
    stable conformation
  • Domains
  • Variable (V)
  • Single V domain in H and L chains
  • Constant (C)
  • Single C domain in L chains
  • Three to four (C) domains in H chains

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ANTIGEN BINDING SITES OF IMMUNOGLOBULINS
  • Antigen binding sites formed from hypervariable
    regions
  • Heavy chain V domain
  • Light chain V domain
  • Hypervariable regions of V domains
  • Amino acid sequence differences concentrated
  • Flanked by less variable framework regions
  • Three hypervariable regions in each V domain
  • Hypervariable regions also called
  • Complementarity-determining regions (CDR)

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ANTIGEN BINDING SITES OF IMMUNOGLOBULINS
  • Antigen binding sites vary with size and shape of
    antigen
  • Part of antigen to which antibody binds
  • Antigenic determinant (Epitope)
  • Antigen-Antibody binding based on non-covalent
    forces
  • Hydrogen bonds
  • Affinity
  • Strength of binding of one molecule to another by
    a single binding site
  • Avidity
  • Overall strength of binding between two molecules

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ANTIBODIES AS DIAGNOSTIC AND THERAPEUTICS AGENTS
  • Based on specificity and affinity of antibodies
  • Both applications require large quantities of
    identical antibodies
  • Monoclonal antibodies
  • Monoclonal antibodies are produced using
    hybridoma cell line
  • Hybridoma cell line
  • Derived from single antibody producing cell fused
    with myeloma cell (neoplastic plasma cell)

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IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE
ENCOUNTER WITH ANTIGEN
  • Immune system capable of producing a limitless
    number of different immunoglobulins/antibodies
  • Mechanism
  • Genes for IG organized differently
  • Genes exist as nonfunctional segments
  • Variable (V), Joining (J), Diversity (D),
    Constant (C)
  • Genes are inherited in this form
  • Germline form (germline configuration)

25
IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE
ENCOUNTER WITH ANTIGEN
  • Expression
  • Gene segments must be rearranged into functional
    gene
  • Gene Rearrangement
  • Takes place during development of B-cells
  • Mechanism of somatic recombination
  • Genes for IG located at 3 chromosomal locations
  • Heavy chain locus on chromosome 14
  • Kappa light chain locus on chromosome 2
  • Lambda light chain locus on chromosome 22

26
GERMLINE ORGANIZATION OF HUMAN IG HEAVY CHAIN AND
LIGHT CHAIN LOCI
  • Lambda light chain locus
  • Gene segments
  • 30 (V), 4 (J) and 4 (C)
  • Kappa light chain locus
  • Gene segments
  • 40 (V), 5 (J) and 1 (C)
  • Heavy chain locus
  • Gene segments
  • 65 (V), 27 (D), 6 (J) and 9 (C)

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CONSTRUCTION OF LIGHT CHAIN AND HEAVY CHAIN
VARIABLE REGIONS
  • Light chain
  • Constructed from 2 segments
  • 1 (V) segment
  • 1 (J) segment
  • Heavy chain
  • Constructed from 3 segments
  • 1 (V) segment
  • 1 (D) segment
  • 1 (J) segment

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SOMATIC RECOMBINATION
  • Performed by enzymes with cut and rejoin DNA
  • Directed by
  • Recombination signal sequences (RSS)
  • Recombination signal sequences
  • Recognition sites for enzymes
  • Recombination occurs between different types
  • 9 / 12 / 7
  • 9 / 23 / 7
  • Mechanism follows the 12/23 rule
  • Ensures segments joined in correct order

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MECHANISMS OF GENETIC DIVERSITY IN V-REGION OF
IMMUNOGLOBULINS
  • Random combination of
  • V and J segments in light chain genes
  • V, D and J segments in heavy chain genes
  • Addition of P (palindromic) and N (non-templated)
    nucleotides at junctions of gene segments during
    recombination
  • Junctional diversity
  • Association of H and L chains in different
    combinations

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CONSTRUCTION OF B-CELL SURFACE IMMUNOGLOBULINS
  • Following rearrangement of VH gene segments, two
    CH loci are transcribed
  • IgM
  • IgD
  • M and D constant segments
  • Located nearest variable segments
  • M and D transcript processed by
  • Cleavage, polyadenylation and splicing
  • IgM and IgD enter endoplasmic reticulum

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SURFACE IMMUNOGLOBULINS ASSOCIATED WITH PROTEINS
TO COMPLETE ANTIGEN RECEPTOR
  • In ER, IgM and IgD associated with transmembrane
    proteins
  • Ig-alpha
  • Ig-beta
  • Transmembrane proteins
  • Transport M and D to B cell surface
  • Communication of antigen binding to inside of B
    cell
  • Tails interact with intracellular signaling
    molecules
  • Complex of IgM and IgD with Ig-alpha and Ig-beta
    forms
  • B-cell receptor

