Ch4. Antibody Structure and the Generation of B-cell diversity
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Transcript of Ch4. Antibody Structure and the Generation of B-cell diversity
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Ch4. Antibody Structure and the
Generation of B-cell diversity
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B-Cell Receptor (BCR)
• Immunoglobulin(Ig), antibody• specificity: one antibody for one antigen or a few
different antigens• diversity: as high as 1016 of antibody repertoire; in
practice about 109
• B cell is stimulated to differentiate into plasma cells
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B cell activation
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Basic structure of Immunoglobulin
Heterodimers ; 2 light + 2 heavy chains by disulfide and non-covalent bond
• V (variable) region ; Ag binding site (CDR)• C (constant) region ; effector function(host cell receptor binding), glycosylation
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Basic structure of immunoglobulin
• Fab ; fragment antigen binding• Fc ; fragment crystallizable
• H chain ; 50 kDa• L chain ; 25 kDa
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Immunoglobulin fold ; multiple domainHinge : flexible antigen binding
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Immunoglobulin isotypes (5 classes)
• 5 heavy chain types : Isotype – determine the class of antibody• IgM (), IgG (), IgA (), IgD (), IgE ()
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IgG
80% of the total serum IgsDifferent biological activity
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IgM
5-10% of total IgsJ chain ; polymerization
Pentameric structure ; 10 Ag binding sites more efficient than IgG
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IgA
10-15% of total IgsPredominant in external secretion ; mucosal transport
Monomer, polymer (2-4) ; J-chain
Poly-Ig receptor – secretary component
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Resist to protease
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IgE
Immediate hypersensitivity reaction ; AllergyP-K reaction Allergen - IgE/Fc ; degranulation
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IgD
Membrane bound Ig in mature B cell
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Structure of heavy and light chain
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Hypervariable regions (HV)
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Hypervariable regions
• CDR : complementarity determining region
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Epitope : antigenic determinant
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Epitopes vs. Ab
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Monoclonal antibody
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Production of hybridoma
Select in HAT(hypoxanthine, aminopterin, thymidine) medium
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Clinical use of mAbs
• Diagnostic reagent ; pregnancy, pathogen, blood level of drugs, tumor Ag• Imaging ; detect tumor metastasis• Therapeutic reagents ; immunotoxin• Abzyme ; catalytic antibody (Ab + enzyme)
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FACS
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chimeric: mouse V region + human C regionhumanized: mouse CDR region + all other human region
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Genetic regulation Immunoglobulin gene expression
Recombination : • genetic material is broken and then joined to different region • heavy and light chain gene rearrangement • isotype switching (class switching)
Alternative splicing: • different combinatorial use of exons • IgM and IgD switching
Alternative polyadenylation: • different use of polyadenylation sites; transmembrane region
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Genetic model for Ig structure
• Vast diversity of antibody specificities• Maintaining the constancy in constant (C) region• Isotypes with the same antigenic specificity
• Germ-line vs. somatic variation • Germ-line theory ; genome of germ cells contains
a large repertoire of Ig genes• Somatic-variation theory ; relatively small number of Ig gene on genome
mutation and gene rearrangement
• Two gene model ; two genes encode a single polypeptide many V region + 1 copies of C region class gene
• Ig gene rearrangement during th B cell differentiation (by Tonegawa)
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Organization of imunoglobulin genes
leader (L) peptide ; signal for transport to ERV (variable), J (joining), D (diversity), C (constant)
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Gene rearrangement
Somatic recombination of gene segments
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Gene rearrangement
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Multigene organization of Ig genes
Gene segment (coding sequence)
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Recombination signal sequences (RSS)
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Recombination signal sequences (RSS)
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Joining the gene segments by recombination
RAG : recombination activating gene
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Generation of antibody diversity
• Multiple germ-line gene segments
• Combinatorial V-(D)-J joining
• Junctional flexibility
• P (palindromic) nucleotide addition (P-addition)
• N (nontemplated) nucleotide addition (N-addition)
• Somatic hypermutation
• Combinatorial association of light and heavy chains
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Junctional flexibility
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Junctional flexibility
P- & N- addition ; different reading frame
Productive rearrangement ; increase antibody diversity
Non productive rearrangement
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Generation of junctional diversity : 3 X 107
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Somatic hypermutation increase diversity
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Membrane-bound vs. Secreted form of Ig ; alternative polyadenylation
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Heavy chain gene expression by RNA processing ; alternative splicing
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Class switching (Isotype switching)
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Function and properties of the human Ig isotypes
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Immature B cell ; gene rearrangement, RNA processing, mIgM, mIgD
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Mature B cell ; Antigen stimulation Activation & differentiation, Class switching
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Fine tuning of antibody function
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• Neutralization: – for masking
• opsonization: – for efficient phagocytosis
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Membrane-bound immunoglobulin (BCR)
Intracellular signaling
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Ig superfamily
Common evolutionary ancestor ; gene duplication & divergenceImmunoglobulin fold (~110 a.a) domain
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Fc receptors (FcR)
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Allelic exclusion
Product of Ig gene inhibit further gene rearrangement of counter alleleAntigenic specificity
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