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 of antibody repertoire; in practice about 10 9 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 domain Hinge : 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 Igs Different biological activity

IgM 5-10% of total Igs J chain ; polymerization Pentameric structure ; 10 Ag binding sites more efficient than IgG

IgA 10-15% of total Igs Predominant in external secretion ; mucosal transport Monomer, polymer (2-4) ; J-chain Poly-Ig receptor – secretary component

Resist to protease

IgE Immediate hypersensitivity reaction ; Allergy P-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 region humanized: 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 ER V (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)

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

P- & N- addition ; different reading frame Productive rearrangement ; increase antibody diversity Non productive rearrangement

Generation of junctional diversity : 3 X 10 7

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 & divergence Immunoglobulin fold (~110 a.a) domain

Fc receptors (FcR)

Allelic exclusion Product of Ig gene inhibit further gene rearrangement of counter allele Antigenic specificity