GENETIC BASIS OF ANTIBODY DIVERSITY

BY,

REKHA.M.S

GENETIC BASIS OF ANTIBODY DIVERSITY

OBJECTIVES: Define the following terms: allelic exclusion, isotype switching,

affinity maturation, antibody repertoire, alternative RNA splicing, recombination signal sequence.

Describe the genes that encode Ig Heavy and Light chains. Describe the sequence of Ig gene rearrangement that occurs

during B-cell differentiation. Discuss how diversity in antibody specificity is achieved. Discuss the mechanisms of heavy chain class switching. Calculate the number of possible Igs which can be produced

from given number of V, J, D, and C genes.

ANTIBODY STRUCTUREAn antibody molecule is composed of two identical Ig heavy chains (H) and two identical light chains (L), each with a variable region (V) & constant region (C).

One of the important feature of the vertebrate immune system is its ability to respond to an apparently limitless array of foreign antigens.

As immunoglobulin (Ig) sequence data accumulated,virtually every antibody molecule studied was found to contain a unique amino acid sequence in its variable region but only one of a limited number of invariant sequences in its constant region.

The genetic basis for this combination of constancy and tremendous variation in a single protein molecule lies in the organization of the immunoglobulin genes.

An Ab combining site is made up of one VL and one VH.

The specificity of any combining site is determined by its amino acid sequence.

There exist at least 106 unique combining sites .

Three families of Ig genes exist in mammals, one encoding HEAVY chains, another KAPPA chains, and the third LAMBDA chains.

Each of these clusters contains one or more constant region genes and a number of variable region gene segments.

The formation of a complete variable region of a light or heavy chain requires the joining of two or three separate genetic elements by a process of GENE REARRANGEMENT; a separate DNA rearrangement in the heavy-chain complex is required for subsequent CLASSSWITCHING.

Both germ-line and somatic events contribute to antibody diversity, including COMBINATORIAL JOINING, SOMATIC MUTATION and COMBINATORIAL ASSOCIATION.

THEORIES Germ-Line Model Somatic-Variation Model Two-Gene Model

GERM-LINE THEORY – For every kappa-chain V-region there exists one

unique germ-line gene. A particular antibody-forming cell selects one of these and expresses it in unmodified form.

SOMATIC THEORY – Only a single germ-line gene exists for all kappa-

chain V-regions. A particular antibody-forming cell expresses this

gene following a process of somatic mutation, which results in each cell expressing a different version of this gene.

TWO-GENE MODEL

Dreyer and Bennett proposed the Two-Gene Model. In 1965-Proposed that two separate genes encode a

single immunoglobulin heavy or light chain, one gene for the V region (variable region) and the other for the C region (constant region).

They suggested that these two genes must somehow come together at the DNA level to form a continuous message that can be transcribed and translated into a single Ig heavy or light chain.

Moreover, they proposed that hundreds or thousands of V-region genes were carried in the germ line,whereas only single copies of C-region class and subclass genes need exist.

STRUCTURE AND EXPRESSION OF IMMUNOGLOBULIN GENES

Three families of immunoglobulin genes exist, each on a separate chromosome. kappa genes- chromosome 2 lambda genes- chromosome 22 heavy chain genes- chromosome 14

Each family consists of a series of V-regions genetically linked to one or more C-regions.

3 IG GENES

STRUCTURE OF THE KAPPA CHAIN COMPLEX

This complex consists of a large number of V-region genes (about 55) genetically linked (by a long stretch of DNA) to a single copy of the constant region gene.

An additional cluster of five short gene segments called J-segments is located a few thousand base pairs upstream (5’ direction) of the C-region gene, and each of these codes for the last 13 amino acids of the variable region.

EXPRESSION OF KAPPA GENE: The first step in expression of this gene is DNA REARRANGEMENT-

involving the joining of one V-region and one J-segment, each chosen at random in any given B-cell.

The resulting structure have the DNA which was originally between the selected V and J genes is cut out and lost in the form of a closed circular molecule.

V-region gene segments which happen to reside outside this excised segment are retained, although they are no longer relevant to expression.

This process is unique to immunoglobulin (and T-cell receptor) genes.

The process of TRANSCRIPTION starts at the beginning of the rearranged V-region and continues past the end of the C-region, resulting in an immature mRNA.

