Chapter 5 Organization and Expression of Ig Genes

Oct 26 & 31, 2006

你需要學習的課題 :

1.抗體基因是如何組成的？

2.抗體基因重組 (rearrangement) 的機制

3.抗體的多樣性 (diversity) 是如何產生的？

4.細胞膜上的抗體如何轉變為分泌性抗體？

5.抗體的類別 (class) 如何變換？ - class switching

Central Feature of Ab Molecules:

1. Vast diversity of Ab specificities

2. A variable (V) region at the N-terminal end and a constant (C) region at the C-terminal end of Ab molecules

3. Different classes (or isotypes) of Ab (e.g., IgG and IgM) with identical V-region sequences (antigenic specificity)

The Two-gene model of Dryer and Bennett

(1965)

Two separate genes encode a single Ig H or L chain, one gene for the V region

and the other for the C region.

The suggestion that two genes encoded a single polypeptide contradicted the existing one gene-one polypeptide principle and was without precedent ( 先例 ) in any known biological system.

Verification of the Dryer and Bennet Hypothesis (by Tonegawa and Hozumi, 1976)

First direct evidence that separate genes encode the V and C regions of Ig and that the genes are rearranged in the course of B-cell differentiation.

- Tonegawa was awarded the Nobel Prize for this work in 1987.

Demonstration of DNA Deletion at an Ig Locus

Non-B cells:

sperm or liver cells

B-cells

Demonstration of DNA Deletion at an Ig Locus

deleted sequence

比 大，因此在電泳時跑得比較慢

Multigene organization of Ig genes

-Chain Multigene Family

Mouse:

V region: 2 VV gene segments 4 JJgene segments (3 are functional) C region: 4 CCgene segments

Human: 30 VV, 4 JJ and 4 CC segments

κ-Chain Multigene Family

Mouse:

V region: ~ 85 VV gene segments 5 JJgene segments (4 are functional) C region: 1 CCgene segment

Human: 40 VV, 5 JJ and 1 CC segments

-Chain Multigene Family

Mouse:

V region: ~ 134 VV gene segments 13 DDHH gene segments 4 JJHHgene segments C region: 8 CCgene segments

Human: 51 VV, 27 DDHH, 6 JJ and 9 CC segments

V-Region Gene Rearrangements

- The H-chain V-region genes rearrange first, then the L-chain V-region genes.

- The rearrangements occur in an ordered sequence, but they are random events.

- The arrangements of Ig and TCR genes are the only known site-specific DNA rearrangements in vertebrates.

H-Chain DNA Undergoes V-D-J Rearrangements

(1st rearrangement)

(2nd rearrangement)

A mature , immunocompetentB cell expresses both IgM & IgD with identical antigenic specificity on its surface.

L-Chain DNA Undergoes V-J Rearrangements

introns are removed

Mechanism of V-region DNA Rearrangements

Two unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment

One-turn RSS: located at 3’ to each V5’ to each Jand both sides of each DH gene segment

Two-turn RSS: located at 3’ to each V & VH and

5’ to each J & JH gene segment

Recombination Signal Sequences (RSS)

CACAGTG

23

nt

ACAAAAACC

GTGTCAC

12

nt

TGTTTTTGG

Vλ Jλ

//

One turn/two-turn joining rule

The rule ensures that VH, DH, and JH segments join in proper order and that segments of the same type do not join each other.

Gene Segments Are Joined by Recombinases

- Recombination-Activating Genes: RAG-1, RAG-2

- The proteins encoded by RAG-1 and RAG-2 act synergistically and are required to mediate V-(D)-J joining.

- Terminal deoxynucleotidyl transferase (TdT), another lymphoid-specific gene product, is also involved in V-(D)-J rearrangement.

Process of Recombination of Ig Gene Segments

Double Strand Break Repair

Terminal deoxy-nucleotidyl Transferase

Defects in Ig-Gene Rearrangements

RAG-1-/- or RAG-2-/- mice: - lack RAG-1 or RAG-2 - cannot start the recombination process

SCID (severe combined immunodeficiency) mice: - lack double strand break repair (DSBR) enzymes - can carry out synapsis, introduce d.s. breaks - cannot properly join the coding sequences

Imprecise Joining - productive and nonproductive rearrangements

- productive rearrangement in one allele is enough

- If rearrangement is not produced, the B cell dies by apoptosis.

