ch4. antibody structure and the generation of b-cell diversity
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Ch4. Antibody Structure and the Generation of B-cell diversity. B-Cell Receptor (BCR). Immunoglobulin(Ig), antibody specificity: one antibody for one antigen or a few different antigens diversity: as high as 10 16 of antibody repertoire; in practice about 10 9 - PowerPoint PPT PresentationTRANSCRIPT
Ch4. Antibody Structure and the
Generation of B-cell diversity
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
B cell activation
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
Basic structure of immunoglobulin
• Fab ; fragment antigen binding• Fc ; fragment crystallizable
• H chain ; 50 kDa• L chain ; 25 kDa
Immunoglobulin fold ; multiple domainHinge : flexible antigen binding
Immunoglobulin isotypes (5 classes)
• 5 heavy chain types : Isotype – determine the class of antibody• IgM (), IgG (), IgA (), IgD (), IgE ()
IgG
80% of the total serum IgsDifferent biological activity
IgM
5-10% of total IgsJ chain ; polymerization
Pentameric structure ; 10 Ag binding sites more efficient than IgG
IgA
10-15% of total IgsPredominant in external secretion ; mucosal transport
Monomer, polymer (2-4) ; J-chain
Poly-Ig receptor – secretary component
Resist to protease
IgE
Immediate hypersensitivity reaction ; AllergyP-K reaction Allergen - IgE/Fc ; degranulation
IgD
Membrane bound Ig in mature B cell
Structure of heavy and light chain
Hypervariable regions (HV)
Hypervariable regions
• CDR : complementarity determining region
Epitope : antigenic determinant
Epitopes vs. Ab
Monoclonal antibody
Production of hybridoma
Select in HAT(hypoxanthine, aminopterin, thymidine) medium
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)
FACS
chimeric: mouse V region + human C regionhumanized: mouse CDR region + all other human region
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
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)
Organization of imunoglobulin genes
leader (L) peptide ; signal for transport to ERV (variable), J (joining), D (diversity), C (constant)
Gene rearrangement
Somatic recombination of gene segments
Gene rearrangement
Multigene organization of Ig genes
Gene segment (coding sequence)
Recombination signal sequences (RSS)
Recombination signal sequences (RSS)
Joining the gene segments by recombination
RAG : recombination activating gene
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
Junctional flexibility
Junctional flexibility
P- & N- addition ; different reading frame
Productive rearrangement ; increase antibody diversity
Non productive rearrangement
Generation of junctional diversity : 3 X 107
Somatic hypermutation increase diversity
Membrane-bound vs. Secreted form of Ig ; alternative polyadenylation
Heavy chain gene expression by RNA processing ; alternative splicing
Class switching (Isotype switching)
Function and properties of the human Ig isotypes
Immature B cell ; gene rearrangement, RNA processing, mIgM, mIgD
Mature B cell ; Antigen stimulation Activation & differentiation, Class switching
Fine tuning of antibody function
• Neutralization: – for masking
• opsonization: – for efficient phagocytosis
Membrane-bound immunoglobulin (BCR)
Intracellular signaling
Ig superfamily
Common evolutionary ancestor ; gene duplication & divergenceImmunoglobulin fold (~110 a.a) domain
Fc receptors (FcR)
Allelic exclusion
Product of Ig gene inhibit further gene rearrangement of counter alleleAntigenic specificity