The large intervening sequence (“intron”) between the J-segments and theC-region is removed by the process of RNA SPLICING, resulting in the mature mRNA.

MOLECULAR BASIS OF KAPPA GENE EXPRESSION

Germ-line configuration:

Following DNA rearrangement:

Following transcription:

Following RNA splicing:

Following translation:

Following peptide processing:

L V J C

COOHNH2

V J C

COOHNH2

2) Rearranged DNA

3) Precursor mRNA

4) Mature mRNA

5) Precursor PEPTIDE

6) Mature PEPTIDE

1) Germ-line DNA

J1J2J3J4J5 CV1 V2 V3 . . .3'5'

V2 J3 CV13'5'

V2 J3 C

3'5'

V2 J3 C

5' 3'3'utAAAAA

transcription of mRNA

The structure and mechanism of expression of lambda chains and heavy chains are similar to what we have just described for kappa chains--all have J-SEGMENTS, all show DNA REARRANGEMENT, TRANSCRIPTION, RNA SPLICING, TRANSLATION and proteolytic cleavage of the LEADER POLYPEPTIDE.

Heavy chain gene structure is somewhat more complex- there exists an additional cluster of gene segments (known as “D”, for “diversity”, segments) which each encodes four amino acids between the V-region cluster and the J-segments.

DNA rearrangement for H-chains thus involves two events, joining of a V with a D, and joining of the D with a J-segment.

Transcription and the other processes discussed above take place as they do for kappa genes.

In each case, the end result is a polypeptide whose amino acid sequence has been determined by three or four separate genetic elements, and which is incorporated into the final immunoglobulin molecule

ALTERNATE SPLICING IN B-CELLS

the simultaneous synthesis of IgM and IgD by a single B-cell. This is the only example of a normal cell simultaneously producing

two different kinds of immunoglobulin. The explanation derives from the fact that-mu (μ) and delta (δ)

constant region genes are adjacent to one another in the heavy chain gene complex.

Using the same rearranged heavy chain V/D/J complex, a B-cell can make two kinds of mRNA--it can transcribe from the V-region through the end of the Cμ gene and make IgM, or it can transcribe all the way through the Cδ gene and make IgD by splicing out the Cμ region together with the intervening sequence during RNA splicing.

Two different mRNAs can thus be made from a single gene complex. It should be emphasized that alternate splicing of RNA is a mechanism

used by many other genes to generate diverse protein products.

mu-chain mRNA

delta-chain mRNA

Dashed lines in RNA indicate an intervening sequence removed during RNA processing

(Rearranged DNA)V J C CDL

SIMULTANEOUS SYNTHESIS OF IgM AND IgD IN B-CELLS BY ALTERNATE RNA SPLICING

V JDL CAAAAAAA

V JDL CAAAAAAA

IMPORTANCE IgM and IgD synthesis in B-cells can occur

simultaneously and continuously. The mu and delta chains are produced from the same

chromosome, and not from the two different allelic copies.

This is an extension of allelic exclusion is known as haplotype exclusion.

In the choice of whether a secreted versus a membrane-bound form of Ig is produced.

MECHANISMS FOR GENERATING ANTIBODY DIVERSITY

Multiple germ-line gene segments Combinatorial V-(D)-J joining Junctional flexibility P-region nucleotide addition (P-addition) N-region nucleotide addition (N-addition) Somatic hypermutation Combinatorial association of light and heavy

chains.

MULTIPLE GERM-LINE GENE SEGMENTS

There are 51 VH, 25 D, 6 JH,40 V, 5 J, 31 V, and 4 J gene segments.

Many psuedogenes also influence. Multiple germ-line V, D, and J gene segments

clearly do contribute to the diversity of the antigen-binding sites in antibodies.

COMBINATORIAL V-(D)-J JOINING

The ability to create many different specificities by making many different combinations of a small number of gene segments.

Each V, D and J segments of DNA are flanked by special sequences (RSS—recombination signal sequences) of two sizes.

Single turn and double turn sequences (each turn of DNA is 10 base pairs long).

Only single turn can combine with a double turn sequence.

Joining rule ensures that V segment joins only with a J segment in the proper order.

Recombinases join segments together.

COMBINATORIAL DIVERSITY

Specificantibody

VH

VL

V≈200

J5

V≈200

D12

J6

V≈100

J6

1000600

1600

14,400

2.3 X 107

JUNCTIONAL FLEXIBILITY

The enormous diversity generated by means of V, D, and J combinations is further augmented by a phenomenon called junctional flexibility.

