Generation of Antibody Diversity

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We are surrounded by countless bacteria, viruses, fungi, and other microorganisms that are constantly seeking entry into the favorable growth medium that human bodies provide. Antigens can be eliminated by the inflammatory response, the action of protein complement and also the engulfment of macrophages. The major barrier to infection include those allow the host to remove only specific antigens quickly and efficiently by recognizing each antigen with tremendous specificity, and this makes the diversity of antibody most important.

There are seven ways of antibody diversification that have been identified in mice and humans lately. They are: 1. Multiple germ-line gene segments; 2. Somatic recombination: Combinatorial V-(D)-J joining; 3. Junctional diversity; 4. P-region nucleotide addition (P-addition); 5. N-region nucleotide addition (N-addition); 6. Somatic hypermutation; 7.Combinations of light and heavy chains.

Multiple germ-line gene segments:

In germ-line DNA the H-chain gene is consists of V (variable).D (diversity) and J (junction) gene segments. An inventory research reveals 51 VH, 25 D, 6 JH, 40 Vκ, 5 Jκ, 31 Vλ, and 4 Jλ gene segments can be presented in one human.

The immunoglobulin loci of different individuals may contain different amount of gene segments types. Those numbers of the gene segments in the mouse are different from that of human and less precision. About 85 Vκ , 134 VH gene segments, as well as 4 functional JH, 4 functional Jκ, 3 functional Jλ, and an estimated 13 DH gene segments, and only three Vλ gene segments are presented.

Somatic recombination: Combinatorial V-D-J joining

The possibility of gene rearrangements can be achieved by the calculation of the random combination of any of the 51 VH , with the 27 DH and with the 6 JH gene segments in humans (51 27 6 = 8262 possible combinations). Similarly, the random combination of any 40 Vκ with 5 Jκ gene segments and 30 Vλ with 4 Jλ gene segments can generate 200 and 120 possible combinations at the κ locus and λ locus, respectively. However, though the potential of diversity of antibody combining-site in humans can be really high, different individual only carries a particular subset that is not affected by the magnitude.

Junctional diversity

Antibody diversity can be increased further by the imprecision of joining that occurs at the DH-JH, VHDHJH, and VL-JL boundaries.

The signal sequences are always joined precisely, however the joining of the coding sequences can be imprecise. Junctional diversity can leads to nonproductive rearrangements (e.g. frame shift mutation that produce non-functional protein), or productive combinations such as the insertion of different number of amino acid into the third hyper-variable region (CDR3) in immunoglobulin H-chain and L-chain DNA where it is mainly contribution to antigen binding, or by the insertion of extra nucleotides during joining process, thus, the generation of antibody diversity occurs.

P-region nucleotide addition

The cleavage of the hairpin structure that was formed by the turning of the end of the variable-region and attached signal gene sequence, can leaves a short single strand, which can be complemented by P-nucleotides, thus a palindromic sequence can be generated in the coding joint. The sequence variation of the coding joint which is caused by the variation of hairpin cutting position, can contribute to the diversity of anybodies.

N-region nucleotide addition

The addition of N-nucleotides also involves the asymmetrical cleavage of the hairpin ends generated in recombination. The rearranged heavy-chain genes (in CDR3) that contain the variable-region coding joints have some short amino acid sequences that are encoded by nucleotides. Those nucleotides were added during the V, D and J joining process catalyzed by the reaction that a terminal deoxynucleotidyl transferase (TdT) was involved, which is responsible for the addition of N-nucleotides (≤15) at the coding joints of DH-JH and VH-DHJH. Since N regions are consisting of random sequences, the additional heavy-chain diversity generated can be quite large.

Somatic Hypermutation

Somatic hypermutation is a kind of additional antibody diversity which is generated in rearranged variable-region gene. The mutation may cause individual nucleotides in VJ or VDJ units replaced with alternatives. The frequency of mutation among the A,T,C,G are all the same. The frequency can be approached to 10-3 per base pair per generation, which is about 105 times higher than that of the spontaneous mutation rate. Normally, somatic mutation occurs in hyper-variable regions within germinal centers, and occurs during secondary immune response after class switching (the mature resting B cells begin to synthesis the H-chain of different classes).

Somatic hypermutation is targeted to rearranged V regions located within a DNA sequence, which is containing about 1500 nucleotides, thus it introduces large numbers of incompletely substitutions

Claudia Berek and Cesar Milstein's experiment* reveals that in the CDR1 and CDR2 hyper-variable regions (mice), the number of mutations and the progressively increased total affinity of the antibodies for phOx hapten (experimental material) is due to the primary, secondary, and tertiary immunizations.

Combinations of light and heavy chains

According to the previous discussion, the theoretical number of the potential combinations of heavy-chain and light-chain is 2,644,240 in humans (8262 H-chain genes320 L-chain genes). This number can be inaccurate in an individual. The recombination process of VH and VL is not completely random, since the gene segments of VH, D, or VL are not used at the same frequency.

To conclude, the new sequences generated by junctional diversity, P-nucleotide and N-nucleotide addition, as well as somatic hypermutation together make an significant contribution to antibody diversity, and the antigen specificity can reach to the number of 109-1011 in total (in mouse).

*Refers to Thomas J Kindt, Richard A Goldsby, Barbara A Osborne, and Janis Kuby. (2007) Immunology. 6th edition, W.H.Freeman and Company. Chapter5. Figure 5-14