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