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DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS
ENCOUNTER ANTIGEN
  • Mature, naïve B cell has membrane bound IgM and
    IgD antigen receptors
  • Binding of antigen initiates proliferation and
    differentiation of B-cells into plasma cells
  • During differentiation, B cells switch from
    making immunoglobulin to antibody M and D
    isotypes
  • IgM
  • Produced in large amounts
  • Provides protective immunity
  • IgD
  • Produced in small amounts
  • No known function

41
MECHANISM OF SWITCHING FROM IMMUNOGLOBULIN TO
ANTIBODY
  • Surface and secreted forms derived from same
    heavy chain gene by alternative RNA processing
  • Each heavy chain C gene has
  • Membrane coding (MC) region
  • Secretion coding (SC) region
  • Mechanism involves a switch in cleavage,
    polyadenylation and splicing
  • From pAm region to pAs region

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DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS
ENCOUNTER ANTIGEN
  • Following antigen activation of B-cells,
    additional diversification occurs in V domain by
  • Somatic hypermutation
  • Somatic hypermutation
  • Introduction of random single nucleotide
    substitutions (point mutations) throughout V
    regions of H and L chains
  • Mechanism poorly understood
  • More common in hypervariable regions (CDRs)

44
OUTCOME OF SOMATIC HYPERMUTATION
  • Gives rise to some antibodies with higher
  • Affinity for antigen
  • Affinity
  • Strength of binding of one molecule to another by
    a single binding site
  • Higher affinity antibodies are produced as immune
    response proceeds
  • Affinity maturation

45
THE PRIMARY HUMORAL IMMUNE RESPONSE
  • Immune response initially produces IgM antibodies
    then switches to IgG antibodies
  • Question
  • Why switch from IgM to IgG?
  • Answer
  • Limited effector mechanisms for IgM
  • Range of effector mechanisms for IgG
  • Mechanism
  • Isotype or class switching

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ISOTYPE OR CLASS SWITCHING
  • Process by which B cell changes class of IG
    produced while preserving antigenic specificity
  • Involves somatic recombination which attaches
    different heavy chain constant region to variable
    region
  • Occurs only during active immune response
  • Mechanisms involves recombination between
  • Switch sequences (regions)

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CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF
IMMUNOGLOBULINS
  • Subclasses are numbered according to plasma
    concentration

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FUNCTIONS AND PROPERTIES OF ANTIBODY
  • Neutralization
  • Direct inactivation of pathogen or toxin thereby
    preventing its interaction with human cells
  • Opsonization
  • Coating of pathogens for more efficient
    phagocytosis
  • Activation of complement
  • More efficient phagocytosis
  • Direct killing

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IgM ANTIBODY OF THE IMMUNE RESPONSE
  • First isotype produced in primary response
  • May or may not be produced in secondary response
  • Produced before B cells undergo somatic
    hypermutation
  • Occurs as pentamer with J chain
  • Found primarily in blood and lymph
  • Multiple binding sites confers high avidity and
    compensates for low affinity of monomers
  • Highly effective in complement activation
  • Functions as rheumatoid factor

54
IgG ANTIBODY OF THE IMMUNE RESPONSE
  • Second isotype produced in primary response
  • Primary isotype of
  • Secondary immune response
  • Memory immune response
  • Represents approximately 75 of total serum IG
  • Four subclassses (1-4)
  • Different effector functions
  • Transported across placenta
  • Functions as rheumatoid factor

55
IgA ANTIBODY OF THE IMMUNE RESPONSE
  • Two subclasses (IgA1 and IgA2) and two forms
    (monomeric and dimeric)
  • Monomeric
  • Located in blood and extracellular spaces
  • Predominately IgA1
  • Ratio of IgA1 to IgA2 is 101
  • Functions as rheumatoid factor
  • Dimeric
  • Located in mucous membranes and secretions
  • Predominately IgA2
  • Ratio of IgA2 to IgA1 is 32
  • J chain like IgM

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IgE AND IgD ANTIBODIES OF THE IMMUNE RESPONSE
  • IgE
  • Binds with high affinity to receptors on mast
    cells, basophils and activated eosinophils
  • Longer half-life when cell bound
  • Initiates a strong inflammatory reaction to
    parasites
  • Involved in allergic reactions
  • IgD
  • Antigen receptor on mature B-cells
  • No other known function

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