Chapter 5 Organization and Expression of Immunoglobulin Genes Dr. Capers

Kindt • Goldsby • Osborne

Kuby IMMUNOLOGY

Sixth Edition Chapter 5

Organization and Expression of Immunoglobulin Genes

Copyright © 2007 by W. H. Freeman and Company

 How does antibody diversity arise?

 What causes the difference in amino acid sequences?

 How can different heavy chain constant regions be associated with the same variable regions?

 In germ-line DNA, multiple gene segments code portions of single immunoglobulin heavy or light chain  During B cell maturation and stimulation, gene segments are shuffled leaving coding sequence for only 1 functional heavy chain and light chain ○ Chromosomal DNA in mature B cells is not the same as germ-line DNA

 Dreyer and Bennett – 1965  2 separate genes encode single immunoglobulin heavy or light chain ○ 1 for the variable region  Proposed there are hundreds or thousands of these ○ 1 for the constant region  Proposed that there are only single copies of limited classes  Greater complexity was revealed later  Light chains and heavy chains (separate multi gene families) are located on different chromosomes

 DNA rearrangement: produces variable region  Later mRNA splicing: produces constant region

 Kappa (κ) and lamda (λ) light chain segments: ○ L – leader peptide, guides through ER ○ V VJ segment codes for variable region ○ J ○ C – constant region  Heavy chain ○ L ○ V VDJ segment codes for variable region ○ D ○ J ○ C

Variable-region gene rearrangements

 Variable-region gene rearrangements occur during B-cell maturation in bone marrow ○ Heavy-chain variable region genes rearrange first ○ Then light-chain variable region ○ In the end, B cell contains single functional variable-region DNA sequence ○ Heavy chain rearrangement (“class switching”) happens after stimulation of B cell

Mechanism of Variable-Region DNA rearrangements  Recombination signal sequences (RSSs) ○ Between V, D, and J segments ○ Signal for recombination ○ 2 kinds 12 base pairs (bp) – 1 turn of DNA 23 bp – 2 turns of DNA 12 can only join to 23 and vice versa

Mechanism of Variable-Region DNA rearrangements  Catalyzed by enzymes ○ V(D)J recombinase  Proteins mediate V-(D)-J joining ○ RAG-1 and RAG-2

Same transcriptional orientation Opposite transcriptional orientation  Gene arrangements may be nonproductive ○ Imprecise joining can occur so that reading frame is not complete ○ Estimated that less than 1/9 of early pre-B cells progress to maturity  Gene rearrangement video:  http://www.youtube.com/watch?v= AxIMmNByqtM

Allelic Exclusion

 Ensures that the rearranged heavy and light chain genes from only 1 chromosome are expressed

Generation of Antibody Diversity

 Multiple germ-line gene segments  Combinatorial V-(D)-J joining  Junctional flexibility  P-region nucleotide addition  N-region nucleotide addition  Somatic hypermutation  Combinatorial association of light and heavy chains ○ This is mainly in mice and humans – other studied species differ in development of diversification

Ab diversity – Multiple gene-line segments AND combination of those segments

Ab diveristy – junctional flexibility

 Random joining of V-(D)-J segments ○ Imprecise joining can result in nonproductive rearrangements ○ However, imprecise joining can result in new functional rearrangements

Ab diversity – P-addition and N-addition

Ab diversity – somatic hypermutation

 Mutation occurs with much higher frequency in these genes than in other genes  Normally happens in germinal centers in lymphoid tissue

Class Switching

 Isotype switching  After antigenic stimulation of B cell  V H D H J H until combines with C H segment gene  Activation-induced cytidine deaminase (AID)  Somatic hypermutation  Gene conversion  CLASS-SWITCH recombination  IL-4 also involved

μ→δ→γ→ε→α IgM→IgD→IgG→IgE→IgA

Ig Gene Transcripts

 Processing of immunoglobulin heavy chain primary transcript can yield several different mRNAs ○ Explains how single B cell can have secreted and membrane bound Ab

Regulation of Ig-Gene Transcription  2 major classes of cis regulatory sequences in DNA regulate   Promoters – promote RNA transcription in specific direction Enhancers – help activate transcription  Gene rearrangement brings the promoter and enhancer closer together, accelerating transcription

Antibody Engineering

   Monoclonal Abs used for many clinical reasons (anti- tumor Ab, for instance) If developed in mice, might produce immune response when injected ○ Can be cleared in which they will not be efficient ○ Can create allergic response Creating chimeric Abs or humanized Abs are beneficial