Question

Explain (with as much detail as possible) the genetic mechanisms that account for the diversity in antibodies.

Answer 1

Antibodies are molecules that bind with antigen to present antigen to immune cells for their elimination from the system. Almost every antibody has a different amino acid sequence in its variable region but only a limited number of invariant sequences in its constant region. A single immunoglobulin heavy or light chain is encoded by multiple gene segments; these gene segments cannot be transcribed and translated until they are rearranged into functional genes. There are seven mechanisms of antibody diversification-

1. Multiple germ-line gene segments

The kappa and lambda light chains, and the heavy chains of immunoglobulin are encoded by different gene segments present in different chromosomes. The light chain family contain V (variable), J (junction) and C (constant) segments and the heavy chain family contains V, D (diversity), J and C segments. Variable region of light chains are encoded by rearranged VJ segments, and VDJ segments for heavy chains. The human germ-line DNA reveals that there are 51VH, 25D, 6 JH, 40Vkappa, 5Jkappa, 31Vlambda and 4Jlambda gene segments. When these many gene segments are rearranged, it is understood that the diversity will be immense.

2. Combinatorial V-D-J joining

The VDJ gene segments are arranged randomly by recombination to increase the antibody diversity. Variable region gene rearrangement produces mature immunocompetent B cells which are committed to produce antibody with a binding site encoded by the rearranged variable sequences. Rearrangement of the heavy chain constant region genes will generate changes in the immunoglobulin class type (the isotypes). DNA sequences studies have revealed the presences of recombination signal sequences (RSSs) are present flanking the V-D-J gene segments. VDJ recombination is catalyzed by VDJ recombinase. David Schatz, Marjorie Oettinger and David Baltimore identified recombination activating genes (RAG-1) and (RAG-2) in 1990. Recombination results in the formation of a coding joint (falling between the coding sequences) and a signal joint (between the RSSs).

3. Junctional flexibility

The immense diversity resulting from VDJ recombination is further augmented by junctional flexibility, where productive gene rearrangements encode alternative amino acids at each coding joint. Functional diversity generated by junctional flexibility falls in the third hypervariable region (CDR3) which makes a major contribution to antigen binding by antibody.

4. P-region nucleotide addition (P-addition)

Reaction catalyzed by RAG-1 and RAG-2 produces a hairpin structure at the cut end of the coding sequence and a flush, 5’ phosphorylated, double strand break at the signal sequence. The hairpin is cleaved that leaves a short single strand at the end of the coding sequence, subsequent addition of complementary sequences (P nucleotides) to this strand generate a palindromic sequence in the coding joint. Variation in the cleavage site of hairpin increases diversity of the antibodies.

5. N-region nucleotide addition (N-addition)

Addition of N nucleotides at the cut ends of V, D and J coding sequences of the heavy chain by the enzyme terminal deoxynucleotidyl transferase contributes towards increasing antibody diversity.

6. Somatic hypermutation

Additional antibody diversity is regenerated by somatic hypermutation in the rearranged variable region genes. Somatic hypermutations are base pair substitutions in the VJ or VDJ regions, thereby affecting the antibody affinity for antigen. Following exposure to antigen, B cells expressing antibodies with higher affinity towards the antigen will be preferentially selected by a process called affinity maturation.

7. Combinatorial associations of light and heavy chains

Humans have the potential to generate (51V x 27D x 6J = 8262 heavy chain genes) and total 320 light chain genes (40V x 5J = 200 kappa chain genes and 30V x 4J = 120 lambda chain genes. These genes can be rearranged to produce significant diversity in antibodies.