Recombination involves both the joining of recombination signal sequences to form a signal joint and the joining of coding sequences to form a coding joint.

joining of the coding sequences is often imprecise.

Cause addition/deletion of nucleotides in CDR.

Resulting in antibody diversity.

Junctional Diversity

Joining of V and the J, or V, D and J segmentsTo form CODING JOINT is Imprecise

Addition and Deletion of Nucleotides

Diversity of the Hypervariable region 3 (CDR3)

Deletions

Additions

Terminal deoxyribonuceotidyl transferase (TdT)

P-REGION NUCLEOTIDE ADDITION (P-ADDITION),N-REGION NUCLEOTIDE ADDITION(N-

ADDITION)

P-Addition:-Adds Diversity at Palindromic Sequences.

During recombination some nucleotide bases are cut from or add to the coding regions (p nucleotides).

Up to 15 or so randomly inserted nucleotide bases are added at the cut sites of the V, D and J regions (N nucleotides).

By TdT (terminal deoxynucleotidyl transferase) a unique enzyme found only in lymphocytes.

N NUCLEOTIDE ADDITION AT JOINING SEGMENTS: THE ADDITION OF RANDOM BASES

SOMATIC HYPERMUTATION ADDS EVEN MORE VARIABILITY B cell multiplication introduces additional

opportunities for alterations to rearranged VJ or VDJ segments

These regions are extremely susceptible to mutation compared to “regular” DNA, about one base in 600 is altered per two generations of dividing (expanding) lymphocyte population

COMBINATION OF HEAVY AND LIGHT CHAINS ADDS FINAL DIVERSITY OF VARIABLE REGION

8262 possible heavy chain combinations 320 light chain combinations Over 2 million combinations P and N nucleotide additions and subtractions

multiply this by 104

Possible combinations over 1010

LOCATION OF VARIABILITY OCCURS WITHIN CDR REGIONS OF V DOMAINS (ANTIGEN BINDING SITES)

CLASS/ISOTYPE SWITCHING

Is the conversion of an immunoglobulin from one isotype to another(e.g. IgG to IgE) while retaining the same antigen specificity.

Switching is dependent on antigenic stimulation and is induced by cytokines released by helper T cells and requires engagement of CD40L. [e.g. IL-4 triggers switching from IgM to IgE or IgG4 (humans); IFN-γ

triggers switching from IgM to IgG2a (mice)]. Cyokines are thought to alter chromatin structure making switch

sites more accessible to recombinases for gene transcription. Involves switch sites located in introns upstream of each CH

segment (except Cδ). Switch sites consist of multiple copies of conserved repeat

sequences [(GAGCT)n GGGGGT)]; where n can vary from 3-7. Class switching occurs usually in activated B cells (including

memory cells) and not in naïve B cells and involves heavy chain genes.

These cells (you will recall) already have rearranged VDJ genes at the DNA level and were producing IgM and IgD.

CLASS SWITCHING AMONG CONSTANT REGIONS: GENERATION OF IGG, IGA AND IGE WITH SAME ANTIGENIC DETERMINANTS—IDIOTYPES

ISOTYPE SWITCHING CAN OCCUR BY:

Switch recombination (Deletion ofDNA) -primary mechanism of isotypeswitching -is irreversible Alternative splicing of primary RNA transcript (rare) -Explains co-expression of multipleisotypes by a single B cell.

SYNTHESIS, ASSEMBLY AND SECRETION OF IMMUNOGLOBULINS

REGULATION OF IG GENE TRANSCRIPTION

Each lymphocyte rearranged gene has regulatory sequences that control gene expression

Promoters: initiation sites of RNA transcription Enhancers: upstream of downstream that

transcription from the promoter sequence Silencers: down-regulate transcription in germline

cells Gene rearrangement brings enhancer and

promoter regions close together and eliminates silencer regions allowing transcription

UNDERSTANDING OF IMMUNOGLOBULIN STRUCTURE AND FORMATION HAS OPENED UP A NEW WORLD OF POSSIBILITIES

Monoclonal antibodies Engineering mice with human immune systems Generating chimeric and hybrid antibodies for

clinical use Abzymes: antibodies with enzyme capability

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