Ig-gene Rearrangements May Be Nonproductive

!!

!!

Only 1/3 attempts at VL – JL joining, and 1/3 subsequent attempts at VH – DHJH joining, are productive.

As a result, < 1/9 (11%) of the early-stage pre-B cells in the bone marrow progress to maturity and leave the bone marrow as mature immunocompetent B cells.

Allelic Exclusion Ensures a Single Antigenic Specificity

A single B cell is only specific for a single epitope !!!

Once a productive rearrangement is attained, its encoded protein is expressed and the presence of this protein acts as a signal to prevent further gene rearrangement.

(1) (2)

* active alleles

Generation of Ab Diversity

Antibody Diversity

Seven means of generation of Ab diversity:

1. Multiple germ-line V, D, and J gene segments 2. Combinatorial V-(D)-J joining 3. Junctional flexibility 4. P-region nucleotide addition (P-addition) 5. N-region nucleotide addition (N-addition) 6. Somatic hypermutation 7. Combinatorial association of light and heavy chains

Junctional Flexibility Adds Diversity- 4 different joinings of V21- J1 in pre-B cell lines

(Flexible) (Precise)

Since CDR3 makes a major contribution to Ag binding by the Ab molecule, amino acid changes generated by junctional flexibility can make a major contribution to Ab diversity.

P-Addition Adds Diversity at Palindromic Sequences

{Palindromic sequences}

N-Addition Adds Considerable Diversityby Addition of Nucleotides

add new (N) -nucleotides

- Up to 15 N-nucleotides can be added to both the DH - JH and VH - DHJH joints.

- Thus, a complete H - chain V region is encoded by a VHNDHNJH unit.

- N regions appears to consist of wholly random sequences

P-nucleotide 及 N-nucleotide addition

有些什麼優缺點？

Somatic Hypermutation Adds Diversity in Already-rearranged Gene Segment

- Somatic hypermutation occurs only within germinal centers, structures that form in secondary lymphoid organs within a week or so of immunization with an Ag that activates a T-cell-dependent B-cell response.

- Somatic hypermutation occurs at a frequency approaching 10-3/bp/generation. This rate is at least 100,000-fold higher than the spontaneous mutation rate, about 10-8/bp /generation, in other genes.

- B cells with higher-affinity Ig receptors will be preferentially selected for survival because of their greater ability to bind to the Ag. ----- Affinity MaturationAffinity Maturation

Experimental Evidence for Somatic Mutation in V region of Ig Genes

Most of the mutations are clustered in the CDR1 and CDR2 hypervariable region.

Antibody Diversity

Seven means of generation of Ab diversity:

1. Multiple germ-line V, D, and J gene segments 2. Combinatorial V-(D)-J joining 3. Junctional flexibility 4. P-region nucleotide addition (P-addition) 5. N-region nucleotide addition (N-addition) 6. Somatic hypermutation – after Ag stimulation 7. Combinatorial association of light and heavy chains

Class Switching Among C-Region Genes

Organization of H chain

V region C region

After antigenic stimulation of a B cell, the H-chain DNA can undergo a further rearrangement in which the VHDHJH unit can combine with any CH gene segment. This process is called class switching.

Class (isotype) switching

- Class-specific switch recombinases may bind to switch regions and facilitate DNA recombination.

- Cytokines secreted by activated TH cells have been shown to induce B cells to class switch to a particular isotype.

- IL-4, for example, induces class switching from C to C1 and then from C1 to C.

Switch regions Class Switching from C to C1

Class Switching from C1 to C a circular excision product

Expression of Ig Genes

Co-expression of membrane forms of and H-chains by Alternative RNA Processing

Expression of Membrane or Secreted Ig mRNAs

先暫時不考慮 C 的表現

(sIgM) (mIgM)

Expression of Membrane or Secreted Ig mRNAs

Expression of Membrane or Secreted IgM Molecules

Therefore, processing of an Ig H-chain primary transcript can yield different mRNAs, which explains how a single B cell can produce secreted or membrane-bound forms of a particular Ig and simultaneously express IgM and IgD.

Synthesis, Assembly, and Secretion of Igs

Membrane Form of Igs Are Anchored to the Membrane

Regulation of Ig-Gene Transcription

Overview of B-cell Development and Ig Expression