Genetic basis of antibody diversity

1. GENETIC BASIS OF ANTIBODY DIVERSITY BY, REKHA.M.S

2. 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.

3. ANTIBODY STRUCTURE An 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).

4. 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 V L and one V H. The specificity of any combining site is determined by its amino acid sequence. There exist at least 10 6 unique combining sites .

5. 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.

6. Theories Germ-Line Model Somatic-Variation Model Two-Gene Model

7. 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.

8. TWO-GENE MODEL Dreyer and Bennett p roposed 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.

9. 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.

10. 3 Ig genes

11. 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.

12. 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.

13. 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.

14. 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

15. 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.

16. 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 .

17. 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.

18. 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.

19. 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.

20. Combinatorial Diversity Specific antibody V H V L V ≈200 J 5 V ≈200 D 12 J 6   V ≈100 J 6 1000 600 1600 14,400 2.3 X 10 7

21. 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.

22. Junctional Diversity Joining of V and the J, or V, D and J segments To form CODING JOINT is Imprecise Addition and Deletion of Nucleotides Diversity of the Hypervariable region 3 (CDR3)

23. Deletions

24. Additions Terminal deoxyribonuceotidyl transferase (TdT)

25. 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.

26. N nucleotide addition at joining segments: the addition of random bases

27. 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

28. 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 10 4 Possible combinations over 10 10

29. Location of variability occurs within CDR regions of V domains (antigen binding sites)

30. 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 C δ). 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 .

31. Class switching among constant regions: generation of IgG, IgA and IgE with same antigenic determinants—idiotypes

32. Isotype switching can occur by: Switch recombination (Deletion of DNA) - primary mechanism of isotype switching - is irreversible Alternative splicing of primary RNA transcript (rare) - Explains co-expression of multiple isotypes by a single B cell.

33. Synthesis, assembly and secretion of immunoglobulins

34. 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

35. 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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